4.7 Principles of transplantation immunology 392

4.7 Principles of transplantation immunology 392

ESSENTIALS The survival of transplanted organs remains limited by the body’s immune responses, which are designed to discriminate between ‘self’ and ‘non-​self’ or ‘altered-​self’, and many of the complications of transplantation result from the crude nature of our attempts to suppress these. The immune response to transplanted tissue The immunological response that follows transplantation of tissue between genetically non​identical individuals is complex. (1) Inflammatory signals are generated at the site of transplantation and activate cells of the innate immune system, promoting the pres- entation of alloantigens—​particularly molecules of the major histo- compatibility complex—​to recipient T cells. (2) Activation and clonal expansion of alloreactive recipient T cells results in the production of populations of effector lymphocytes. (3)  Activated lympho- cytes, along with macrophages and neutrophils, migrate to the graft. (4) Many effector mechanisms contribute to graft destruction, including the delayed-​type hypersensitivity response, direct cytotox- icity, and B-​cell alloantibody production. Clinical features of allograft rejection In clinical practice allograft rejection is categorized by the timing in relation to the transplantation procedure and the dominant arm of the immune system involved, cellular or humoral. (1) Hyperacute re- jection—​caused by preformed complement-​fixing antibodies against allogeneic major histocompatibility complex molecules or ABO antigens (modern cross-​match techniques have made this extremely rare). (2) Acute rejection—​may be predominantly due to acute cel- lular or acute antibody-​mediated rejection; leads to a sudden de- terioration in graft function over days to weeks. (3) Chronic graft dysfunction (‘chronic rejection’)—​may be partly due to chronic ac- tivation of the immune system; causes gradual deterioration in graft function occurring over weeks to month. Immunosuppressive therapy Many different immunosuppressive regimens for solid organ
transplantation are in clinical use. The agents employed in- clude glucocorticoids, antiproliferative agents (e.g. azathioprine, mycophenolate mofetil), calcineurin inhibitors (e.g. ciclosporin, tacrolimus), mammalian target of rapamycin (mTOR) inhibitors (e.g. sirolimus, everolimus), depleting antibodies (e.g. antithymocyte globulin, alemtuzumab), and other biological agents (e.g. daclizumab and basiliximab, both of which are directed against CD25, the high-​ affinity IL-​2 receptor α-​chain, and belatacept, a fusion protein linked to CTLA-​4 (CD152) that blocks T-​cell costimulation). Clinical perspective and future prospects Modern immunosuppressive therapy has improved 1-​year graft survival to above 90% for kidney and liver recipients, and above 80% for most other solid organ transplants, but late graft loss and the adverse effects of chronic immunosuppression remain a signifi- cant problem. Challenges include improving donor organs prior to transplantation and the development of better assays to monitor the immune response following transplantation and make it easier to individualize immunosuppressive therapy. Ongoing trials of novel therapies provide the exciting possibility of donor-​specific hyporesponsiveness or even operational transplantation tolerance. Introduction Since the first successful transplant of a kidney between identical twins in 1954, transplantation has progressed from being an experi- mental procedure to a routine clinical therapy offering immense benefits for patients with organ failure. However, the survival of transplanted organs remains limited by the body’s immune re- sponses, and many of the complications of transplantation result from the crude nature of our attempts to suppress these. The immune system has evolved to protect the individual from invasion by pathogenic microorganisms as well as malignant or premalignant mutation of the individual’s own cells, hence strin- gent discrimination of ‘self’ from ‘non-​self’ or ‘altered-​self’ is cru- cial to ensure immunological responses are directed correctly and to limit damage to self tissues that might otherwise occur during the response to a foreign pathogen. To complicate matters, the immune system is continually exposed to new antigens, and since many of these are harmless or even beneficial (e.g. bacteria composing the in- testinal flora), the immune response generated must be proportional to the threat that each new antigen indicates. 4.7 Principles of transplantation
immunology Elizabeth Wallin and Kathryn J. Wood

4.7  Principles of transplantation immunology 393 The character of an immune response is determined partly by the particular antigens involved and partly by the context in which these antigens are encountered. In particular, priming of T cells with antigen from sites of inflammation is much more likely to produce an aggressive immune response aimed at clearing the antigen than priming with antigen in the absence of inflammatory signals. In the context of the highly inflammatory microenvironment sur- rounding transplantation, the introduction of tissue from a genet- ically disparate (allogeneic) individual (see Table 4.7.1) containing a multitude of non-​self peptides (alloantigens) triggers a vigorous immune response that almost invariably results in destruction of the transplanted tissue unless active steps are taken to suppress the im- mune system. Acute allograft rejection is a complex T-​cell-​dependent process, summarized in Fig. 4.7.1. Inflammation triggered by the retrieval of the donor organ or tissue and the transplant procedure itself generates so-​called ‘danger’ signals that activate both the innate and adaptive immune systems. Triggering of the innate immune system creates an environment that promotes activation of the adaptive response, which includes T cells and B cells. T-​cell in- tegration of signals delivered through recognition of alloantigen, costimulation, and inflammation leads to activation, clonal ex- pansion, and differentiation of donor-​reactive lymphocytes into effector cells. While these events begin in the transplant itself, activation of the adaptive immune system occurs predominately in the lymphoid tissue draining the transplant site. Innate and in- flammatory changes in the graft then provide homing signals for the migration of mature effector lymphocytes to orchestrate the destruction of the transplant. While T cells, both cytotoxic and helper, are absolutely required for transplant rejection, recent evidence has shown that the roles played by B cells and cells of the innate immune system, including monocytes and natural killer (NK) cells, are more important than previously thought, and can increase the speed and magnitude of the response to the graft, as well as initiating long-​term immunological memory directed against the donor antigens. In clinical practice allograft rejection is frequently categorized depending on the timing in relation to the transplantation procedure or the dominant arm of the immune system involved (Table 4.7.2). Internationally accepted scoring systems have been developed for most solid organ transplant biopsies to characterize rejection and fa- cilitate the decision-​making process regarding the implementation of antirejection therapy. The best known of these is the Banff scoring system for renal allografts, but similar systems are in place for liver, heart, lung, and vascularized composite allografts. This chapter will outline the cellular and molecular events in- volved in alloantigen recognition and subsequent allograft rejection, as well as the mechanism of action of the current pharmacological agents available in clinical practice to suppress graft rejection. Initiation of response to graft The initiation of a response to transplanted tissue is complex and multilayered, involving all aspects of the immune system—​ inflammation, innate immune cells, and an adaptive response. Multiple factors, both donor and recipient, interact and affect the magnitude of this response. Broadly these can be divided into innate and adaptive responses, with the innate consisting of both inflam- mation and innate immune cell responses. Innate Both the physical process of removing and reimplanting tissue for transplantation, also the state of the donor organ, can have a pro- found impact on the initiation of responses against the graft. The type of donation (cadaveric heart beating, cadaveric non-​heart Table 4.7.1  Transplantation terminology Autograft Tissue transplanted from one part of an individual’s body to another (e.g. skin grafts in patients with burns; vascular grafts for coronary or peripheral vascular disease) Isograft Tissue transplanted between genetically identical (syngeneic) members of the same species (e.g. grafts between monozygotic twins; grafts between members of the same inbred strain of mouse or rat) Allograft Tissue transplanted between genetically disparate (allogeneic) members of the same species (e.g. grafts between unrelated humans; grafts between different inbred strains of mice or rats) Xenograft Tissue transplanted between individuals of different species (e.g. pig to human, rat to mouse) Generation of DAMPs, ROS, HSPs, cytokines, complement products Ischaemia/ reperfusion injury Surgical injury (retrieval/ implantation) Brain death, donor factors, e.g. infection Invasion of innate cells increasing inflammation Migration of APCs to SLOs, setting up adaptive immune response Macrophages, neutrophils, NK cells, complement CD8 cytotoxic T-cell damage CD4 T cell help B-cell antibody responses GRAFT REJECTION Fig. 4.7.1  Overview of allograft rejection. Multiple cell types and effector mechanisms contribute to graft destruction, including both innate and adaptive arms of the immune system. DAMPS, damage associated molecular patterns; ROS, reactive oxygen species; HSPs, heat shock proteins; APCs, antigen-​presenting cells; SLOs, secondary lymphoid organs; NK cells, natural killer cells.

394 SECTION 4  Immunological mechanisms beating or living donor) as well as the age and physical health of the donor can all affect the degree of preimplantation damage that oc- curs within the graft. Ischaemia from hypoxia and hypoperfusion, as well as the cytokine storm associated with brainstem death, initiates a sequence of changes in gene expression within the donor tissue that have a profound influence on the immunological response of the recipient. Adding to this the injury from direct surgical trauma, and further injury from reactive oxygen species released when the ischaemic tissue is reperfused, and the graft environment becomes profoundly proinflammatory. The initial responders to this inflammatory milieu are innate im- mune cells, which express pattern-​recognition receptors. These en- able them to recognize markers of tissue injury and to detect the presence of pathogens. The best characterized of these are the Toll-​ like receptors (TLRs) which bind to phylogenetically conserved mo- lecular features unique to microorganisms, as well as endogenous molecules that are produced as a consequence of tissue injury, such as reactive oxygen species, activated complement components, and heat shock proteins. In the context of transplantation, damage associated molecular patterns (DAMPs) lead to graft infiltration with innate immune cells and the local production of inflammatory mediators, chemokines (chemoattractant cytokines), and P-​selectin (CD62P), an adhesion molecule necessary for leucocyte transmigration into tissue. This identifies the transplanted tissue as a site of inflammation and trig- gers the migration of donor-​derived antigen-​presenting cells (APCs) to recipient lymphoid tissue, as well as the recruitment of recipient immune cells into the graft. Recently, particular interest has fallen on the role of monocytes, which are one of the earliest invaders (Fig. 4.7.1), and their role in initiating an alloimmune response in the case of a genetically non​identical donor. Many of the events initiated by the transplant operation are a non​specific response to tissue damage, and the invasion of recipient cells occurs regardless of whether the transplantation procedure occurs between genetically identical or genetically disparate indi- viduals. However, recent work has shown that recipient monocytes invading the graft respond differently to genetically disparate in- dividuals and are capable of differentiating self from non-​self. The proinflammatory milieu is sensed by TLRs, but in the case of gen- etically identical individuals, the stimulation is insufficient to fully activate these monocytes and the response subsides, leading to reso- lution of inflammation and healing. In allogeneic transplants, the monocytes differentiate into inflammatory dendritic cells, capable of presenting antigen and providing both costimulation and inflam- matory cytokine production. Adaptive In addition to the innate immune response, an adaptive response is initiated in the context of allogeneic transplants. Although the innate immune reaction occurs in auto-​, iso-​ and allo-​grafts, only in the case of genetically non​identical individuals is an adaptive re- sponse both initiated and sustained by the alterations in the innate response. There are several components to this (see Fig. 4.7.1) al- though T cells remain the key players. T-​cell allorecognition Animals that lack T cells do not reject fully major histocompatibility complex (MHC)-​mismatched allografts or xenografts, but adop- tive transfer of wild-​type T cells to these animals is able to restore allograft rejection. In contrast, B-​cell deficient mice reject cardiac allografts at control rates. In clinical transplantation, therapies that deplete peripheral T cells are highly effective at reversing episodes of acute rejection. These observations highlight the role of T cells in transplant rejection. Several different approaches have been used to evaluate the rela- tive contribution of CD8+ and CD4+ T cells to allograft rejection. The available animal data indicate that, in general, CD4+ cells are essential for allograft rejection. However, CD8+ cells contribute to rejection and in certain circumstances are capable of rejecting MHC class I mismatched allografts in the absence of CD4+ cell help. There are many cell surface and intracellular molecules that are variable, or polymorphic, between different members of the same species, and recognition of this genetic variation by T cells is a cru- cial step in initiating transplant rejection. One of the reasons that transplantation induces such a dynamic immune response is the high precursor frequency of T cells able to respond to mismatched Table 4.7.2  Characterizing the immune response following transplantation Time after transplant Cellular mechanisms Humoral mechanisms Innate mechanisms Hyperacute—​ minutes to hours If preformed complement-​fixing antibodies against allogeneic MHC molecules or ABO antigens are present at the time of transplantation, catastrophic rejection occurs. In solid organ transplantation this is characterized by rapid widespread vascular thrombosis leading to infarction of the graft Complement Phagocytes Acute—​days to weeks Infiltration of the graft by activated allospecific CD8 T cells, causing acute TCMR Pre-​existing or de-​novo generated antibodies against allogeneic MHC molecules causes acute ABMR Infection may trigger TLRs via DAMPs or PAMPs, creating a proinflammatory environment and triggering adaptive responses Chronic graft dysfunction—​ weeks to months Repeated episodes of acute TCMR may contribute to fibrosis and scarring, although CD8 responses are no longer thought to be prominent in chronic graft dysfunction Now thought to be largely due to chronic antibody-​ mediated damage, histologically this is typically associated with fibrointimal proliferation in intragraft arteries and interstitial fibrosis NK cells may be involved in the response to antibodies. Maladaptive macrophage responses to injury can switch healing and resolution of inflammation to fibrosis and scarring ABMR, antibody-​mediated rejection; DAMP, damage-​associated molecular pattern; MHC, major histocompatibility complex; PAMP, pathogen-​associated molecular pattern; TLR, Toll-​like receptors; TCMR, T-​cell mediated rejection.

4.7  Principles of transplantation immunology 395 MHC molecules, which can be as high as 1 in 10, orders of mag- nitude higher than the proportion of T cells that are able to react to a nominal peptide antigen–​self-​MHC complex (1 in 20 000 to 1 in 100 000). However, although MHC molecules are the most im- portant alloantigens, transplants between siblings with identical MHC molecules are still vulnerable to rejection, albeit at a slower tempo than MHC-​mismatched transplants. Rejection in this set- ting is a result of T-​cell recognition of other antigens. These include polymorphic non-​MHC molecules called minor histocompati- bility antigens (miH) that are derived from a wide variety of pro- teins and not necessarily expressed by cells of the immune system. Additionally, responses to endothelial antigens and other non-​MHC molecules have been described. Pathways of allorecognition Transplantation is a unique immunological situation in which priming of recipient T cells with antigen can occur via three dis- tinct pathways. Direct allorecognition is the interaction of recipient T cells with intact allogeneic MHC–​peptide complexes on the sur- face of donor-​derived APCs. Indirect allorecognition occurs when peptides derived from donor antigens are degraded and presented by recipient APCs. The final pathway, semi-​direct allorecognition, involves the exchange of intact MHC–​peptide complexes between recipient and donor APCs (Fig. 4.7.2). Direct allorecognition Transplanted tissue contains bone marrow-​derived haematopoi- etic cells of donor origin that have the characteristics of immature dendritic cells. In response to the inflammatory milieu that follows transplantation, the donor-​derived passenger leucocytes rapidly leave the graft and migrate to the secondary lymphoid tissues of the recipient. During migration the passenger leucocytes acquire the phenotype and functional characteristics of mature dendritic cells, expressing high levels of MHC class I and II molecules, as well as other cell-​surface costimulatory molecules necessary to fully acti- vate naive CD4+ and CD8+ T cells. Once in the secondary lymphoid tissues they act as professional antigen-​presenting cells, presenting donor-​derived MHC molecules in the transplanted tissue to re- cipient T cells, which recognize them as non-​self. Antigen presentation via the direct pathway was originally thought to play a dominant role in initiating the response to a trans- plant as T cells that recognize alloantigen in the context of donor MHC constitute 90% of the total alloreactive T cell repertoire. Evidence for the dominance of this pathway is variable: some animal models have suggested that depletion of donor APCs from the tissue prior to transplantation leads to long-​term graft survival without immunosuppression, but other studies show that donor APCs are rapidly killed within secondary lymphoid tissue by recipient NK cells, before they are able to directly activate T cells. Models showing that the expanded T-​cell repertoire post-​transplant is comprised of T cells stimulated by indirect allorecognition have supported this observation. It is therefore likely that direct allorecognition plays a much smaller role that originally thought. Semi-​direct allorecognition With new evidence suggesting a much more limited role for direct allorecognition in the transplant response, but older evidence sup- porting the role of donor derived APCs, it has been suggested that, rather than acting as direct APCs, the main role of donor-​derived DCs is to transport antigen from the graft to lymphoid tissue. It has been shown that MHC–​peptide complexes can be directly passed from one DC to another on membrane fragments, in this case permitting donor MHC-​peptide to be passed to recipient DCs, allowing semi-​direct allorecognition of non-​self by alloreactive T cells. There is increasing evidence to support this pathway of allorecognition as being the pathway that dominates early in the Recipient APC Direct pathway Semi-direct pathway Donor MHC Recipient MHC TCR Recipient T cell Donor APC Donor MHC TCR Recipient T cell Recipient APC Recipient T cell Indirect pathway Fig. 4.7.2  Pathways of allorecognition. In the direct pathway donor, APCs present donor peptide to recipient T cells in the context of donor MHC. In the semi-​direct pathway, intact donor MHC–​peptide complexes are passed from donor APCs to recipient APCs, and presented to recipient T cells. In the indirect pathway, which dominates over time, donor MHC is taken up by recipient APCs, processed and presented on recipient MHC to recipient T cells in the classical pathway of antigen presentation.

396 SECTION 4  Immunological mechanisms response to transplanted tissue, but detailed mechanisms are yet to be elucidated. Indirect allorecognition Whatever the precise mechanism of early allorecognition, the long-​ term alloresponse is dominated by antigen presented via the indirect pathway. This is the standard pathway for recognition of non-​self, where donor peptides are taken up by recipient APCs, processed and presented on self-​MHC to T cells. While antigen capture can take place both in the graft and transplant-​draining lymphoid tissue, presentation of donor antigen-​self-​MHC occurs in the secondary lymphoid organs (SLOs). As in direct and semi-​direct pathways, the dominant antigenic peptides presented by the indirect pathway are the hypervariable peptide-​binding regions of MHC molecules, but unlike direct and semi-​direct pathway allorecognition, the in- direct pathway is available for antigen presentation for as long as the graft remains in situ and therefore becomes the dominant mode of allorecognition in the long term. T-​cell activation Regardless of the method of antigen presentation, T cells cannot be activated by recognition of peptide-​MHC complex alone. This signal, delivered via the TCR-​CD3 complex, is known as ‘signal 1’ and, in isolation, suggests recognition of self-​antigen, which will lead to deletion or anergy rather than activation of the naive T cell. T cells therefore require additional signalling—​‘signal 2’—​in the form of costimulation, and ‘signal 3’ in the form of cytokines. Signal 2 is pro- vided by the interaction of pairs of cell-​surface molecules present on T cells and antigen-​presenting cells called costimulatory molecules. Many of these molecules are homologous and in general they can be divided into two families: the CD28/​B7 family, best characterized by the T-​cell costimulatory molecules CD28 and CD152 (CTLA-​4), which both interact with the APC molecules CD80 and CD86; and the tumour necrosis factor (TNF)/​tumour necrosis factor receptor (TNFR) family of which the prototype receptor–​ligand pair are CD40 and CD154 (CD40L) (Table 4.7.3). T-​cell costimulatory molecules CD28 is constitutively expressed by T cells and binds to the B7 family molecules CD80 and CD86 on antigen-​presenting cells. Signalling via CD28 lowers the threshold for T-​cell activation, increases expression of IL-​2, and promotes T-​cell proliferation and resistance to apoptosis. During an immune response, activated T cells upregulate expres- sion of CD152 (CTLA-​4), a molecule that has close homology to CD28. CD152 also binds to CD80 and CD86, but it has an inhibitory effect on T-​cell activation and is able to attenuate immune responses by competing with CD28 for ligation of these molecules. The im- portance of CD152 as a negative regulator of immune responses was demonstrated by the generation of CD152 knockout mice, which develop a fatal disorder characterized by massive proliferation of lymphocytes. Another effect of CD28 signalling during T-​cell activation is to upregulate expression of other costimulatory molecules such as CD154 (CD40L). CD154 is the ligand for CD40 expressed by antigen-​presenting cells, and as well as delivering a positive signal to the T cell, CD40–​CD154 ligation activates APCs, leading to in- creased expression of B7 family molecules and therefore an en- hanced ability to activate further T cells. As more novel costimulatory molecules are identified, it is becoming clear that the outcome of interaction between T cells and antigen-​presenting cells is determined both by the avidity of the cognate TCR–​MHC–​peptide interaction and the balance of positive and negative signals delivered by the costimulatory molecules present on the surface of the participating cells (see Table 4.7.3). Transduction of costimulatory signals occurs in parallel to TCR–​CD3 complex signalling and can be blocked independently, and the development of pharmaceutical agents able to interrupt costimulation has provided evidence of the importance of these pathways in transplant rejection. Administration of a fusion protein constructed from the extracellular domains of CD152 (CTLA-​4Ig or CTLA-​4Fc; see ‘Immunosuppression in trans- plantation’, to follow), or monoclonal antibodies directed against CD80 and CD86, are able to block costimulation via CD28, and this is sufficient to prevent allograft rejection in animal models. Monoclonal antibodies directed against CD154 are also able to prevent acute allograft rejection. Both of these examples of costimulation blockade lead to long-​term rejection-​free survival of vascularized and non​vascularized allografts in experimental rodent models, but only CD152 fusion proteins have made it into clinical practice. Table 4.7.3  T-​cell costimulation molecules and their ligands T cell Antigen-​presenting cell Effect on T cells CD28 CD80 (B7–​1) CD86 (B7–​2) Activatory CD27 CD70 CD154 (CD40-​L) CD40 ICOS (inducible costimulator) ICOS-​L (ICOS ligand) OX40 (CD134) OX40-​L (CD252) 4–​1BB (CD137) 4–​1BB-​L (CD137L) CD152 (CTLA-​4—​cytotoxic T lymphocyte antigen-​4,) CD80 (B7–​1) CD86 (B7–​2) Inhibitory PD-​1 (programmed death-​1, CD279) PD-​L1 (programmed death ligand 1, CD274, B7-​H1) PD-​L2 (programmed death ligand 2, CD273, B7-​DC)

4.7  Principles of transplantation immunology 397 TCR signal transduction and ‘signal 3’ The intracellular signalling pathway downstream of the TCR is complex. Briefly, TCR–​MHC–​peptide engagement results in the recruitment and phosphorylation of several signalling mol- ecules. These phosphorylation events initiate several intracel- lular biochemical processes, resulting in activation of the Ras-​ and Rac-​mitogen-​activated protein (MAP) kinase pathways and hy- drolysis of membrane phosphatidylinositol 4,5-​biphosphate to generate the secondary messengers, inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 leads to the release of stored calcium from the endoplasmic reticulum and activation of the phosphatase calcineurin, which in turn dephosphorylates the transcription factor NFAT (nuclear factor of activated T cells), allowing it to translocate to the nucleus. Generation of DAG results in the activation of an- other transcription factor, NF-​κB, and a third transcription factor, AP-​1, is generated by the MAP kinase cascades. The action of these transcription factors alters expression of many genes, in particular leading to upregulation of the T-​cell growth factor IL-​2 and the high-​affinity IL-​2 receptor α-​chain (CD25). Soon after activation the generation of large amounts of IL-​2 and other proproliferative cytokines act in an autocrine and para- crine fashion to provide what has been described as ‘signal 3’ (see Fig. 4.7.3). Transduction of signals delivered by IL-​2 promotes cell-​cycle progression and initiates the clonal expansion and dif- ferentiation of activated T cells. B-​cell activation For many years, transplantation immunology has focused on T cells as the key cells in both initiation and maintenance of a re- sponse to transplanted tissue. However, interest has grown in the role of B cells, both in terms of rejection and, more recently, poten- tial protection of the graft. B cells have long been defined by their main antibody-​producing role, but they are also known to function as APCs, providing costimulation and activation signals to T cells. Several groups have described B cells with the potential to regulate immune responses in a similar way to regulatory T cells, although not as powerfully. The role of B cells in transplantation is clearly multifaceted, and they are by no means bystanders in the early re- sponses to the graft. B-​cell activation also occurs in secondary lymphoid organs. Mature naive B cells continuously sample antigen, either that held on follicular dendritic cells within the B-​cell zone or if presented on subcapsular sinus macrophages. When the B cells recognize antigen via their B-​cell receptor (BCR) they can take it up, process it, and then combine peptide fragments into the binding site of class  II MHC. The antigen-​MHC complex is then displayed on the B-​cell surface and presented to CD4 T cells. B cells therefore recognize antigen when intact, whereas the peptide they present will be a much smaller fragment, introducing additional layers of complexity into the recognition and attack of non-​self by T cells. For full activation of B cells, they then require help and recip- rocal signals from the CD4 T cells (see Fig. 4.7.4), a process which usually occurs at the border between the T-​cell zone and the B-​cell zone in the SLOs. Following antigen recognition there are several fates for B cells: those recognizing non-​self can either go on to form extrafollicular responses, leading to the rapid but short-​lived pro- duction of low affinity antibody, or they can receive signals that allow them to migrate further into the B-​cell zone and form clusters called germinal centres where they undergo recombination of their BCR, with each round of somatic hypermutation an attempt to im- prove receptor specificity for the antigen. Those B cells that increase their affinity in this process compete with specialized CD4 T cells, which have also entered the B-​cell follicle, to receive survival and Upregulation of gene transcription TCR-CD3 complex Calcineurin Antigen presenting cell NFAT NF-κB MAP kinases AP-1 Protein kinase C Nucleus mTOR Signal 1 Signal 2 Signal 3 IL-2 production IL-2 CD4 Costimulatory molecules IL-2 receptor T cell Cell cycle progression MHC molecule + peptide Upregulation of gene transcription Fig. 4.7.3  Three-​signal model of T-​cell activation. Signalling through the TCR is not sufficient for activation of T cells, and in isolation leads to anergy or deletion of cells. For activation of cells and development of a pathogenic response, T cells also require signal 2 in the form of costimulation. Signal 3 is required to drive the differentiation of the activated cell into a subset (e.g. Th1, Th2), and thus the downstream response. B B T T Tfh Tfr GC APC APC Memory B cell Plasma cell B-cell follicle T-cell zone Fig. 4.7.4  The germinal centre reaction. B cells are activated by APCs and migrate to the T-​B border. Here they interact with recently activated CD4 T cells, reciprocal signals allow the B cells to move deeper into the B-​cell follicle and form germinal centres, and induce CXCR5 expression in CD4 T cells, allowing them to enter the germinal centre as T follicular helper (Tfh) cells and support B-​cell differentiation into plasma and memory cells. T follicular regulatory (Tfr) cells control the size of this reaction.

398 SECTION 4  Immunological mechanisms differentiation signals allowing them to differentiate into antibody secreting plasma cells, or memory B cells. This interaction between cells of the innate immune system (macrophages, dendritic cells) and adaptive immune system (T cells, B cells) leads to a complex and multilayered response to the graft, providing both a rapid and less specific response but setting up im- munological memory and refining responses to be highly targeted against non-​self-​antigens. This long-​lived response is the biggest hurdle in terms of transplantation. Ongoing responses and immunological memory While the early response to pathogens is predominately innate, the high frequency of alloantigen-​specific T cells means the early re- sponse to transplanted tissue is more aggressive and more specific than that to pathogens. These initial responses also set in motion the development of a long-​lived response to alloantigens which, if left unchecked, will rapidly destroy the graft. The actions of recipient and donor-​derived innate cells with allospecific T cells play key roles in the development of the ongoing response. Additional factors modify the character of the immune response to an allograft, including the site of transplantation, the organ or tissue transplanted, the antigen-​ presenting cells involved in T-​cell priming, and the immune status of the recipient at the time of transplantation. Innate immune system in transplant rejection The innate immune system comprises a group of cells and mol- ecules (Table 4.7.4) that provide a first line of defence against pathogens and which also play an important role in allograft re- jection. Primary adaptive immune system responses, which rely on the activation and expansion of antigen-​specific lymphocytes, take several days to reach maturity. In contrast, the innate im- mune system represents a ‘preformed’ defence that is immediately available to defend the host until either the dangerous stimulus is cleared or the adaptive immune system is able to mount an antigen-​specific response. As just described, the physical process of graft retrieval and im- plantation generates ‘danger signals’ in the form of heat shock pro- teins, complement breakdown products, and reactive oxygen species that activate cells of the innate immune system via TLR ligation. Macrophages and other phagocytic cells ingest necrotic tissue and, when activated, release cytokines such as tumour necrosis factor α (TNFα), interleukin 1 (IL-​1), and interleukin 6 (IL-​6) that con- tribute to the local inflammatory environment. Complement Activated complement components constitute a proteolytic cas- cade that generates a range of effector molecules (see Fig. 4.7.5). The anaphylatoxins C5a and C3a are chemoattractant molecules that as- sist leucocytes to home to the graft, while other soluble mediators are able to opsonize cells, targeting them for destruction by phago- cytes. Recognition of C3b, C4b, or their fragments covalently bound to target cells by complement receptors on the surface of leucocytes, facilitates antigen presentation and T-​cell activation. Generation of the terminal components of the complement cascade (C5b-​9) re- sults in formation of the membrane attack complex (MAC) within the target cell membrane and initiation of target cell lysis. The al- ternative pathway, mediated by C3, has been shown to play a key role in ischaemia-​reperfusion injury, with C3 knockout mice being resistant to the damage associated with this insult. The production of the MAC also rises following reperfusion and components of the terminal complement pathway, particularly C5a and C5a receptor, have been shown to be upregulated in donors with brainstem death. The proinflammatory effects of complement can play a signifi- cant role in activating the adaptive immune system, particularly in deceased donors, and despite strategies to reduce ischaemia-​ reperfusion injury (see later paragraphs on therapeutics), this re- mains a significant hurdle in improving outcomes for deceased donors compared to live donors. Innate lymphoid cells NK cells are large granular lymphocytes, comprising 5–​20% of lymphocytes in spleen and blood, which have natural cytotoxic ac- tivity against virus-​infected or mutated host cells. They also produce several cytokines and mediate antibody-​dependent cellular cytotox- icity (ADCC), so have multiple roles in the response to allograft. NK cells express invariant cell-​surface receptors, including activating receptors that bind to widely expressed carbohydrate residues on self cells and inhibitory receptors that bind self-​MHC class I mol- ecules. Some malignant or virally infected cells down-​regulate MHC class I expression or express altered class I molecules as a strategy to evade CD8+ T-​cell cytotoxicity. As a result, they are unable to stimu- late inhibitory receptors and are vulnerable to NK cell killing, in the same way as transplanted tissue expressing non-​self-​MHC class I. In addition, some cells express both a TCR and a NK phenotype, and are known as NKT cells. These cells recognize glycolipids pre- sented by the MHC class I-​like molecule CD1d, and are divided into type I  (iNKT), expressing an invariant TCR reactive to α-​ galactosylceramide, and type II, with diverse TCR specificity. Interest has grown in the role of both NK and NKT cells in the past few years. It has long been known that NK cells are respon- sible for the early killing of donor-​derived APCs, which would otherwise present antigen through the direct pathway, and therefore they may play a role in tolerance induction by limiting the presen- tation of donor antigen. However, in solid organ transplantation, ischaemia-​reperfusion injury has been shown to upregulate ligands to NK activating receptors, and NK cells have been shown to mi- grate into grafts prior to CD8 T cells, producing IFNγ and thereby inducing upregulation of MHC I and MHC II on graft endothelial cells. This makes grafts more susceptible to cytotoxic T-​cell attack, Table 4.7.4  Components of the innate immune system Component Primary function Macrophage/​neutrophil Phagocytosis; secretion of cytokines, enzymes, and inflammatory mediators Dendritic cell Antigen uptake and presentation to lymphocytes Natural killer (NK) cell Cytotoxic to virally infected or mutated cells Complement Opsonization, target cell lysis, and chemoattraction Eosinophil Killing of antibody-​coated parasites

4.7  Principles of transplantation immunology 399 and the proinflammatory cytokines secreted by NK cells can both drive DC maturation and promote the activation of allospecific T cells, enhancing the response against donor tissue. It is therefore not clear whether NK cell responses are positive or negative in the con- text of transplantation. Other innate cells The role of DCs and other APCs has been discussed earlier in terms of leucocyte activation, but many other components of the innate immune system contribute to inflammation (neutrophils, macro- phages), activation of the adaptive immune system (macrophages, NK cells, NKT cells, complement) and tissue damage (NK cells, macrophages). In practice, the response to the graft represents a con- tinuum between early non​specific responses, and later allo-​specific and subsequently donor-​specific responses, with both innate and adaptive components involved at all time points. Adaptive responses to the graft CD8 T-​cell responses Following activation in SLOs, activated CD8 T cells migrate to the graft site, recruited by chemotactic cytokines called chemokines produced by the inflammatory process ongoing in the graft. Inflammatory signals also affect blood vessels in the vicinity of the transplant, causing vasodilation and endothelial activation. This causes upregulation of the adhesion molecule P-​selectin, as well as endothelial surface expression of chemokines released from the graft. Leucocytes are usually conveyed within the fast laminar flow at the centre of blood vessels, but when activated leucocytes reach postcapillary venules close to the graft they are able to leave this rapid flow and move towards the edge of the vessel. Here they bind to P-​selectin and begin the process of extravasation into the graft it- self, moving along the chemokine gradient. Once in the graft, activated CD8 T cells recognize allogenic class I MHC on donor cells and form an immunological synapse with their intended target. The CD8 T-​cell releases granules con- taining cytotoxic molecules such as perforin and granzyme B, as well as up-​regulating cell-​surface expression of Fas ligand (FasL) and secreting soluble mediators such as TNF. Perforins polymerize and insert into the target cell membrane, forming a pore that facili- tates the entry of granzyme B and other compounds into the cell. Granzyme B is a protease that is able to initiate apoptosis by several mechanisms, including activation of caspase cascades. These mech- anisms are designed to lead to cell death by apoptosis. Binding of FasL to Fas on the target cell surface is also able to trigger apoptosis by activating caspases. CD4 T-​cell responses CD4 T cells have multiple and very diverse roles in transplant- ation. Alloantigen-​specific CD4+ T cells (typically T helper 1 cells) can contribute to inflammation and the effector phase of allograft C3a C3b C5 convertases Classical pathway Lectin pathway Alternative pathway C1q, C1r, C1s C4 C2 MBL/ficolin, MASPs C4 C2 C3i B D C3 C3 convertases C5a C5b C5b C6 C7 C8 C9 (n) Terminal pathway Lysis Sublytic effects C5 C3b Membrane-attack complex Antigen-antibody complexes pathogen surfaces Apoptotic cells—infected cells Carbohydrate residues Activating surfaces (e.g. pathogen surfaces, tumour cells) Fig. 4.7.5  A simplified overview of the complement cascade. For more detail see Chapter 4.2. In the context of organ transplantation, all three pathways have been implicated in damage to organs, with the classical pathway being most relevant in ABMR, but both lectin and alternative pathways implicated in ischaemia-​reperfusion injury. The use of drugs that interfere with the complement cascade represents a promising new therapeutic avenue in transplantation.

400 SECTION 4  Immunological mechanisms rejection via a non​specific effector mechanism referred to as the delayed-​type hypersensitivity (DTH) response. DTH reactions are characterized by the release of multiple soluble mediators, including the proinflammatory cytokines IL-​1, interferon-​γ, and TNFα. These induce infiltration of the graft by of activated leucocytes, including monocytes, macrophages and eosinophils, and the production of non​specific mediators such as nitric oxide, reactive oxygen spe- cies, and inflammatory arachidonic acid derivatives (prostaglandin E2, thromboxane, and leukotrienes). Although this activity is trig- gered in an antigen-​specific manner by the T helper cell, the effector ­mechanisms that lead to the destruction of the graft are non​specific. DTH reactions have been shown to directly affect graft physiology by altering cell permeability and vascular smooth muscle tone and play a role in both acute and chronic allograft rejection. CD4 T cells also play a key role in the humoral response to alloantigen (Fig. 4.7.4) by providing survival signals to activated B cells and receiving reciprocal signals, allowing them to migrate into the B-​cell follicle of SLOs and participate in the germinal centre (GC) response. These reciprocal interactions maintain the GC and allow the formation of long-​lived memory B cells and plasma cells. CD4 T cells can also have a regulatory role, suppressing immune responses to antigen in order to control the size of the response. The best studied regulatory or suppressor T cells (Treg) are a popula- tion of naturally occurring CD4+ cells that constitutively express the IL-​2 receptor α-​chain (CD25). These cells develop within the thymus under the direction of the transcription factor Foxp3 and have a critical role in limiting immune responses, particularly to self-​antigens. Mutation of Foxp3 in humans is responsible for the IPEX syndrome (immune dysregulation, polyendocrinopathy, en- teropathy, X-​linked), which causes severe disease and death by aged two in most cases. Mouse models of Treg deficiency also lead to overwhelming autoimmune disease. Tregs are known to play key roles in downregulating immune responses to all antigens, as well as prevention of autoimmunity, but can also prevent responses to malignant cells. B-​cell responses The B-​cell response to alloantigen following transplantation results in the generation of alloantigen-​specific antibodies (alloantibodies—​ also referred to as donor-​specific alloantibodies (DSA)), which play an important role in allograft rejection. B cells use surface immuno- globulin as their antigen receptor (B-​cell receptor or BCR) and are able to internalize antigens, which are then degraded and presented in conjunction with class  II MHC molecules. Early antibody re- sponses to the graft do not require significant interaction with T cells, but long-​lived antibody responses require B cells to go through the GC reaction and are absolutely dependent on interactions with CD4 T cells (Fig. 4.7.4). When B cells recognize their antigen in SLOs they migrate to the edge of the B-​cell follicle, where they interact with recently primed CD4 T cells. If two cells recognize the same antigen, the T cell pro- vides the B-​cell with survival and activation signals, allowing it to migrate further into the B-​cell follicle. The B-​cell provides recip- rocal signals to the CD4 T cell, causing upregulation of CXCR5 via the transcription factor Bcl6, a chemokine receptor that allows the CD4 T cell to follow the B-​cell into the follicle and become a follicular helper T cell, or Tfh. Here the Tfh and B cells interact to form a germinal centre. This process is absolutely required for the formation of memory B cells and plasma cells, and involves random mutation of the variable region of their BCR. With each mutation, B cells alter the affinity of their BCR for the antigen and compete with each other for survival and maturation signals from the Tfh. There are also regulatory cells within the GC (T follicular regulatory cells or Tfr) that control the size of the GC response by limiting the interactions between B cells and Tfh and ensure that only the cells with the highest affinity BCR for antigen go on to become plasma or memory cells (Fig. 4.7.4). As in the T-​cell response to alloantigen, the predominant anti- genic targets of DSA are mismatched MHC molecules, but anti- bodies that recognize other antigens such as miH or blood group antigens also contribute to rejection. The development of de-​novo DSA after transplantation in kidney transplant recipients is strongly associated with graft decline and failure. The mechanism of antibody-​mediated rejection is primarily via complement fixation and membrane attack complex formation leading to target cell lysis. NK cells and macrophages express recep- tors that bind to the non-​antigen-​specific (Fc) portion of antibodies, stimulating them to kill target cells that have antibody bound on the surface (a process called antibody-​dependent cell-​mediated cyto- toxicity or ADCC), and this provides a second mechanism by which alloantibodies can induce donor cell death. Antibody-​mediated rejection Antibody-​mediated rejection (ABMR) can occur immediately in patients with preformed antibodies in bouts of acute rejection, or in a more chronic and insidious manner. Hyperacute rejection, now exceedingly rare, occurs if patients have preformed DSA at the time of transplantation and results in graft destruction within minutes of organ reperfusion. In this situation the antibodies (which are usually directed at allogeneic MHC molecules or ABO blood group antigens expressed on graft endothelium) cause local activation of the coagu- lation and complement cascades, resulting in extensive thrombosis within the vascular supply to the graft and culminating in infarction. Modern cross-​matching techniques screen for preformed DSA and in deceased donor transplants this is considered an absolute contra- indication to transplantation. Some living donor kidney transplant recipients can undergo desensitization protocols where preformed ABO or HLA antibodies are removed prior to the operation, which only goes ahead if antibody levels are within acceptable limits: these procedures require careful planning. Humoral responses to transplanted tissue are more commonly seen in both acute and chronic rejection of grafts. Acute antibody-​ mediated rejection is uncommon with modern cross-​matching techniques: it is very rare in liver transplantation, very uncommon in lung and renal transplantation, and moderately uncommon in cardiac transplantation, although rates vary as it is rarely screened for outside of renal transplantation. The presence of DSA does not in itself indicate antibody-​mediated rejection, and many scoring systems have been used to define histological indicators of ABMR. In the Banff renal histology scoring system, positive staining for complement component 4d (C4d) in biopsies allows indirect iden- tification of antibody deposition and complement fixation, and peritubular C4d staining is strongly associated with early as well as late graft failure. C4d staining is also used in lung and cardiac transplantation as an indicator of ABMR, but there is no clear con- sensus on its use.

4.7  Principles of transplantation immunology 401 More recently, evidence has emerged that the presence of DSAs, even without histological evidence of rejection, is associated with late graft loss, and it is becoming clear that antibodies may play a more complex role than originally thought, with suggestion that the subclass of and ability of antibodies to fix complement may indicate how pathological they are, but this is an emerging field and much needs to be elucidated. As with T cells, evidence has emerged that suggests B cells can also have a regulatory role. These cells have been identified in both murine models and human peripheral blood and shown to have the capacity to suppress immune responses. However, regulatory B cells are less well characterized than regulatory T cells (see Table 4.7.5) and the mechanisms underlying their suppressive action are yet to be fully elucidated. Immunological memory in transplantation As just described, the generation of memory is a key step in the re- sponse to non-​self-​antigen. Following primary antigen exposure, long-​lived antigen-​specific memory T and B cells are generated which are able to deliver a larger and more rapid immune response if the same antigen is encountered on a subsequent occasion. Memory lymphocytes have a reduced activation threshold and are less de- pendent on costimulation. As a result they are able to upregulate effector function and cytokine secretion more rapidly than naïve lymphocytes. With increasing age, the proportion of memory T cells within an individual’s peripheral T-​cell pool increases, reflecting cu- mulative antigen exposure, and can be as high as 50% in adult hu- mans. While the generation of immunological memory is beneficial for protection against infectious pathogens, in transplantation the presence of allospecific memory produces an accelerated or ‘second-​ set’ rejection response. In clinical transplantation, evidence of prior sensitization to donor antigens is associated with increased risk of acute rejection episodes and premature graft failure. Memory-​type responses towards alloantigens are frequently a result of exposure to alloantigens at the time of a previous blood transfusion, pregnancy, or transplantation. However, it is now rec- ognized that memory-​type responses may also be generated as a consequence of antigen receptor cross-​reactivity (heterologous im- munity) or by homeostatic proliferation of lymphocytes following an episode of lymphopenia. Sensitization Patients on the transplant waiting list have their HLA genotype charac- terized and are regularly monitored for the development of anti-​HLA antibodies as a marker of sensitization to potential donor antigens. This was previously carried out using a ‘panel’ of random donors with known HLA types and testing the serum of potential recipients to see what proportion of donor lymphocytes was lysed. This would give the percentage of panel reactive antibodies, or PRA. In practice, this was a crude measure of reactivity as it depended on large numbers of donors or immortalized cell lines, was work intensive, and only de- tected complement-​fixing antibodies. In most centres it has been re- placed with solid phase assays, which use purified HLA antigens to detect antibodies, whether complement fixing or not, in the serum of recipients. These assays are extremely sensitive, but also give an idea of antibody levels, allowing thresholds to be set for low, medium, and high-​risk transplants, depending on the amount of antibody present, thus allowing patients with extremely high (i.e. broad) sensitization to receive transplants, where previous PRA methodology would limit their options. Sensitization is now given as either a calculated PRA or a calculated reaction frequency, both indicating the proportion of the population to which a patient is likely to react. Many events can lead to sensitization. In most cases antibodies develop in response to previous transplants, paternal antigens in multiparous women, and in patients who have received blood product transfusions. The risk of sensitization from blood product transfusion is highly variable, although overall sensitization rates are low. No obvious sensitizing event can be identified in some cases and it has been proposed that heterologous immunity or homeostatic proliferation after lymphopenia may contribute to memory cell for- mation and hence antibody production. Heterologous immunity Experiments in mice have found that sequential viral infections can generate populations of alloreactive memory-​phenotype T cells, possibly through epitope sharing or TCR cross-​reactivity, and the presence of these cells prevented transplant tolerance induction by protocols that usually result in reliable long-​term graft acceptance. It is therefore possible that memory lymphocytes generated by ante- cedent viral infections in humans may be able to cross-​react with epitopes presented following transplantation, resulting in memory-​ phenotype responses towards the graft without prior sensitization to donor alloantigen. Homeostatic proliferation The size of the peripheral T-​cell pool, as well as the relative ratios of CD4+ to CD8+ and naive to memory cells, are tightly regulated by homeostatic mechanisms. A consequence of this is that reduction of the overall T-​cell population, either during illness or following med- ical intervention, strongly induces the residual T cells to proliferate, whether or not cognate antigen is present. A proportion of T cells that have undergone homeostatic proliferation exhibit memory T-​ cell-​like phenotype and behaviour. Homeostatic proliferation there- fore presents a particular concern in transplantation both in terms of Table 4.7.5  Characteristics of regulatory T and B cells (Tregs and Bregs) Tregs Bregs Surface markers (not exhaustive) CD4+, CD25hi, IL-​7Rlo, ICOS+, CD152+ (CTLA-​4) CD19+, CD24hi, CD38hi, CD5+, CD1d+, CD27+, IgD+, IgM+ Transcription factor Foxp3 Unknown Cytokines produced TGF​β, IL-​10 IL-​10 Mechanism of action Contact dependent inhibition of T-​cell activation via inhibitory receptors and cytokine dependent suppression Unknown—​possibly via IL-​10, possibly via induction of Tregs

402 SECTION 4  Immunological mechanisms pretransplant sensitization, but also as many patients receive mono- clonal or polyclonal antibody therapy designed to deplete leucocytes and/​or T cells either as part of induction immunosuppression or as therapy for an acute rejection episode. Since as many as 10% of naive T cells are able to respond to alloantigen, it is likely that homeo- static expansion in this setting could generate alloreactive memory-​ phenotype T cells. Cross-​matching In all except liver transplants, a ‘cross-​match’ of recipient reactivity towards donor is carried out to exclude the presence of complement-​ fixing and binding antibodies directed against donor HLA. This is normally done immediately prior to the transplant, with labora- tory tests including the complement-​dependent cytotoxicity (CDC) assay and/​or flow cytometric cross-​matching. This combination al- lows detection of complement-​fixing alloantibodies which would cause hyperacute rejection (where the CDC is positive) as well as non-​complement fixing or lower level antibodies that may cause problems in future with acute or chronic rejection (CDC negative, flow positive cross-​match). In patients with no antibodies, or long-​standing and well-​ characterized HLA-​specific antibodies, current guidelines suggest a ‘virtual’ cross-​match is possible. In the virtual cross-​match the donor HLA genotype is compared with the potential recipient’s HLA genotype and any known antibodies to ensure that the donor genotype is suitably matched and avoids any known antibodies. In practice, most transplants proceed after a pre-​emptive laboratory cross-​match, and in those where a virtual cross-​match is performed, a laboratory cross-​match or solid phase assay will be carried out within 24–​48 h to confirm the lack of reactivity (see Fig. 4.7.6). Patients who have become highly sensitized to HLA antigens have a reduced chance of transplantation and may remain on the transplant waiting list for a long time. Patients with renal failure who have a living kidney donor can sometimes be successfully transplanted following a programme of ‘desensitization’. Typically this involves a strategy of removing existing alloantibodies by plasma exchange or immunoadsorption. However, in isolation this is insufficient to prevent antibody rebound, and additional strategies include the use of IVIg, and reducing alloantibody pro- duction by the administration of B-​cell depleting agents (such as rituximab) in combination with other immunosuppressive agents (see ‘Immunosuppression in transplantation’, next). There is no clear consensus on the best therapeutic strategy at present. Similar strategies have also been employed to allow transplantation be- tween ABO-​incompatible donor–​recipient pairs (Table 4.7.6). However, desensitization requires profound immunosuppression, which cannot be maintained for long periods without deleterious effects on the recipient. As deceased donor transplants are unpre- dictable in terms of timing, these approaches are currently only applicable to live-​donor recipients. Immunosuppression in transplantation The dramatic improvements in allograft survival that have occurred since the first successful transplants were performed in the 1950s have been achieved by the development of potent immunosuppres- sant medications able to inhibit the aggressive immune response to the transplant. These agents work by depleting lymphocytes, interfering with lymphocyte signal transduction pathways, and/​or altering lymphocyte trafficking. The risk of acute graft rejection is greatest during the initial 3 months after transplantation and therefore most solid organ trans- plant recipients initially receive strong immunosuppression, often consisting of an induction agent alongside triple therapy with gluco- corticoids, a calcineurin inhibitor (CNI), and an antiproliferative agent. Provided there are no episodes of acute rejection, the doses of these agents are gradually reduced and then maintenance immuno- suppression is continued indefinitely. Liver transplant patients tend to have a much lower risk of rejection compared to other solid organ transplants and therefore generally receive low-​dose CNI with an antiproliferative agent. Kidney-​pancreas patients tend to be given a steroid-​sparing regimen, and—​given their plethora of side effects—​ there is a move towards reduction or cessation of glucocorticoids in most patients where feasible. Chronic immunosuppression is associated with several undesir- able sequelae, in particular an increased relative risk of infections and malignancy. Recognized side effects of particular immunosup- pressive agents are listed in Table 4.7.7. Many different immunosuppressive regimens for solid organ transplantation are in clinical use, and a detailed discussion of these is beyond the scope of this chapter. What follows is an introduction to the immunosuppressive agents in widespread clinical use cur- rently (Fig. 4.7.7). Glucocorticoids Corticosteroids were developed in the 1950s and have complex immunosuppressive as well as anti-​inflammatory effects. They act principally by binding to cytoplasmic glucocorticoid receptors, although at higher doses they can exhibit receptor-​independent effects as well. The steroid–​receptor complex translocates to the nucleus where it is able to alter the expression of multiple cyto- kines through DNA-​binding and by targeting transcription factors such as AP-​1 and NF-​κB. Corticosteroids reduce the expression of many molecules important in the immune response, including interleukins 1, 2, 3, and 6, TNFα, interferon-​γ and chemokines. By inhibiting cyclooxygenase, corticosteroids are also able to reduce the production of inflammatory mediators such as leukotrienes and prostaglandins. Glucocorticoids are used as induction and main- tenance immunosuppression, as well as the first line of therapy for rejection episodes. Antiproliferative agents Azathioprine and mycophenolate mofetil interfere with DNA syn- thesis and prevent cell-​cycle progression. In the context of allograft rejection this impairs the clonal expansion of alloreactive T cells. The introduction of azathioprine into clinical practice in the 1960s and its use in conjunction with corticosteroids allowed transplantation to progress from an experimental procedure into a practical therapy for patients with organ failure. It is widely metabolized (mostly in erythrocytes and the liver) to the purine analogue 6-​mercaptopurine and incorporated into DNA. By inhibiting purine nucleotide synthesis (and therefore DNA and RNA synthesis), azathioprine reduces gene transcription and

Donor cells Donor cells Recipient serum with donor-specific antibodies Antihuman globulin (AHG) Complement Complement binding and cell death Recipient serum with donor-specific antibodies Fluorescently tagged AHG Stained with T- and B-cell markers Negative CDC Positive CDC Flow cytometry- positive peak in red (a) (b) (c) Luminex: Serum is added to a cocktail of microbeads, each coated with a purified HLA antigen and with a different fluorescence ELISA: Serum is added to a plate of purified HLA antigens, then a secondary antibody is added to allow measurement of levels Solid assays phase Fig. 4.7.6  Methods used in cross-​matching. (a) CDC cross-​match. Recipient serum is added to donor leucocytes: if there are donor-​specific antibodies (DSA) present, these will bind to the cells. An antihuman globulin is then added to enhance the effects of complement. Complement is added and, if antibodies are bound, the classical pathway is activated and the cells are lysed. (b) Flow cross-​match. This is similar to the CDC except the antihuman globulin is labelled with a fluorescent tag, which can be detected by a flow cytometry machine. Additional fluorescently labelled antibodies are added to divide the leucocytes into B cells (anti-​CD19) and T cells (anti-​CD3). Positive fluorescence is seen on the flow cytometer. (c) Solid phase assays. These are not routinely used in the immediate pretransplant cross-​match but are used regularly to assess sensitization to HLA antigens. The antigens are purified and can be coated on a plate where individual wells contain different antigens, and antibody binding is then measured using enzyme-​linked immunosorbent assay (ELISA). Alternatively, and more sensitively, the HLA antigens can be used to coat microbeads, each with a slightly different fluorescence, and antibody binding of these can then be measured using a Luminex machine. Both give read outs of the antibodies in a recipient’s serum, but they cannot say whether it is complement fixing or not, and hence cannot accurately predict whether an antibody is likely to cause rejection. In general, the higher the level of antibody, the more likely it is to cause a problem, and each laboratory will have a level that they consider to be high risk, intermediate risk, and low risk. Luminex results reproduced with the kind permission of the Transplant Immunology Laboratory, Churchill Hospital, Oxford.

404 SECTION 4  Immunological mechanisms prevents cell-​cycle progression. The effects of azathioprine are not lymphocyte-​specific and patients must be monitored closely for bone marrow suppression. Mycophenolate mofetil is metabolized in the liver to mycophenolic acid, which is a non​competitive, reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH). Cells are able to gen- erate purines either de novo by converting inosine monophosphate to guanosine monophosphate (catalysed by IMPDH), or from guanine via the salvage pathway. The salvage pathway is less active in lymphocytes and therefore they are relatively dependent on the de-​ novo pathway of purine synthesis compared to other cell types. As a result, the effects of mycophenolate are more lymphocyte-​specific than azathioprine, and it is less myelosuppressive although it is com- monly associated with diarrhoea, which can be severe. Calcineurin inhibitors The introduction of ciclosporin in the early 1980s was a great step forward in transplant immunosuppression as this was the first drug able to selectively block T-​cell activation. Subsequently a second calcineurin inhibitor, the macrolide antibiotic tacrolimus, was de- veloped. Both drugs bind cytoplasmic immunophilins (cyclophilin in the case of ciclosporin and FK506-​binding protein 12 in the case of tacrolimus) to form complexes that can inhibit the calcium-​ dependent phosphatase calcineurin, a rate-​limiting enzyme in the T-​cell receptor signal transduction pathway. By preventing trans- location of the transcription factor NFAT to the nucleus, calcineurin inhibition impairs upregulation of many molecules important for T-​cell proliferation and the generation of an effective immune re- sponse, including the cytokines IL-​2, IL-​4, TNFα, and interferon-​γ, and costimulatory molecules such as CD154 (CD40L). In practice, ciclosporin is gradually being phased out in favour of tacrolimus, which is more immunosuppressive with fewer side effects. mTOR inhibitors Sirolimus and everolimus bind to the same immunophilin as tacrolimus (FKBP12), although the complexes they form are un- able to interact with calcineurin. Instead they bind to the regulatory kinase mammalian target of rapamycin (mTOR), which has a critical role in cytokine receptor signal transduction. The usual actions of mTOR are to activate the ribosomal enzyme p70 S6 kinase and block an inhibitory protein 4E-​BP1, both of which are required for trans- lation of proteins necessary for progression from the G1 (growth) phase to the S (DNA synthesis) phase of the cell cycle. Inhibition of this pathway (‘signal 3’) in T cells therefore blocks the action of cyto- kines such as IL-​2, IL-​4, and IL-​15, preventing cell-​cycle progression and clonal expansion. Depleting antibodies Polyclonal antithymocyte globulin (ATG) is produced by im- munizing either rabbits or horses with human lymphocytes. Immunoglobulins directed against lymphocyte epitopes can then be harvested from the animal’s serum for clinical use. ATG causes profound lymphocyte depletion that takes many months to recover, and is highly effective as an induction agent and in the treatment of steroid-​resistant rejection. CAMPATH-​1H or alemtuzumab is a humanized monoclonal anti- body directed against human CD52, a surface molecule that is ex- pressed by most nucleated bone-​marrow-​derived cells, including T and B cells, monocytes, macrophages, and eosinophils. Administration of alemtuzumab causes profound lymphopenia that recovers over months to years, although patients can remain CD4 lymphopenic for many years. It is used routinely in induction for simultaneous kidney-​ pancreas transplant recipients as part of a steroid-​sparing regimen, and widely used for high immunological risk transplants as an alter- native induction agent to basiliximab (discussed next). It can also be used for the treatment of steroid-​resistant rejection Administration of either ATG or alemtuzumab can result in the massive release of cytokines caused by the initial activation of lympho- cytes prior to their depletion. Clinical manifestations of this range from fever and flu-​like symptoms to a potentially fatal severe systemic inflammatory response syndrome characterized by hypotension, rigors, and pulmonary oedema. This is more common with ATG. Rituximab is a monoclonal antibody licensed for use in B-​cell lymphoma and rheumatoid arthritis. It is directed against CD20, which Table 4.7.6  ABO compatibility for transplantation Blood group Can receive transplant from: Can donate transplant to: O O O, A, B, AB A O, A A, AB B O, B B, AB AB O, A, B, AB AB Table 4.7.7  Principal side effects of immunosuppressive agents Corticosteroids Glucose intolerance, osteoporosis, weight gain, hyperlipidaemia, cosmetic changes Ciclosporin Nephrotoxicity, hypertension, glucose intolerance, gingival hyperplasia, hyperlipidaemia, hirsutism, neurological sequelae (including tremor) Tacrolimus Nephrotoxicity, hypertension, new-​onset diabetes after transplantation (NODAT), alopecia, neurological sequelae (including tremor) Azathioprine Bone marrow suppression, macrocytosis, hepatotoxicity Mycophenolate Diarrhoea, bone marrow suppression Sirolimus Hyperlipidaemia, thrombocytopenia, mouth ulcers, interstitial lung disease Anti-​CD25 (IL-​2 receptor α-​chain) antibodies Occasional hypersensitivity reactions ATG Cytokine release syndrome (fever, influenza-​like symptoms, hypotension), leukopenia, thrombocytopenia, serum sickness Alemtuzumab Cytokine release syndrome, cytopenias, infections

4.7  Principles of transplantation immunology 405 is expressed on most B cells (except plasma cells). In transplantation practice rituximab is rarely used outside of desensitization protocols and occasionally in ABMR. Attempts to use it for induction therapy had to be abandoned due to a high incidence of acute cellular rejection. Other biological agents Basiliximab is a monoclonal antibody directed against CD25 (the high-​ affinity IL-​2 receptor α-​chain). The use of basiliximab as induction therapy was shown to reduce 6-​month acute rejection rates by one-​ third, with minimal side effects, and it is now in widespread use as part of induction immunosuppression for many solid organ transplants. Another biological agent now in routine clinical use is belatacept (LEA29Y). This is a fusion protein consisting of the extracellular domain of CD152 (CTLA-​4, cytotoxic-​T-​lymphocyte-​associated antigen 4)  combined with the Fc (non-​antigen-​binding) portion of IgG. As discussed earlier, CTLA-​4 has a higher affinity for the costimulatory molecules CD80 and CD86 than does CD28, and belatacept is therefore able to block CD28:CD80/​86 costimulation, thus increasing the T-​cell activation threshold. Belatacept is used as maintenance therapy in cases where patients are intolerant of CNIs and are not able to take mTOR inhibitors, and has shown non​inferiority to ciclosporin when combined with mycophenolate mofetil and steroids, although is yet to be directly compared to tacrolimus. Ongoing clinical trials and future possibilities in transplantation Advances in the management of transplant recipients have been accompanied by both improved 1-​year graft survival and reduced acute rejection rates, offering many patients with organ failure great improvements in both morbidity and mortality. However, these ad- vances still rely on the continuous administration of potent non-​ specific immunosuppressive medication, which is associated with increased risk of infection and malignancy as well as drug toxicity. The improvement in short-​ to medium-​term outcomes has un- fortunately not been followed by comparable improvement in long-​ term graft survival, with considerable late attrition of grafts resulting from the complex interaction of drug toxicity and chronic immune activation. A further major problem facing patients on transplant waiting lists is organ availability, with average waiting list time stable or increasing for most organs in the United Kingdom at present, and rates of cadaveric donation remain insufficient to meet demand. There is therefore considerable interest in strategies that might allow reduced exposure to immunosuppression medication with improved long-​term outcomes (which in itself would decrease the pressure on scarce organ resources), as well as improvement in organ quality, allowing more offered organs to be utilized. While there are many therapies that are not currently in routine use, the past few years have seen a plethora of new drugs and ther- apies enter into clinical trials. Some therapies that were considered purely experimental are now in early trials, and the targets for therapy are being extended. Treatments aimed at immunosuppres- sion reduction (e.g. regulatory cell therapy), new drugs targeting the innate immune system (e.g. complement and TLRs) and ther- apies aimed at improving the quality of donor organs are all under- going trials. There are still many hurdles to overcome, which will be discussed next. Adjunctive cell therapies Perhaps the most exciting development in recent years is the moving of regulatory cell therapy from an experimental technique into early clinical trials. Trials have begun in several centres across the United States and Europe using regulatory T cells as well as regulatory macrophages and dendritic cells. Currently these phase I/​IIa trials in living donor kidney transplants are aimed at establishing safety, not efficacy, but it is hoped that future studies will allow assessment of efficacy and reduction of immunosuppression. The central concept behind the use of regulatory cell therapy is the fact that regulatory cells are able to switch immune responses off and convert the response to an antigen from immunogenic to tol- erant. The hope with infusion of regulatory cells is that the immune system can be ‘persuaded’ to see the transplant as non​threatening by inhibiting the proliferation of T cells in SLOs (Treg) or by rendering alloantigen-​specific cells anergic (Mreg or tolerogenic DCs). Interest has also grown in the idea of using regulatory B cells, a relatively newly described subset of B cells, which may work by dir- ectly suppressing responses, or by inducing Tregs. Present work is entirely experimental, but cell therapy appears to be a promising av- enue towards the ultimate goal, which is transplant tolerance. Transplantation tolerance In healthy individuals, autoimmunity is avoided by specific mech- anisms that maintain immunological tolerance to self-​antigens (Table 4.7.8). The optimal outcome for patients after transplant- ation would be to harness these mechanisms to induce specific tol- erance to the graft. Transplantation tolerance can be defined as the Upregulation of gene transcription Calcineurin Antigen presenting cell NFAT NF -κB MAP kinases AP-1 Protein kinase C Nucleus mTOR Signal 1 Signal 2 Signal 3 IL-2 production IL-2 CD4 Costimulatory molecules IL-2 receptor T cell Cell cycle progression MHC molecule + peptide Upregulation of gene transcription Basiliximab Sirolimus Belatacept CD52 Alemtuzumab CD52 CNI Azathioprine Mycophenolate Corticosteroids Fig. 4.7.7  Molecular targets of common immunosuppressive drugs.

406 SECTION 4  Immunological mechanisms lack of a destructive immune response towards the graft without the requirement for indefinite non​specific immunosuppressive therapy, while preserving immune responses to pathogens. The science of immunological tolerance and the technology of transplantation have evolved hand in hand. The first description of acquired tolerance to foreign antigen in mice by Billingham, Brent, and Medawar in 1953 (for which Medawar was awarded the Nobel Prize for medicine in 1960)  actually preceded the first successful renal transplant (between identical twins) by Murray and colleagues in 1955. Billingham, Brent, and Medawar demonstrated that it was possible to acquire immunological tolerance to foreign antigen, and much effort has gone into defining strategies that would lead to toler- ance to alloantigens in transplantation. Many successful experimental techniques can produce durable hyporesponsiveness to mismatched allografts in rodent models, but so far few of these have been success- fully translated into large animal models or clinical trials. Progress in the field of transplantation tolerance is also hampered by the lack of definitive laboratory parameters able to give a clear indication of whether a particular recipient is tolerant of their graft. Of the proposed strategies for inducing transplant tolerance, few have been adopted into clinical practice. Regulatory cell therapy re- mains the most promising at present, although other strategies have been trialled on a smaller scale. Peripheral depletion Therapies that deplete circulating leucocytes, and in particular T cells, have been used successfully both as induction immunosup- pression and in the treatment of acute allograft rejection episodes for many years. The efficacy of this strategy in preventing allograft rejec- tion led to the hypothesis that profound lymphocyte depletion at the time of transplantation may allow the development of donor-​specific immunological hyporesponsiveness. Trials have been undertaken using peripheral T-​cell depletion with several poly and monoclonal antibodies, the most widely studied of which is alemtuzumab. Initial reports are excellent and show efficacy of alemtuzumab with low rates of early rejection. However, there is counter evidence that sug- gests the homeostatic proliferation of cells following therapeutic lymphopenia may paradoxically increase the response to the trans- plant. Patients who were given alemtuzumab with no or little main- tenance therapy have a higher incidence of late rejection, and there is some evidence to suggest that those given alemtuzumab induction are at higher risk of DSA development in the longer term, even when given maintenance triple therapy, hence this strategy does not seem likely to induce transplant tolerance. Macro-​ and mixed chimerism The most robust experimental strategies for the induction of toler- ance to foreign antigen utilize the mechanisms of central deletion to eliminate T-​cell clones with specificity for the foreign antigens in question, thereby preventing them from entering the periphery. This can be reliably achieved by the establishment of haematopoietic chimerism through bone marrow transplantation. Stable engraft- ment of donor haemopoietic stem cells results in repopulation of the recipient thymus with donor-​type thymic dendritic cells, with the result that developing T cells with antidonor specificity are deleted by negative selection. In experimental models, full donor chimerism induced by myeloablative therapy (frequently a combination of total body irradiation and cytotoxic medication) followed by donor bone marrow transplantation produces tolerance to a subsequent allo- graft from an identical donor. There are several patients who have undergone successful bone marrow transplantation for haem- atological indications and have subsequently been successfully transplanted with a kidney from the same donor, without the re- quirement for increased immunosuppression. However, the es- tablishment of full donor chimerism is not acceptable for most recipients on the transplant waiting list as the risk profile (i.e. the toxicity and mortality associated with myeloablative condi- tioning regimens and the high incidence of graft-​vs.-​host disease in patients), is not acceptable when compared to the alternative (i.e. lifelong immunosuppression with the agents just described). However, this may be an option for young patients who are highly sensitized and who may not otherwise be suitable for transplant- ation, for whom the risks of long-​term dialysis may be more sig- nificant than those associated with myeloablative conditioning. Data from North America in 12 patients treated with this combin- ation therapy has shown that 7 of these are immunosuppression free by 4 years post-​transplant, so this seems a promising option for those with limited alternatives. Mixed chimerism may be a less toxic alternative, where recipi- ents receive donor stem cell infusion aimed at transient mixed chimerism, rather than full donor chimerism, just around the trans- plant period. The aim is to transiently fool the immune system, al- lowing a tolerogenic rather than pathogenic response to be initiated towards the graft, which is then maintained indefinitely. Mixed chimerism regimens are less toxic than full chimerism regimens and generally involve costimulatory blockade or non​myeloablative treat- ment such as cyclophosphamide with high dose of donor stem cells. This approach is still experimental in nature. Targeting of the innate immune system As interest grows in the role of the innate immune system in trans- plantation, new therapeutics are being trialled. As previously dis- cussed, there is renewed interest in the role of complement in transplantation, in particular with regards to the damage caused by ischaemia-​reperfusion injury. Eculizumab, a C5a inhibitor, is Table 4.7.8  Mechanisms of immunological tolerance Deletion Deletion of autoreactive T cells occurs in the thymus during T-​cell development (central deletion). Peripheral deletion of T cells can also occur if antigen is encountered under suboptimal condition (such as the absence or blockade of costimulatory signals) Anergy Anergy is the functional inactivation of T cells following antigen encounter and another possible outcome of antigen encounter under suboptimal conditions Immunoregulation Several populations of lymphocytes are able to suppress or regulate the ability of other lymphocytes to respond to a particular antigen Ignorance T cells do not have access to antigens present at certain sites of immune privilege (such as the anterior chamber of the eye)

4.7  Principles of transplantation immunology 407 used with success in kidney transplantation in patients with atyp- ical haemolytic uraemic syndrome (aHUS) to prevent immediate disease recurrence, and it has also been used in cases of severe antibody-​mediated rejection, particularly in patients who have undergone desensitization. There is potential for using eculizumab in deceased donor transplants, where complement components are known to be upregulated. Studies are in progress to investigate the possibility of blocking Toll-​like receptors, and a phase II study of a monoclonal antibody to TLR2 in extended-​donor criteria organs. The hope is that blocking receptors to DAMPs may prevent activation of phagocytes and hence reduce responses to inflammatory stimuli and it is likely that other therapeutics will be introduced in the coming years. Strategies to improve donor organs While many trials of therapeutics are limited to recipients of living kidney donors, particularly regulatory cell therapies which must be prepared in advance, most kidney donors are deceased, and outcomes for deceased donors are worse than for living donors. Furthermore, some solid organs have no possibility of being do- nated by living donors. Strategies are therefore required to increase the donor pool, both by sociological intervention and encouraging donation, but also by extending the criteria for accepting organs and improving organs retrieved from deceased donors. For many years, the gold standard of retrieval was perfusion of the organ with ice cold fluid at the time of organ harvesting. However, it is known that prolonged ischaemia leads to anaerobic metabolism and production of damaging and proinflammatory molecules on reperfusion. Trials into the use of ex-​vivo, hypothermic machine perfusion in kidney transplantation, and ex-​vivo normothermic machine perfusion in liver transplantation are ongoing, with prom- ising early results. In addition to potentially improving outcomes by reducing ischaemic time, the ex-​vivo perfusion allows surgical teams to assess function of the potential transplant, looking at bile and urine production, which may allow assessment of the potential for delayed graft function and guide decisions about which organs are suitable for implantation (see Fig. 4.7.8). Xenotransplantation Since the 1960s attempts have been made to use animals as an alter- native source of organs for transplantation. Initial attempts to trans- plant kidneys from non​human primates to humans resulted in renal function for weeks to months, although non​human primates are too scarce to be a realistic long-​term source of organs. Instead, most re- search has focused on other mammals, and in particular pigs, which can be raised in sufficient numbers in pathogen-​free facilities, can be genetically engineered, and have organs that are a suitable size. However, there remain significant practical as well as ethical hur- dles to overcome before clinical xenotransplantation becomes a possibility. The practical considerations include the robust immune re- sponse to xenografts. Although genetic engineering has been employed to remove Galα1, 3Gal, a carbohydrate determinant ex- pressed by cells of pigs and other lower mammals that commonly causes hyperacute rejection, other molecules have emerged to cause problems with acute rejection and further genetic manipu- lation is under investigation. There also remains a risk of viral transmission, which is theoretical but could present a significant public health problem. Xenografts are used for heart valve replacement, but no other xenografts are in routine clinical use or trials outside of non​human primates. Bioengineering At present a very theoretical possibility, the interest in bioengineered grafts is very considerable. The theory is that organs could be grown using the intended recipient’s own stem cells, seeded onto an organ scaffold (usually a decellularized donated organ, harvested up to 6 hours after death) and then implanted. This would allow transplant- ation of an entirely self-​derived organ, with no risk of rejection and thus no requirement for immunosuppression. A few case studies have given proof of concept, but they are currently limited to simple, avascular tissues, such as tracheas, bladders, and blood vessels, which rely on simple diffusion for oxy- genation and nutrients. Bioengineering of more complex grafts has been attempted, and some success has been achieved in producing animal liver, kidney, pancreas, and intestinal grafts, but implant- ation of these has had limited success. One major concern is the malignant potential of the seeder cells, as the reprogramming of stem cells can cause genetic alterations with the potential to be- come malignant. It is likely to be many years before bioengineered organs become a reality for human transplantation. However, with the shortage of donor organs, the increased lifespan of transplant recipients leading to problems with immunosuppressive burden and the limited pro- gress in the field of xenotransplantation, the possibility of bioengin- eering remains attractive. Fig. 4.7.8  Ex-​vivo liver perfusion. The OrganOx machine was designed by transplant surgeon Professor Peter Friend and biomedical engineer Professor Constantin Coussios as a way of supporting the retrieved liver prior to implantation. The liver is perfused with oxygenated blood at 37oC and supported by infusions of nutrients, bile salts, prostacyclin, heparin, and insulin. Sensors allow assessment of haemodynamic, metabolic, and synthetic liver performance during preservation. The machine is intended to allow assessment of liver function, longer preservation times, and use of extended criteria donors. Image and information reproduced with the kind permission of Professor Peter Friend.

408 SECTION 4  Immunological mechanisms FURTHER READING Abecassis M, Kaplan B (2015). Transplantation:  biomarkers in transplantation—​the devil is in the detail. Nat Rev Nephrol, 11, 204–​5. Batista FD, Harwood NE (2009). The who, how and where of antigen presentation to B cells. Nat Rev Immunol, 9, 15–​27. Benichou G, et al. (1992). Donor major histocompatibility complex (MHC) peptides are presented by recipient MHC molecules during graft rejection. J Exp Med, 175, 305–​8. Billingham RE, Brent L, Medawar PB (1953). Actively acquired toler- ance of foreign cells. Nature, 172, 603–​6. Carney EF (2015). Transplantation: induction of chimerism and im- mune tolerance using belatacept. Nat Rev Nephrol, 11, 66. Clatworthy MR (2013). B cell responses to allograft—​more common than we thought? Am J Transplant, 13, 1629–​30. Clatworthy MR (2014). B-​cell regulation and its application to trans- plantation. Transpl Int, 27, 117–​28. Crotty S (2015). A brief history of T cell help to B cells. Nat Rev Immunol, 15, 185–​9. Edwards JK (2015). Immunology: clonal deletion contributes to allo- graft tolerance. Nat Rev Nephrol, 11, 196. Feng G, et al. (2009). Donor reactive regulatory T cells. Curr Opin Organ Transplant, 14, 432–​8. Gandhi AM, et al. (2008). Costimulation targeting therapies in organ transplantation. Curr Opin Organ Transplant, 13, 622–​6. Gasteiger G, Rudensky AY (2014). Interactions between innate and adaptive lymphocytes. Nat Rev Immunol, 14, 631–​9. Girmanova E, Hruba P, Viklicky O (2015). Circulating biomarkers of tolerance. Transplant Rev (Orlando), 29, 68–​72. Gosset C, Lefaucheur C, Glotz D (2014). New insights in antibody-​ mediated rejection. Curr Opin Nephrol Hypertens, 23, 597–​604. Grakoui A, et al. (1999). The immunological synapse: a molecular ma- chine controlling T cell activation. Science, 285, 221–​7. Hu Q, Liu Z, Zhu H (2014). Pig islets for islet xenotransplantation: cur- rent status and future perspectives. Chin Med J (Engl), 127, 370–​7. Juvet SC, et al. (2014). Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning. Am J Transplant, 14, 750–​63. Kawai T, et al. (2018). Summary of the third international workforce on clinical tolerance. Am J Transplant, doi: 10.1111/​ajt.15086. Kinnear G, Jones ND, Wood KJ (2013). Costimulation blockade: current perspectives and implications for therapy. Transplantation, 95, 527–​35. Kolev M, Le Friec G, Kemper C (2014). Complement—​tapping into new sites and effector systems. Nat Rev Immunol, 14, 811–​20. Kugelberg E (2014). Neutrophils:  bugging transplantation. Nat Rev Immunol, 14, 430–​1. Land WG (2013). Chronic allograft dysfunction: a model disorder of innate immunity. Biomed J, 36, 209–​28. McDonald-​Hyman C, Turka LA, Blazar BR (2015). Advances and challenges in immunotherapy for solid organ and hematopoietic stem cell transplantation. Sci Transl Med, 7, 280rv282. Meier-​Kriesche HU, et al. (2004). Lack of improvement in renal allo- graft survival despite a marked decrease in acute rejection rates over the most recent era. Am J Transplant, 4, 378–​83. Mella A, et al. (2014). Complement cascade and kidney transplant- ation: the rediscovery of an ancient enemy. World J Transplant, 4, 168–​75. Messner F, Etra JW, Dodd-O JM, Brandacher G (2019). Chimerism, transplant tolerance and beyond. Transplantation, doi: 10.1097/ TP.0000000000002711. Michel SG, et al. (2015). Current progress in xenotransplantation and organ bioengineering. Int J Surg, 13, 239–​44. Montgomery RA, et  al. (2018). HLA in transplantation. Nat Rev Nephrol, 14, 558–​70. Nasralla D, et al. (2018). A randomized trial of normothermic preserva- tion in liver transplantation. Nature, 557(7703), 50–6. Nayak DK, Mohanakumar T (2014). Transplantation:  recognizing self versus non-​self: new territory for monocytes. Nat Rev Nephrol, 10, 548–​9. Oberbarnscheidt MH, Lakkis FG (2014). Innate allorecognition. Immunol Rev, 258, 145–​9. Ochando J, Ordikhani F, Boros P, Jordan S (2019). The innate immune response to allotransplants: mechanisms and therapeutic potentials. Cell Mol Immunol, 16(4), 350–6. Pratschke J, Stauch D, Kotsch K (2009). Role of NK and NKT cells in solid organ transplantation. Transpl Int, 22, 859–​68. Quezada SA, et al. (2004). CD40/​CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol, 22, 307–​28. Reddy UG, Danovitch GM (2014). Transplantation. T-​cell depletion—​ balancing acute rejection and cancer risk. Nat Rev Nephrol, 10, 301–​2. Resch T, et al. (2015). The role of natural killer cells in humoral rejec- tion. Transplantation, 99, 1335–​40. Rickert CG, Markmann JF (2019). Current state of organ trans- plant tolerance. Curr Opin Organ Transplant, doi: 10.1097/MOT. 0000000000000670. Sachs DH, Kawai T, Sykes M (2014). Induction of tolerance through mixed chimerism. Cold Spring Harb Perspect Med, 4, a015529. Saidi RF, Hejazii Kenari SK (2014). Clinical transplantation and toler- ance: are we there yet? Int J Organ Transplant Med, 5, 137–​45. Schinstock CA, Stegall M, Cosio F (2014). New insights regarding chronic antibody-​mediated rejection and its progression to trans- plant glomerulopathy. Curr Opin Nephrol Hypertens, 23, 611–​8. Schroder PM, et al. (2019). The past, present, and future of costimulation blockade in organ transplantation. Curr Opin Organ Transplant, doi: 10.1097/MOT.0000000000000656. Stolp J, Turka LA, Wood KJ (2014). B cells with immune-​regulating function in transplantation. Nat Rev Nephrol, 10, 389–​97. Thomson AW (2018). A view of the future or regulatory immune cell therapy in organ transplantation. Curr Opin Organ Transplant, 23, 507–​8. Turka LA, Lechler RI (2009). Towards the identification of biomarkers of transplantation tolerance. Nat Rev Immunol, 9, 521–​6. Willis JC, Lord GM (2015). Immune biomarkers: the promises and pit- falls of personalized medicine. Nat Rev Immunol, 15, 323–​9. Wood KJ, et al. (2014). Bridging innate with adaptive immunity in transplantation: triggers, sensors, and modulators. Transplantation, 98, 1021–​4. Yolcu ES, Leventhal JR, Ildstad ST (2015). Facilitating cells in tolerance induction for kidney transplantation. Curr Opin Organ Transplant, 20, 57–​63. Zachary AA, Leffell MS (2014). Desensitization for solid organ and hematopoietic stem cell transplantation. Immunol Rev, 258, 183–​207. Zhuang Q, Lakkis FG (2015). Dendritic cells and innate immunity in kidney transplantation. Kidney Int, 87, 712–​8. Suggested websites British Transplantation Society. http://​www.bts.org.uk ESOT. http://​www.esot.org Immune Tolerance Network. http://​www.immunetolerance.org NHS Organ Donation. http://​www.organdonation.nhs.uk Project HLA Sequence Database. http://​www.ebi.ac.uk/​imgt/​hla The ONE Study. http://​www.onestudy.org The Transplantation Society. http://​www.tts.org

SECTION 5 Principles of clinical oncology Section editors:  John D. Firth, Christopher P. Conlon, and Timothy M. Cox 5.1 Epidemiology of cancer  411 Anthony Swerdlow and Richard Peto 5.2 The nature and development of cancer:
Cancer mutations and their implications  445 James D. Brenton and Tim Eisen 5.3 The genetics of inherited cancers  456 Rosalind A. Eeles 5.4 Cancer immunity and immunotherapy  471 Charles G. Drake 5.5 Clinical features and management  487 Tim Eisen and Martin Gore 5.6 Systemic treatment and radiotherapy  497 Rajesh Jena and Peter Harper 5.7 Medical management of breast cancer  505 Tim Crook, Su Li, and Peter Harper

ESSENTIALS The epidemiology of cancer is the investigation of the incidence and causes of the disease in people under different conditions of life. Such investigations have generally been the way in which reliable evidence about causal agents for cancer, and the magni- tude of the risks from these agents, have been found. They have shown that any type of cancer that is common in one population is rare in some other, and that the differences between popu- lations are mostly not genetic, but rather the consequences of behaviours and circumstances of life. In principle, cancers are therefore largely preventable. The range of incidence rates between geographical and ethnic groups is more than 10-​fold for each of the common cancers, and for some cancers is more than 100-​fold. Large changes in rates of many tumours can occur in migrants compared with rates in their homeland, and large changes have occurred in rates within popula- tions over time, indicating the scope for prevention. The causes of cancer These can be divided into nature (biological factors), nurture (envir- onment and behaviours), and chance. Biological factors—​important biological factors are genetic suscep- tibility, age, and sex. Tobacco smoking—​this is the most important extrinsic factor causing cancer in developed countries, and is a major cause of can- cers of the mouth, pharynx (other than nasopharynx), oesophagus, larynx, lung, pancreas, renal pelvis, and bladder (and it also causes a proportion of several other types of cancer). In 2010, smoking is esti- mated to have caused 27% of all fatal cancers in the United Kingdom. Other extrinsic causes—​include (1) alcohol—​a cause of at least six types of cancer, including liver, various upper aerodigestive sites, and breast; (2) ionizing radiation—​which can cause cancer in most tis- sues; in the United Kingdom the main sources of exposure are natural sources including radon, and medical uses; (3) ultraviolet radiation—​ causes skin cancer; (4) infection, principally viral, but also bacterial and parasitic—​a major cause of cancer of several sites, especially in developing countries; (5)  immunosuppression—​patients with per- sistent immunosuppression from therapeutic, infective, or genetic causes have raised risks of certain cancers, notably non-​Hodgkin’s lymphoma; (6) chemotherapeutic agents—​about 20, used for treat- ment of specific diseases and including several that are used to treat cancer, have been shown themselves to cause cancer, of dif- ferent anatomical sites according to the agent; (7)  other drugs—​ hormone replacement therapy and oral contraceptives, both widely used in the general population, affect the risk of certain female reproductive-​related malignancies, increasing risk for some cancers but (for combined steroid contraceptives) decreasing it for others; (8) occupation—​numerous occupational groups have been found to be at increased risk of cancer, mainly of the respiratory tract, and es- pecially the lung; (9) air, water, and food pollution—​these are probably responsible for a small percentage of cancers in Western countries; (10) diet—​this may well have an effect on the aetiology of a substantial proportion of cancers, but there is considerable uncertainty on the figure and specific dietary associations are largely unknown. Other factors of particular note are (1) menstrual and reproductive history—​these affect the risks of breast, endometrial, and ovarian cancers in women; (2) obesity—​relates to increased risks of cancers of the breast (postmenopausally), endometrium, colon, kidney, and gallbladder, adenocarcinoma of the oesophagus, and possibly other cancers; (3)  physical inactivity—​relates to increased risk of breast, endometrial, colonic, and possibly other cancers. Epidemiology and aetiology of particular cancers The most common cancers worldwide are those of the lung, breast, and colorectum, and the most common causes of cancer death are lung, liver, and stomach cancers. Descriptive and aetiological epidemiological information is given about 33 types of cancer in this chapter. Lung cancer—​the major cause is smoking tobacco, particu- larly cigarettes. Lung cancer became epidemic in men in Western countries during the mid-​20th century, with rates rising later in women, and in Western countries there have been consider- able decreases in men in recent years. In developing countries, however, the epidemic has arrived later, with rising rates to be expected in future years as a consequence of current smoking levels. Occupational causes of lung cancer include exposures to asbestos, polycyclic hydrocarbons, and radon. Air pollution in towns may have also been a factor, largely in smokers, and radon in indoor air contributes to a small percentage of cases. 5.1 Epidemiology of cancer Anthony Swerdlow and Richard Peto

412 SECTION 5  Principles of clinical oncology Breast cancer—​the most common cancer worldwide in women. It has greatest incidence in Western countries, where rates have tended to increase slowly over decades; rates have generally been much lower in Asia and Africa. Hormonal and reproductive factors are important to risk: early menopause, late menarche, nulliparity, and older age at first full-​term pregnancy all increase risk, as do postmenopausal hormone replacement therapy and combined
oral contraceptives, while tamoxifen treatment of unilateral breast cancer decreases risk in the unaffected breast. There is also raised risk of breast cancer in relation to a history of benign breast disease, alcohol consumption, lack of physical exercise, postmenopausal obesity, taller height, and ionizing radiation exposure at young ages, as well as genetic predisposition. Introduction Cancer is a major cause of death and an increasing one: in England and Wales (Fig. 5.1.1) the proportion of deaths attributed to cancer has risen inexorably for over a century. All cancers have certain pathological and clinical characteristics in common, but those arising in different organs often have very different causes. The epidemiology of cancer, by which is meant the study of the incidence and causes of the disease in people under different conditions of life, is, therefore, the epidemiology of spe- cific types of cancer, usually—​but not always—​defined as cancers of specific organs. In this sense, the subject has a history dating back nearly 300 years to Ramazzini’s observation that cancer of the breast occurred more often in nuns than in other women of similar age and to Pott’s observation, over 200 years ago, that scrotal cancer in young men occurred characteristically in chimney sweeps. The high risk in nuns (which largely reflected the protective effect of multiple pregnancies in the general population) helped the real- ization that hormonal factors can substantially affect the incidence of several types of cancer, while the latter led to the recognition that the combustion products of coal to which sweeps had been exposed could cause cancer on any part of the skin with which they came into repeated contact and to the isolation of the first specific chemical carcinogen. Many other similar observations were made over the next 150 years, mostly as a result of the acumen of individual doctors who noticed clusters of cases of a particular type of cancer occurring in patients with a similar occupational or cultural background. Lip and tongue cancers were found in pipe smokers; bladder cancer in certain aniline dye workers; buccal cancer in those who habitually chewed mixtures of tobacco and betel in India; lung cancer in miners of particular ores (who, it was subsequently realized, were heavily exposed to radon and its daughter products); and skin cancer in the early radiologists and radiographers who were heavily exposed to X-​rays and in farmers and seamen heavily exposed to sunlight. Gradually, however, clinical anecdotes were replaced by statistics as epidemiological methods began to be applied to the study of cancer and other non​infectious diseases. As a result, many other causes were identified with sufficient certainty to justify preventive action and data were obtained to suggest hypotheses that could be tested in the laboratory. Preventability of cancer Perhaps the most important result of such observations has been the realization that any type of cancer that is common in one population is rare in some other, and that the differences between populations are mostly not genetic. Hence, where they are common these can- cers occur, in large part, as a result of the way people behave and the circumstances in which they live and they are, therefore, at least in principle, preventable. This does not mean that we can at present en- visage a society in which any of the common cancers are completely eliminated (although this may prove to be possible when we under- stand more clearly the mechanisms by which they are produced). What it does mean is that we can envisage a society in which the age-​specific risk of developing any type of cancer is low. Differences in incidence between communities Variation in incidence of cancer between different ethnic and geo- graphical groups around the world can be ascertained from data provided by population-​based cancer registries. Table 5.1.1 shows, for selected types of cancer, the range of variation recorded by cancer registries that have produced data sufficiently reliable for the purpose of international comparison, or, in one instance, the range determined by special surveys. Types of cancer have been included if they are common enough somewhere to have a cumu- lative incidence among men or women of at least 2% by 75 years of age. The range of variation is never less than 14-​fold and is sometimes more than 100-​fold. Despite the selection of reason- ably reliable registries, some of this tabulated variation may still be an artefact due to differing standards of medical service, case registration, and population enumeration. In many cases, how- ever, the true ranges will be greater, because much of the world is not covered by reliable cancer registries, and because the data generally refer to cancers of whole organs and do not distinguish between different histological types or different locations within an organ, for which greater variation may apply. 40 30 20 10 0 1910 1920 1930 1940 1950 1960 Year Females Males Percentage of all deaths due to neoplasms 1970 1980 1990 2000 2010 2020 Fig. 5.1.1  Percentage of all deaths due to neoplasms, England and Wales, 1911–​2014.

5.1  Epidemiology of cancer 413 The variation in incidence is not limited to the common cancers. Burkitt’s lymphoma, for example, never affects more than 1 in 1000 of the population, but it is at least 100 times as common among chil- dren in parts of Uganda as it is in Europe and North America; while Kaposi’s sarcoma, which was extremely rare in most of the world until the advent of AIDS, was so common in children and young adults in parts of Central Africa, even before 1970, that it accounted for 10% of all tumours seen in one of the African hospitals surveyed by Cook and Burkitt. Some few cancers occur with approximately the same frequency in all communities, but all are relatively un- common. Acute myeloid leukaemia at 15–​25 years of age is an ex- ample; nephroblastoma is another, except that it appears to be only half as common in Japan as elsewhere. The aforementioned figures refer to cancer incidence in commu- nities defined by geography, but substantial differences are found be- tween communities defined in other ways such as by ethnic origin, religion, or socioeconomic status. Jewish women, for example, have a low incidence of cervical cancer irrespective of the country in which they live, and Mormons and Seventh Day Adventists living in the United States of America have low incidence of cancers of the respiratory, gastrointestinal, and genital systems. Few of the large differences between communities can be ex- plained by genetic factors, apart from some of the differences in the incidence of cancer of the skin, the risk of which is much greater for whites than for blacks, and possibly also for some of those in the incidence of testis cancer, which rarely affects black popula- tions, and in chronic lymphocytic leukaemia, which rarely affects people of Chinese or Japanese descent. Genetic factors cannot ex- plain the differences observed on migration or with the passage of time, which are discussed in the next sections of this chapter, nor can they explain the correlations observed between the national rates for particular types of cancer and aspects of the lifestyle in different countries. Changes in incidence in migrant groups That changes in the incidence of cancer occur on migration is cer- tain. Numerous groups have been studied, particularly migrants from many countries to Australia, Israel, and the United States. These show, for example, that Afro-​Americans experience in- cidence rates for internal cancers that are generally much more like those of white Americans than those of the black populations in West Africa from which most of their ancestors came, while Japanese in Hawaii have experienced rates that are much more like those of the white residents of Hawaii than those of Japanese living in Japan (Table 5.1.2). The ancestors of black Americans and Hawaiian Japanese will have come from many different parts of Africa and Japan, some of which are likely to have cancer rates somewhat different from those cited in Table 5.1.2. Nevertheless, the contrasts are so great that there can be no serious doubt that new factors were introduced with migration. Table 5.1.1  Range of incidence rates of common cancers Site of origin of cancera High-​incidence areab,c Cumulative incidence (%) 
in high-​incidence aread Low-​incidence area,c same sex Ratio of cumulative rates in high-​ and
low-​incidence arease Non​melanoma skin Australia (Queensland)f

20 Several non​white populations 200 Prostate US (Delaware, black) 28 China (Yancheng) 250 Oesophagus China (Cixian) 24 Kuwait, non-​Kuwaitis 480 Stomach China (Yangcheng) 22 Kuwait, non-​Kuwaitis 92 Breastg Hawaii, Hawaiian 13 China (Yanting) 14 Lung US (Wisconsin, black) 12 India (Dindigul, Ambilikkai) 25 Cervix uterig Zimbabwe (Harare, black) 10 Egypt (Gharbiah) 58 Liver China (Qidong) 8 Brazil (Forteleza) 64 Kaposi’s sarcoma Malawi (Blantyre) 8 Several 500 Colong US (Alaska, American Indian) 6 India (Dindigul, Ambilikkai) 200 Melanoma of skin Australia (Queensland) 6 Several 500 Bladder Italy (Syracuse) 5 India (Sikkim, and Dindigul, Amilikkai) 32 Corpus uterig New Zealand, Pacific Islander 4 Libya (Benghazi) 43 Rectum Czech Republic 3 India (Dindigul Ambilikkai) 20 Kidney Czech Republic 3 China (Yanting) 135 Nasopharynx China (Zhongshan) 3 Several 280 Non-​Hodgkin’s lymphoma US (Washington, Seattle) 2 India (Sikkim) 68 a Sites of cancer are shown if somewhere they reach a cumulative incidence by age 75 of at least 2% in either sex. b The geographic area of highest recorded incidence in either sex by age 75. Since most are males, footnotes to indicate sex are only given where the rate is in females. c Excluding very small cancer registry populations, with unstable numbers. d By age 75 years, in the absence of other causes of death. e By age 65 years, in the absence of other causes of death. f Special survey. g Women.

414 SECTION 5  Principles of clinical oncology Changes in incidence over time Within one population there may be substantial changes in the in- cidence of a particular type of cancer over a period of a few decades that provide conclusive evidence of the existence of preventable fac- tors. Changes in incidence over time may, however, be difficult to assess reliably. This is chiefly because it is difficult to compare the thoroughness of the diagnosis and registration of particular types of cancer at different periods and partly because few incidence data have been collected for long enough, so we often have to fall back on changes in mortality rates even though these may be influenced by changes in treatment, as well as changes in incidence. There are no simple rules for deciding which changes in recorded cancer incidence and mortality rates are reliable indicators of real changes in incidence. Each set of data has to be assessed individu- ally. It is relatively easy to be sure about changes in the incidence of cancer of the oesophagus, as the disease can be diagnosed without complex investigations and its occurrence is nearly always recorded, at least in middle age, because it is nearly always fatal. By contrast, it is much more difficult to be sure about changes in the incidence of basal cell carcinomas of the skin, which, although easy to diagnose, seldom cause death and can be treated effectively outside hospital, and so often escape registration. What appears to be a change in in- cidence may therefore be a change only in the completeness of regis- tration. Cancers of the pancreas, liver, and brain, and myelomatosis, in contrast, usually cause death, but even when they do they may be misdiagnosed as another disease, so that an increased incidence or mortality rate may be wholly or partly due to improvements in diag- nosis or in the availability of medical services. Such changes are par- ticularly likely to affect the rates recorded for people over 65 years of age, as many older people who were terminally ill used not to be intensively investigated. Despite these difficulties, some of the decreases and increases in the recorded rates of particular types of cancer have been so gross that there must have been real changes in their incidence. Examples include the increase in lung cancer throughout most of the world (and its recent large decrease in men in the United Kingdom), the in- crease in mesothelioma of the pleura in men in industrialized coun- tries, the decrease in cancer of the tongue in the United Kingdom, and the decrease in cancers of the uterine cervix and stomach throughout Western Europe, North America, and Australasia. For a fuller account, see Doll et al., 1994 and Swerdlow et al., 2001. Identification of causes More-specific evidence of the preventability of cancer, and of meas- ures to enable this, has come from identification of agents and cir- cumstances that cause the disease. In general, reliable evidence of causality (and particularly of the magnitude of any risks) has come from epidemiology and not from laboratory experiments, although the latter can often provide reinforcement of epidemiological find- ings and understanding of the mechanisms of cancer causation. Reliable epidemiological evidence does not require randomized trials within particular populations, but it does require the study of different individuals within populations and not just the com- parison of incidence rates between populations. Non​randomized epidemiological studies of individuals have often yielded proof of causation beyond reasonable doubt, and have provided the decisive evidence of aetiology for almost all proven carcinogens. Action based on such evidence has, moreover, often been followed by the desired result; for example, a reduction in the incidence of bladder cancer in the chemical industry on stopping the manufacture and Table 5.1.2  Comparisons of cancer incidence rates in migrantsa and residents in homelands and adopted countries (men, unless otherwise specified), mid-​1990s. Cumulative rate to age 65, per 1000 persons Japanb Hawaii West Africab United States Japanese Whites Blacks Whites Oesophagus 6.5 3.0 1.7 1.9 7.4 2.6 Stomach 32.9 7.4 3.6 7.3 6.6 3.1 Colon 14.8 17.2 14.0 0.8 16.3 11.0 Rectum 10.0 11.6 6.6 0.9 6.8 6.4 Liver 17.3 3.5 2.6 32.1 4.9 1.9 Pancreas 4.4 4.0 3.9 1.3 7.0 3.4 Larynx 1.7 1.8 5.2 0.4 6.6 3.2 Lung 15.2 13.4 21.7 1.9 49.5 25.8 Breastc 24.2 61.7 72.2 10.5 59.8 65.1 Uterine cervixc 4.7 3.4 5.7 27.5 7.5 5.3 Corpus uteric 3.0 13.1 9.9 1.9 7.6 12.4 Ovaryc 4.7 7.4 10.2 1.4 5.5 9.1 Prostate 2.0 17.6 40.6 2.2 91.2 47.4 Testis 1.0 1.7 5.1 0.2 0.8 4.1 Non-​Hodgkin’s lymphoma 3.4 4.3 10.4 1.4 11.0 10.1 a ‘Migrant’ rates are based on ethnicity, and hence include all generations of migrant. b Average of rates in two regions. c Women.

5.1  Epidemiology of cancer 415 use of 2-​naphthylamine and, on a national scale, the reduction in the incidence of lung cancer in men in the United Kingdom following the decrease in smoking since the mid-​20th century. The causes of cancer can, briefly, be divided into nature (biological factors), nurture (behaviour and environment), and chance. Biological factors Genetic susceptibility Many common types of cancer tend to cluster in families to some extent. Differences of this sort do not in themselves imply that the familial clusters are genetic in origin; they could be due to familial similarities of behaviour or environment. Nevertheless, various genetic factors are known that affect cancer risks to different ex- tents. Very large risks are seen in patients with certain rare cancer-​ associated genetic syndromes in which bearers of one gene (if the condition is dominant) or two (if recessive) almost invariably de- velop a particular type of cancer. Examples include the dominant genes for polyposis coli that lead to cancer of the large bowel, and the recessive gene for retinoblastoma, and that for xeroderma pigmentosum, which leads to squamous carcinoma and (less com- monly) melanoma of the skin. Similar evidence has shown that other genetic syndromes frequently, but not invariably, lead to cancer, such as von Recklinghausen’s neurofibromatosis leading to fibrosarcoma, the Peutz–​Jeghers syndrome leading to carcinoma of the small bowel, the Wiskott–​Aldrich syndrome leading to non-​ Hodgkin’s lymphoma, and ataxia telangiectasia, Bloom’s syndrome, and Fanconi’s anaemia leading to leukaemia. Very high cancer risks are also present in individuals with various cancer susceptibility genes; for example, raised risks of breast and ovarian cancers in pa- tients with mutations in BRCA1 and BRCA2 genes. The recognition of these genes is important to the individual, as it may provide an opportunity for prophylactic surgery, or enable the diagnosis of ma- lignancy to be made at an early stage when treatment is more likely to be effective, or (rarely) enable precautions to be taken to prevent exposure to the relevant carcinogens, as in the case of sufferers from xeroderma pigmentosum or albinism, who can be protected against sunshine. The proportion of all cancers that occur in people who are highly susceptible to cancer in this way is, however, very small, al- though substantially greater at young than at older ages. Genes conveying less-​raised risks of cancer are far more wide- spread in the population, and are involved in the causation of a much larger proportion of cancers. Large numbers of relatively common susceptibility variants influencing cancer risks have been discovered in the last few years. Discovery of genetic factors that affect particular types of cancer is unlikely to explain much of the social and geographical differences in the distribution of cancer other than skin cancer, but it can help to elucidate mechanisms and to focus health education and early diag- nosis on the sections of the populations that are most at risk. Age Some risk of cancer occurs at every age, but the risk of developing any particular type varies with age. The most common relationship with age is a progressive increase in incidence from near zero in childhood and adolescence to a high rate in old age. This type of relationship is shown by carcinomas of the skin, lung, and gastro- intestinal and urinary tracts, and by myelomatosis and chronic lymphatic leukaemia. The rate of increase is rapid, being typically proportional to the fourth, fifth, or sixth power of age in years, so that the annual incidence may be 100 or 1000 times greater above age 75 than before age 25. It is probable that this reflects the cu- mulative effect of processes that operate steadily throughout life, starting at around the time of birth or at young ages (e.g. for lung cancer, in adolescence). With most of these cancers, the recorded incidence may stabilize, or even decrease, in the oldest age groups, but this is partly or wholly an artefact due to incomplete investiga- tion of the terminal illnesses of old people. A less common pattern is a peak incidence early in life, which may be followed either by a decline virtually to zero or by a slow rise in middle and old age. Retinoblastomas and nephroblastomas occur only in childhood, with peak incidences (respectively) in the first and second years of life. Teratomas and seminomas of the testis have peak incidence rates at about 25 and 35 years of age, respectively, and later almost cease to occur, while osteogenic sarcomas have a peak incidence in adolescence and then show a slow increase with age from a lower rate in young adult life. The remaining cancers show a variety of patterns. Carcinomas of the breast and uterine cervix of women, for example, begin to ap- pear in young adulthood and become rapidly more common up to the menopause. After the menopause the incidence of carcinoma of the breast may remain approximately constant, or may even be- come slightly reduced for a few years, before increasing again with age, though at a slower rate. Carcinoma of the cervix continues to increase fairly steeply for a few years after the menopause, before showing a stable or declining rate. Hodgkin’s disease, on the other hand, appears in childhood but thereafter continues relatively evenly throughout life with only modest peaks in young adult life and at older ages, while connective tissue sarcomas become progressively more common from childhood onwards, but with a much slower rate of increase than is shown by the common carcinomas. Some of these relationships with age, like that for retinoblastoma in early childhood, seem to be invariant everywhere and, as far as is known, at all times. Others vary from community to community, or from time to time. In postmenopausal women, for example, cancer of the breast becomes progressively less common with increasing age in parts of Asia, but more common in Europe, while carcinoma of the lung used to show a peak incidence at about 60 years of age in the United Kingdom, which gradually moved to older ages, as a generation that had not smoked substantial numbers of cigarettes throughout adult life was replaced by one that had, and the same process is now being repeated in many developing countries. These various patterns provide information, either about the period of activity of the stem cells from which the cancers derive, or about the period when the main exposure to causative agents occurs and the duration of that exposure. Some of this variation has already helped to explain some of the causes of cancer, as was the case with the shift in the peak incidence of bronchial carcinoma, but much still awaits elucidation. Sex Cancer used to be more common in women than in men in many countries due to the great frequency of carcinoma of the breast and of the uterine cervix, and to the rarity of bronchial carcinoma. However, now the reverse is generally true except in a few popu- lations in Africa and Asia for which similar conditions to those

416 SECTION 5  Principles of clinical oncology formerly persist. This overall male preponderance hides, however, a variety of sex ratios for cancer of different organs. Aside from sites of cancer that are peculiar to one sex, the sex ratio varies (in Britain) from a male excess of 6 to 1 for pleural mesothelioma and carcinoma of the larynx, through many types of cancer with only a small male preponderance, to carcinomas of the thyroid and gallbladder, which are about twice as common in women, and breast cancer which is over 100 times more common in women. For many types of cancer the sex ratio is much the same in different countries and at different times. For some, however, and particularly for cancers of the mouth, oesophagus, larynx, and bronchus, the sex ratio is extremely variable—​not only between countries and at dif- ferent times, but sometimes also between different ages at the same time and in the same country. The most marked variation is shown by cancer of the oesophagus, which may affect both sexes equally or be 20 times more common in men than in women. As with the pat- terns of incidence with age, these sex ratios and their variation can provide useful clues to the causation of particular types of cancer, not all of which have yet been successfully followed up. Delay between cause and effect One reason why it has been difficult to recognize causes of cancer in humans is the long delay that characteristically occurs between the start of exposure to a carcinogen and the appearance of the clin- ical disease. This ‘latent period’, as it is commonly, but rather mis- leadingly, called is often several decades, although it may be as short as 1 year or as long as 60. The exact relation between the date of exposure and the date of the appearance of different cancers is still uncertain, partly because the interval is subject to random factors, partly because few cancers are induced by a single, brief exposure, and partly because there are still relatively few sets of quantitative data with detailed information about the dates when exposure began and ended. When cancer is induced by short but intensive exposure to ion- izing radiation, as following the explosions of the atomic bombs in Hiroshima and Nagasaki or in patients treated by radiotherapy, the excess incidence of solid tumours rises for 15–​20 years and then may continue to rise, level off, or decline. In the case of acute leukaemia, however, peak incidence occurs much earlier (c.5 years after irradi- ation) and relatively few cases appear after more than 30 years. Short, intensive exposure to a carcinogen is, however, exceptional. The more usual situation is for sporadic or continuous exposure to a carcinogen to be prolonged for years—​a decade or two in the case of occupational exposure, several decades in the case of tobacco smoking, and a lifetime in the case of ultraviolet radiation. In this situation, the incidence of cancer increases progressively with the length of exposure. In the last two cases cited, the incidence appears to increase approximately in proportion to the fourth power of the duration of exposure, so that the effect after (say) 40 years is more than 10 times as great as that after 20 years, and more than 100 times as great as that after 10 years. Whether the same holds for occupa- tional exposure is not known, but it has been shown to hold in some experiments in which chemicals were repeatedly applied to the skin of genetically similar mice and it may prove to be a general biological rule for many types of carcinoma and many carcinogens. There is still less quantitative information about what usu- ally happens when exposure ceases, but in the case of cigarette smoking the rapidly rising annual risk among those who continue to smoke stabilizes for one or two decades after smoking ceases be- fore increasing again slowly. The ex-​smoker consequently avoids the enormous progressive increase in risk suffered by the continuing smoker. These delayed effects accord with the idea that the appearance of clinical cancer is the end result of a multistage process in which sev- eral mutations have to be produced in a single stem cell to turn it into the seed of a growing cancer. From a practical point of view, the important conclusions are that cancer may be very much more likely to occur after prolonged exposure to a carcinogen than after short exposure; that it is seldom likely to appear within a decade after first exposure (except in the case of leukaemia, certain hormone-​related and immune suppression-​related cancers, and the specific cancers of childhood); that it commonly occurs several decades after first exposure; and that some excess risk may continue to occur for dec- ades after exposure has ceased. The exact relationship may, how- ever, differ for different carcinogens and different types of tumour. Bladder tumours, for example, began to appear within 5 years of intensive exposure to 2-​naphthylamine in the dye industry, while mesotheliomas of the pleura have seldom, if ever, appeared within 10 years of exposure to asbestos, but they continue to increase in incidence for up to 50 years after first exposure, even if the exposure was relatively brief. Chance There remains the influence of chance, which is important for the individual as it is the reason why two animals of identical genetic constitution that have been treated in the same way do not, in gen- eral, develop cancer in the same place at precisely the same age. It reflects the element of chance that determines whether a particular series of events all occur in one particular stem cell out of the many thousands of stem cells that exist that do not give rise to a malignant clone. For any one individual, the role of chance in determining the occurrence of cancer may be large, but in a large population it has little net effect on the incidence of cancer and only nature and nur- ture are important. Avoidable factors Tobacco Tobacco is by far the most important single cause of cancer in de- veloped countries. Chewed, it can cause cancers of the mouth and oesophagus; smoked, it is a major cause of cancers of the mouth, pharynx (other than nasopharynx), oesophagus, larynx, lung, pan- creas, renal pelvis, and bladder. For these eight cancers, epidemio- logical evidence indicates that prolonged smoking of average numbers of cigarettes per day increases the risk 3–​20 times. It is, however, now clear that cigarette smoking also causes a propor- tion of several other types of cancer, increasing the incidence up to thrice that in non​smokers: namely, cancers of the lip, nose, naso- pharynx, stomach, colorectum, cervix, liver, and renal body, mu- cinous ovarian tumours, and also myeloid leukaemia. Although the proportional increases are not large, the consistency of the findings in different countries, the evidence of dose–​response re- lationships, the lower mortality in ex-​smokers than in continuing smokers, the lack of evidence for important confounding, and the presence in the smoke of many different carcinogens, provide strong grounds for believing that most or all of these observed as- sociations are causal.

5.1  Epidemiology of cancer 417 In sum, smoking is estimated to have caused 27% of all fatal can- cers in the United Kingdom in 2010, down from 34% 35 years earlier. The reduction was substantial in men (down from 52% in 1975 to 39% in 1995 and 31% in 2010) but it was largely counteracted by the increase in women (from 12% in 1975 to 20% in 1995 and 23% in 2010). Comparable figures from the United States and from some other developed countries are shown in Table 5.1.3. In men, there have in the past decade been decreases in many developed countries, but in each the proportions remain substantial. In women, the pro- portion of cancer deaths attributed to smoking was generally low in 1975, but has subsequently increased in all developed countries and must be expected to increase further. It was, however, still small in countries such as France or Spain, where many young women now smoke but few middle-​aged or elderly women have been smoking for long enough for any material effect to be produced. Fig. 5.1.2 shows the overall trends in UK cancer mortality at ages 35–​69 since 1950, divided by whether or not they were attribut- able (in a statistical sense) to smoking; it can be seen how greatly the trends in men especially have been influenced by changes in smoking-​attributed mortality. In developing countries, the effects of smoking have only recently begun to be studied systematically and much remains unclear. Large, nationally representative studies of smoking and death have, however, been conducted in China and India. In general, women in developing countries do not smoke (although there are particular regions in China and India where they do so). In men, however, Table 5.1.3  Percentage of cancer deaths attributed to smoking, 1955, 1975, 1995, and c.2010, by sex: various countries Country Male Female 1955 1975 1995 c.2010a 1955 1975 1995 c.2010a Australia 20 40 33 26 0 4 14 15 Finland 38 46 37 30 1 1 5 10 France 17 34 37 34 0 0 3 9 Hungary 21 36 51 50 2 5 13 21 Spain 13 28 39 36 0 0 0 4 UK 41 52 39 31 3 12 20 23 USA 23 42 42 37 0 10 25 27 a Most recent year available from WHO (Australia 2006; United States 2008; France, Spain, Hungary 2009; others 2010). 3 2.5 2 1.5 1 0.5 0 1940 1960 Rate per 100 000 age-standardized 1980 Year 2000 2020 (a) Rate per 100000 age-standardized (b) 3 2.5 2 1.5 1 0.5 0 Year 1940 1960 1980 2000 2020 Not attributed to smoking Attributed to smoking Not attributed to smoking Attributed to smoking Fig. 5.1.2  United Kingdom cancer mortality at ages 35–​69, 1950–​2010, attributable to smoking and not attributable to smoking: (a) males, (b) females.

418 SECTION 5  Principles of clinical oncology there has been a very large increase in worldwide cigarette con- sumption over the past few decades, the full effects of which have yet to materialize. China, with 19% of the world’s population, smokes 37% of the world’s cigarettes. By 1987 smoking was responsible for about 20% of male cancer deaths in China, and now accounts for 25%. In India, where many men have smoked ‘bidis’ (small, home-​ manufactured cigarettes) for decades, smoking caused 32% of all cancer deaths in men and 5% in women in the early 2000s (and 20% of all male deaths in India at ages 35–​69 in 2010), partly because smoking can act as a cofactor for the production of cancers of the mouth, oesophagus, or stomach in those who habitually chew quids containing betel and tobacco. In some parts of South America and China the male lung cancer rates from smoking are already as high as in developed countries. Overall, tobacco may be causing about as many cancer deaths in developing as in developed countries, in which case it would be responsible for about 20% of cancer deaths throughout the world. Alcohol At least six types of cancer are caused in part by the consumption of alcohol. One, liver cancer, is produced mainly by the produc- tion of liver cirrhosis and is, consequently, caused mainly by heavy and prolonged consumption. Four are causally related to smoking as well as to alcohol: namely, cancers of the mouth, pharynx (other than nasopharynx), oesophagus, and larynx. The two agents act synergistically, increasing each other’s effect, so that the risk from alcohol in non​smokers or long-​term ex-​smokers is very small, while that in heavy smokers is disproportionately large. The re- maining type, cancer of the breast, has been shown to be related to alcohol more recently. Epidemiological cohort studies show that the risk increases progressively with the amount drunk (at least up to moderately high levels) and laboratory studies that show that al- cohol increases the level of oestrogen in the blood suggest a plaus- ible mechanism. Cancers of the large bowel have also been associated with alcohol in many studies, but the relationship is modest and its nature uncer- tain: it could be due to confounding by diet. Ionizing radiation Ionizing radiations, of whatever type, and whether from external sources or from inhaled or ingested radionuclides, share the char- acteristic of having sufficient energy to damage DNA through ion- ization when they pass through the tissues of the body. It is not surprising, therefore, that they have been found to increase the in- cidence of cancer in practically every organ. The radiosensitivity of different organs varies greatly, however, and particularly large risks, relative to background rates, occur for thyroid cancer in people exposed as children, for myeloid leukaemia, and for can- cers of the breast and bladder. In contrast, there is no good evi- dence that exposure to ionizing radiation can increase the risk of chronic lymphocytic leukaemia, Hodgkin’s disease, or testis cancer. Many exposures to ionizing radiation are specific to certain parts of the body or to certain organs or tissues, and this determines the site of the induced cancer. For example, the sites of cancers after radiotherapy depend on the sites exposed to radiation as a conse- quence of the treatment; Thorotrast (a radioactive contrast agent used in the mid-​20th century) tends to be incorporated in the liver and bone marrow and hence to cause liver cancer and leukaemia; inhalation and ingestion of iodine-​131 was the principal exposure from the Chernobyl accident to the general population in the sur- rounding areas, which led to an increase in thyroid cancer in chil- dren; and inhalation of the natural radioactive gas radon and its progeny gives rise to lung cancer. Estimates of the carcinogenic effect of X-​rays have been derived by following groups of people with unusual but well-​documented exposures, including patients given radiotherapy, or repeatedly screened radiologically, and the survivors of the atomic bombings of Hiroshima and Nagasaki (in whom exposure was principally to γ-​rays, which are high-​energy X-​rays). Estimates of the risk of lung cancer from the inhalation of radon and its progeny have been de- rived both from studies of uranium miners and from studies of in- door radon exposure carried out in the general population. At low doses (<c.20 mGy) it seems probable that the carcino- genic effect of ionizing radiation is linearly proportional to the dose, while at higher doses the same is true for most cancers other than leukaemia, for which the risk is approximately proportional to the square of the dose. For most sites of cancer the risk is higher in people exposed in childhood than those exposed as adults, and it starts within the first 5 years and lasts for several decades. The International Commission on Radiological Protection (2007) has concluded that the lifetime risk of developing a fatal cancer is ap- proximately 10%/​Gy for X-​rays (or per Sievert for other types of radiation) to the whole body if the radiation dose is moderate and given acutely, and about half that if the dose is low and spread out over time (that is, 5 per 100 000 per mGy (or mSv)), with corres- ponding reductions if only part of the body is exposed. It has not been possible to detect by direct observation the effect of very small exposures, including, for example, the effect of a single chest radiograph given to an adult. However, theoretical consider- ations and the dose–​response relationship observed at larger doses both indicate that there is unlikely to be any threshold below which no effect is produced. This conclusion is reinforced by the observa- tion that children who received doses of 10–​20 mGy in utero (be- cause their mothers were irradiated for diagnostic purposes while pregnant) were subject to an added risk of developing cancer in childhood of approximately 1 in 2000. People are exposed to different amounts of radiation in different countries, depending principally on the concentration of radon in indoor air and the medical use of radiation for diagnosis. In the United Kingdom, the average indoor radon concentration is 20 Bq/​m3 and this is estimated to give rise to about 1000 lung cancer deaths each year. In the United States the average radon concentration is about twice that for the United Kingdom and, within the United Kingdom, it varies from one part of the country to another. Most notably, Devon and Cornwall have average indoor radon concen- trations three or four times greater than the national average and there are a few houses with concentrations that are 10 or even 100 times greater. Other sources of radiation are estimated to give rise to an average annual dose of about 1.4 mSv in the United Kingdom, which, on the basis of the risk estimate recommended by the International Commission on Radiological Protection, would lead to about 4200 deaths per year in the national population of about 60 million. This gives an estimated total of 5200 deaths per year from radiation-​induced cancer, or just over 3% of total cancer deaths. The major contributors are radon (20%), other natural sources (55%), and medical uses (24%). In addition, there is a contribution from

5.1  Epidemiology of cancer 419 radiotherapy for cancer, but less than might at first sight be implied from the collective dose, because a substantial proportion is received by people who will not survive long enough for a radiation-​induced cancer to appear. Less than 1% of all radiation-​induced deaths in the United Kingdom can be attributed to occupational exposure, fallout from past nuclear weapons tests, manufactured products, or radioactive waste. Ultraviolet radiation Photon energies in the ultraviolet (UV) range are sufficient to damage DNA and hence to cause cancer mutations. UV does not penetrate much below the skin, so that it is chiefly within the skin that it is directly carcinogenic. Within the skin, however, it is the principal cause of all types of cancer, other than Kaposi’s sarcoma. Whether it has any indirect carcinogenic effect on other tissues (notably the lymphopoietic tissue) remains uncertain, as does the possibility that the effect of UV in raising vitamin D levels may lead to consequent reduction in risks of certain cancers, especially colorectal. The main source of human exposure to UV radiation is sunshine. Infection Infection, principally viral, but also in some cases bacterial and para- sitic, is a major cause of avoidable cancer, especially in developing countries: in sub-​Saharan Africa about 40% of cancers in women and 30% in men, compared with about 10% in developed countries, are attributable to infections. Viral infection Viruses that are known to cause human cancers, or suspected of doing so, are listed in Table 5.1.4, along with the types of cancer with which they are associated. Not all infected people develop the disease. In some cases the proportion doing so is quite small, un- less other factors are also present. Such cofactors include endemic malaria for Burkitt’s lymphoma, the consumption of a type of salted fish for nasopharyngeal cancer, and the consumption of aflatoxin, a metabolic product of fungal infection with Aspergillus flavus, for liver cancer. What they are for the cancers produced by the human papillomavirus is not known. Quantitatively, chronic infection with hepatitis B virus is one of the most important causes of cancer in many parts of the world. In China, for example, liver cancer accounts for about 15% of all cancer deaths, the large majority of which are due to chronic lifelong infec- tion with the virus. Infant vaccination against the virus is now being introduced and will protect those born in the present century, but will not provide retrospective protection for those born in the 20th century. Bacterial infection Only one specific bacterial infection has been closely linked with the development of cancer: Helicobacter pylori. Persistent H. pylori infection acquired early in life leads to chronic gastritis in the an- trum of the stomach and increases the risk of gastric cancer two-​ to threefold. Non​specific chronic infection in the bladder may increase the risk of bladder cancer. Table 5.1.4  Viral causes of cancer Virus Cancer Hepatitis B Cancer of liver Hepatitis C Cancer of liver, non-​Hodgkin’s lymphoma HPV types 16, 18, and others Cancers of cervix, vulva, vagina, penis, anus; oral cavity, oropharynx, tonsil, some skin cancers HHV type 4 (EBV) Burkitt’s lymphoma Post-​transplant lymphoproliferative disease Nasal T-​cell lymphoma Other non-​Hodgkin’s lymphoma Hodgkin’s diseaseb Nasopharyngeal cancer HHV type 8 (Kaposi-​associated herpesvirus) Kaposi’s sarcoma Primary effusion lymphoma Human T-​cell leukaemia type 1 Adult T-​cell leukaemia/​lymphoma HIVa Kaposi’s sarcoma Non-​Hodgkin’s lymphoma Hodgkin’s disease Conjunctival carcinoma Cancers of the cervix and anus EBV, Epstein–​Barr virus; HHV, human herpesvirus; HPV, human papillomavirus. a In most cases, if not in all, by facilitating the effect of other viruses, probably via immunosuppression. b Causal nature of observed association unproved. Simian virus 40 (SV40) has been suspected in the aetiology of mesothelioma and several other types of cancer, without clear proof.

420 SECTION 5  Principles of clinical oncology Parasitic infection In parts of Africa and Asia, parasitic infection is a major cause of cancer. Infection with Schistosoma haematobium, which excretes its eggs through the bladder wall, causes a high incidence of bladder cancer in Egypt and East Africa while infection with S. japonicum, which excretes its eggs through the wall of the large bowel, is respon- sible for a high incidence of intestinal cancer in parts of China. Liver flukes (Clonorchis sinensis and Opisthorcis viverrini) are similarly responsible for the high incidence of cholangiosarcoma of the bile ducts in parts of Southeast Asia. The parasites may not cause cancer directly, but chronic infection may start a chain of events that leads to cancer in other ways, such as chronic bacterial infection and the local formation of nitrites and nitrosamines. Immunosuppression Patients with persistent immunosuppression, either therapeutic (notably immunosuppressive drug treatment for organ transplant- ation) or as a consequence of infection (e.g. HIV) or genetic (e.g. in ataxia telangiectasia and Wiskott–​Aldrich syndrome) have greatly raised risks of non-​Hodgkin’s lymphoma, and often of other viral infection-​related cancers including Kaposi’s sarcoma, although the pattern of malignancy and scale of risk varies according to the type of immunosuppression—​Kaposi’s sarcoma, for instance, predominates after HIV, whereas non-​Hodgkin’s lymphoma is the most common consequence of genetic immunodeficiency and of transplantation. Medical drugs Apart from ionizing radiation, some 20 agents have been used therapeutically that are known to cause cancer in humans. These are listed in Table 5.1.5. That so many carcinogenic agents should have been prescribed medically is not surprising when it is borne in mind that treatment often requires modification of cellular metab- olism and is sometimes intended to interfere with DNA. The hazard of cancer, however, is not necessarily a bar to the use of a drug if this risk is outweighed by the therapeutic benefits, as is commonly the case with antineoplastic agents, immunosuppressive drugs, and radiotherapy. Some of the chemotherapeutic agents listed in Table 5.1.5 were soon abandoned, while others have continued to be used for treat- ment, and the sum of the cancers that these now produce cannot amount to more than 100 or so a year in the United Kingdom. Two of the listed drugs are, however, used extensively in the general population:  hormonal replacement therapy (HRT) for postmenopausal women, and selected steroids for contraception, which increase the risk of breast cancer. Both also increase the risk of endometrial cancer, but HRT does so substantially only when given in the form of oestrogen alone and steroid contraceptives do so only in the form (now abandoned) in which oestrogen and pro- gestogen are given sequentially. The combined steroid contracep- tives currently in use can also rarely cause liver cancer and they may increase the risk of cervix cancer. Combined steroid contra- ceptives, however, also reduce the incidence of endometrial cancer and halve the risk of ovarian cancer for many years after they have been used. Meanwhile, HRT and combined steroid contraceptives are associated with a reduction of some 20% in the risk of colo- rectal cancer, although whether this is causally related to their use remains unknown. Other drugs that may inhibit cancer rather than cause it are non-​ steroidal analgesics, most notably aspirin, the prolonged use of which may somewhat reduce the risk of colorectal cancer and per- haps cancers of certain other sites. Taken altogether, it seems unlikely that medically prescribed drugs can be responsible for more than 1% of all today’s fatal cancers and may, in total, reduce the risk by somewhat more. Occupation In the years that followed Pott’s observation that chimney sweeps tended to develop cancer of the scrotum, many other groups of workers were found to suffer from specific hazards of cancer; indeed, more substances that are known to be carcinogenic to humans have been unearthed by the search for occupational hazards than by any other means. Most of these occupational cancers are in the respira- tory tract, especially the lung. The hazards are listed in Table 5.1.6. Many of the hazards that have been recognized caused large, or at least relatively large, risks, albeit for limited populations, and it may well be that other occupational causes exist that have not yet been detected, either because the added risk is small in comparison with that due to other causes, or because only a few workers have been persistently exposed, or simply because the hazards have not been suspected and so not looked for. It must also be borne in mind that cancer in humans seldom develops until one or more decades after exposure to the carcinogen first occurs and it is, therefore, too soon to be sure whether agents that have been introduced into industry only during the last 20 years are carcinogenic or not. Many groups of workers not listed in Table 5.1.6 have been sus- pected of having a special risk, but it has not been possible to decide whether the risk is real and attributable to their work. Some of these excesses may have arisen by chance alone, especially if the excess has not been confirmed in other studies, and others may be due to confounding; that is, they may have been produced by social factors or behaviours that are associated with the occupation in question rather than by the occupation itself. Given sufficient details and the ability to repeat the observations, it is usually possible to obtain a fairly clear idea of whether an excess incidence in an occupational group reflects an occupational hazard (e.g. by seeing whether the effect is related to the length of employ- ment, the time after first exposure, and a specific type of work within the industry). Unfortunately these details are not always available and the reasons for many of the reported moderate excesses of occu- pational cancer are still uncertain. In addition to the directly occupational cancers discussed earlier, workers may be indirectly exposed to carcinogens while at work (e.g. tobacco smoke from clients or colleagues). At present it seems likely that occupational hazards account for only a few per cent of all fatal cancers in developed countries such as the United Kingdom. The three principal causes are probably as- bestos dust (lung, pleural, and laryngeal cancers), the combustion products of fossil fuels (skin and lung cancer), and ionizing radi- ation (a wide variety of cancers). Pollution The idea that pollution might be an important cause of cancer has been in the forefront of the minds of cancer research workers since it was realized that the incidence of lung cancer tended to be higher in towns than in the countryside and that the combustion products

5.1  Epidemiology of cancer 421 of coal, which used to produce a pall of smoke over all large cities in Britain, contained carcinogenic hydrocarbons. Subsequently, with the rapid expansion of the chemical industry and the discovery that some of its products are mutagenic in vitro and carcinogenic in laboratory animals, anxiety increased about the possible effects of distributing such products ubiquitously in air, water, and food. The effects of pollution of this sort are, however, peculiarly dif- ficult to assess directly by epidemiological methods, as pollutants are likely to be present in most areas, the absolute risk from each is likely to be small, and there may be little difference in the extent to which individuals are exposed over a wide area. Reliance is, there- fore, often placed mainly on two indirect methods: extrapolation from the effects of chronic exposure to much larger amounts in an occupational setting, and prediction of the effects on humans from laboratory tests. Both, however, (but particularly the latter) involve substantial uncertainties. So far as atmospheric pollution is concerned, the epidemiological picture is complicated by the personal pollution produced by to- bacco smoke and the social distribution of smoking habits. Despite this complication, however, the various methods that have been dis- cussed under lung cancer all lead to the conclusion that the pollu- tion of the past may have contributed to the production of a few per cent of all lung cancers in Western countries, but that the levels over the last three decades (principally from the combustion of fossil fuels, but also from asbestos, dioxins, and various other materials) are unlikely to be responsible for more than a fraction of 1% of fu- ture cancers—​although there may be exceptions awaiting discovery in the neighbourhood of particular factories. The greater effect of Table 5.1.5  Carcinogenic agents used in medical practice (other than ionizing radiations) Agent Type of cancer Antineoplastic agents including:   Busulphan Leukaemiaa   Carmustine (BCNU) Leukaemiaa   Chlorambucil Leukaemiaa   Chlornaphazine Bladder   Cyclophosphamide Bladder, leukaemiaa   Lomustine (CCNU) Leukaemiaa   Etoposide in combination with cisplatin and bleomycin Leukaemiaa   Melphalan Leukaemiaa   MOPPb Leukaemia,a probably lungc   Semustine (methyl-​CCNU) Leukaemiaa   Thiotepa Leukaemiaa   Treosulphan Leukaemiaa Arsenic Skin, liver (angiosarcoma), lung Immunosuppressive drugs:   Azathioprine Non-​Hodgkin’s lymphoma, Kaposi’s sarcomad   Cyclosporine Non-​Hodgkin’s lymphoma, Kaposi’s sarcomad Methoxypsoralen (plus UV radiation) Skin Phenacetin Renal pelvis, ureter Plants containing aristolochic acid Renal pelvis, ureter Polycyclic hydrocarbons (coal-​tar ointment) Skin Sex hormones:   Unopposed oestrogens Endometrium, ovary, breast   Transplacental diethylstilboestrol Vagina and cervix (adenocarcinoma)   Diethylstilboestrol during pregnancy Breast   Oxymetholone (an anabolic steroid) Liver (hepatoma)   Oral contraceptives (combined)e Breast, cervix, liver (hepatoma)   Combined oestrogen-​progestogen menopausal therapy Breast, endometrium (diminishing with more days/​month of progestogen use)   Tamoxifene Endometrium a Acute or non​lymphocytic. b Combination of nitrogen mustard, vincristine, procarbazine, and prednisone. c Lung cancer might also be caused by certain other alkylating agents or regimens. d There have been excesses also of several other cancers in transplant patients treated with immunosuppressive drugs. e Oral contraceptives also reduce the risk of ovarian and endometrial cancers and tamoxifen reduces the risk of contralateral breast cancer and of breast cancer in women at high risk.

422 SECTION 5  Principles of clinical oncology the modern type of pollution with ultra-​fine particles and of the in- tense indoor pollution with smoke that occurs in parts of China is examined later under lung cancer and of erionite in certain Turkish villages under pleural cancer. The effect of polluted drinking water and food is more obscure. Modern analytical techniques permit the detection of chemicals at concentrations of less than one part per billion in both food and water and, in consequence, many have been detected that might ar- guably be carcinogenic, including pesticide residues and a variety of halogenated organic materials produced by the chlorination of water supplies. Relationships have been reported between the con- centrations of some of these compounds in water and the mortality from cancers of the bladder and, possibly, the large intestine, in dif- ferent localities, but it is extremely difficult to know what these rela- tionships mean as there are many potentially confounding factors. Mortality rates from cancers of the gastrointestinal and urinary tracts are, for the most part, stable or decreasing in early middle age, when the effects of new agents might be expected to show themselves Table 5.1.6  Occupational causesa of cancer Agent Type of cancer Occupationb Aromatic amines (several including): Bladder Dye manufacturers   4-​Aminodiphenyl Rubber workers   Benzidine Coal-​gas manufacturers   2-​Naphthylamine Some chemical workers Arsenic Skin, lung, bladder Copper and cobalt smelters Pesticide manufacturers Some gold miners Asbestos (all forms) Lung, pleura, peritoneum, larynx Asbestos miners Asbestos textile manufacturers Carpenters and general builders Insulation workers Shipyard workers Benzene Leukaemia Workers with glues and varnishes Berylliumc Lung Beryllium refiners and machiners Bis-​chloromethyl ether and technical-​grade chloromethyl methyl ether Lung Makers of ion-​exchange resins 1, 3-​butadiene Haematolymphatic system Styrene-​butadiene rubber workers Cadmiumc Lung Cadmium refiners Chromiumc Lungd Manufacturers of chromates from chrome ore; pigment manufacturers Dioxin (2,3,7,8–​TCDD) All cancers combined (not yet clear which sites) Exposed chemical workers Formaldehyde Nasopharynx Exposed industrial workers; embalmers Ionizing radiations Lung Uranium and some other miners Bone Luminizers Leukaemia, skin Radiologists, radiographers Mustard gas Larynx, lung Poison-​gas manufacturers Nickelc Nasal sinuses, lung Nickel refiners Polycyclic hydrocarbons in soot, tar, oil Skin, scrotum, lung, and sometimes bladder Coal-​gas manufacturers, roofers, asphalters, aluminium refiners, and many groups exposed to tars and selected oils Silica, when crystalline as quartz or cristobalite Lung Miners, stone workers, refractory brick workers Sulphuric acid mists (strong acid) Nasal sinuses, larynx Many industries, isopropanol manufacture, ‘steel pickling’ Ultraviolet radiation Skin (melanoma and non​melanoma) Farmers, seamen Vinyl chloride Liver (angiosarcoma, hepatocellular carcinoma) PVC manufacturers ? Nasal sinuses Hardwood furniture manufacturers ? Nasal sinuses Leather workers a Additional to those in the table, there have been raised risks of various cancers, of uncertain causation, found in workers in iron and steel founding; painters; and rubber manufacturers. b Typical occupations with the hazard. c Certain compounds or oxidation states. d And possibly nasal sinuses.

5.1  Epidemiology of cancer 423 first. In the absence of more specific evidence, it seems unlikely that chemical pollution of water and food could have a greater effect than the small effect already estimated for pollution of the air. Diet For many years there has been suggestive evidence that most of the cancers that are currently common could be made less so by modifi- cation of the diet, but, with few exceptions, there is still little reliable evidence as to the modifications that would be of major importance. If we define diet to include all materials that occur in natural foods, are produced during the processes of storage, cooking, and diges- tion, or are added as preservatives or to give food colour, flavour, and consistency, the ways in which diet could influence the development of cancer are legion. Ingestion of preformed carcinogens The most obvious is the ingestion of small amounts of powerful carcinogens or precarcinogens. Several have been identified in foodstuffs but only two have been related at all clearly to the produc- tion of cancer in humans. One is aflatoxin, a metabolic product of Aspergillus flavus, which contaminates stored or oily foods such as grains and peanuts in many countries, and is a major cause of liver cancer in the tropics among those individuals who are also chronic carriers of the hepatitis B (or less commonly hepatitis C) virus. Likewise, the salted fish eaten extensively in South China probably acts synergistically with the Epstein–​Barr virus to cause nasopha- ryngeal cancer. A third possible source is bracken fern, an extract of which is carcinogenic in animals. It is eaten extensively in Japan and has been tentatively linked with the development of oesophageal cancer. The polycyclic hydrocarbons and other mutagens that are produced in food by grilling or smoking have often been suspected of playing a role, and there is considerable evidence that processed meat consumption may increase colorectal cancer risk. It therefore seems likely that any effects of diet on the incidence of cancer in the Western world may be mainly by more indirect means, such as affecting the formation, transport, activation, or deactivation of carcinogens in the body or affecting the secretion of hormones. Overnutrition That overnutrition could affect the incidence of cancer was first sug- gested by Tannenbaum’s experiments on mice during the Second World War. These showed that the incidence of various spontaneous tumours and tumours produced experimentally could be halved by moderately restricting the intake of food without modifying the proportions of the individual constituents. This protective effect has subsequently been demonstrated repeatedly. It is now clear, however, that what is considered normal nutrition in developed countries in- creases the risk of breast cancer (by bringing forward menarche and increasing body size). With greater consumption obesity (i.e. a body mass index >25 kg/​m2) has been estimated to be responsible for 5% of all incident cancers in Europe and 10% of all cancer deaths in non​smokers in the United States: most notably, cancer of the breast in women after the menopause and cancers of the endometrium, large bowel, kidney, and gallbladder, oesophageal adenocarcinoma, and perhaps cancers of the prostate and thyroid. The increases in the two female cancers in postmenopausal women are probably at- tributable to the formation of oestrogen from androstenedione in adipose tissue, but for others the explanation is unclear. Meat and fat Figures for food consumption and cancer incidence and mortality rates in different countries show fairly close correlations between the consumption of fat, and to a lesser extent the consumption of meat, and the incidence of several types of cancer. The correlations are closest for breast cancer and cancer of the large bowel and are less strong for cancers of the endometrium, pancreas, and prostate. When, however, attempts are made to associate the consumption of either type of food with the disease in individuals within a country, the evidence is commonly conflicting. This could be because the international correlations are misleading, indicating only that the risks are correlated with something that is itself correlated with fat and meat consumption (e.g. some other aspect of a high gross na- tional product), but it could be partly because of the inaccuracy of dietary histories and partly because people within developed coun- tries eat such similar diets. The published data suggest that a high consumption of fat, and of red meat, is associated with a high risk of colorectal cancer, but these are not established causes. The claim that a high consumption of fat (or of particular types of fat) is associated with high risks of breast and endometrial cancers after the meno- pause, other than by providing a high-​calorie diet leading to obesity, is controversial. Fibre That fibre may play a part was suggested by Burkitt’s observation that several intestinal diseases, including cancer of the colon, were common in countries in which cereals were processed to remove the fibre and rare in rural Africa and Asia where they were not. The idea was attractive, as ‘fibre’ passes through the small bowel unchanged and serves as pabulum for the colonic bacteria, thus increasing faecal bulk and possibly protecting mechanically against the devel- opment of cancer by diluting any carcinogens present and hastening their transit through the bowel. The idea was too simple, however, and has not been confirmed (using the original definition of fibre) by either epidemiological studies on individuals in developed countries or by experiments aimed at reducing the recurrence of colorectal adenomas. In fact, fibre is difficult to define and the term is better replaced by ‘non​starch polysaccharides’ as there are many that share the characteristics of passing through the small bowel unchanged and being, for the most part, partially or wholly degraded by bac- teria in the large bowel. Some starch, moreover, known as ‘resistant starch’ and found in green bananas and cold potatoes, has similar physiological characteristics. It is uncertain whether ‘fibre’ in any of its manifestations can be considered as having a place in protecting against the development of cancer. Retinoids and carotenoids Experiments on animals and on cell cultures in vitro have suggested that vitamin A (retinol) and its esters and analogues (retinoids) may, in appropriate circumstances, reduce the risk of cancer by reducing the probability that partially transformed cells become fully trans- formed and proliferate into clinically detectable tumours, although in other circumstances they appear to have opposite effects. Human studies, however, did not support the idea that serum levels of ret- inol are related to the risk of any type of cancer, at least in countries in which clinical symptoms of vitamin A deficiency seldom or never occur. Such studies suggested that the risks were inversely related to the serum level of β-​carotene, which acts as an antioxidant and

424 SECTION 5  Principles of clinical oncology is broken down to produce retinol. When β-​carotene was put to the test of clinical trials, however, it provided no benefit and the inverse relationship commonly observed in epidemiological studies is pre- sumably due to confounding with some other protective factors in vegetables. Other components Many other components of the diet, including lycopene in tomatoes, indoles in brassicas (e.g. cabbages and sprouts), phyto-​oestrogens (plant chemicals structurally similar to oestradiol), fresh fruit and vegetables, vitamins C, D, and E, and calcium and selenium have also been proposed as protective agents. Conversely, nitrates, ni- trites, secondary amines, and the preservation of food by salting, have been thought to increase the risk of cancer. For some the evi- dence is strongly suggestive:  notably for vitamin C as protective against gastric cancer and for salt-​preserved foods predisposing to it. In general, however, the evidence of benefit or harm is insufficient to justify any firm conclusion. Conclusion Considerable uncertainties remain about the effect of diet on cancer risks. Practicable modifications of the diet may well provide the means for reducing cancer deaths in developed countries by up to one-​third, but the range of uncertainty about this figure is large. Meanwhile the only major dietary changes that can be recommended with confidence in developed countries are a general increase in the use of fresh fruit and vegetables and a sufficient limitation of calories to avoid obesity. Reproduction, other factors affecting secretion of reproductive hormones, and other hormones Epidemiological observations have shown clear relationships be- tween a woman’s menstrual and reproductive history and the risks of cancers of the breast, endometrium, and ovary, which are gen- erally thought to reflect changes in hormonal secretions. Which hormones are concerned, however, and the mechanisms by which they act are, for the most part, still uncertain. An exception is endo- metrial cancer, the risk of which is directly related to the degree of exposure to oestrogen not followed after an appropriate interval by progestogen. Strong evidence that oestrogenic stimulation of the mammary tissues is a cause of most cases of breast cancer in de- veloped countries has been provided by randomized trials of tam- oxifen, an antioestrogenic drug that blocks the oestrogen receptors in the cells of the normal breast. The effect is large and rapid: five years of tamoxifen approximately halves the incidence of contralat- eral breast cancer in a woman who has had previous breast cancer, not only while the drug is being taken but also for some years afterwards. Exogenous oestrogen also increases the risk of breast cancer when given as HRT and endogenous oestrogen accounts for the increased risk associated with adiposity after the menopause, as androstenedione, which continues to be produced by the ad- renals, is converted to oestrogen in adipose tissue. It is presumably oestrogens, too, that cause a small increase in risk of breast cancer during and immediately after pregnancy and the oestrogen com- ponent of the steroid contraceptives that causes a similar small in- crease in risk during their use and for a few years after their use is stopped. It is, however, unclear which hormone-​related processes are involved in reducing the long-​term risk for the rest of a woman’s life that occurs some years after the occurrence of each pregnancy, and it is equally unclear why the use of oral contraception and the consequent suppression of ovulation reduces the long-​term risk of ovarian cancer. Sex hormones, it is thought, may also be involved in produ- cing cancers of the testis and prostate in men. For testis cancer, the strongest evidence has been for an effect of maternal oestrogen levels on the developing testis in utero, whereas for prostate cancer the evidence relates to adult androgen levels, but for neither has caus- ation been established. Randomized trials of the effects of physical or medical castration in men who already have prostate cancer have shown that progression of the disease can be slowed substantially, presumably by the reduction of androgenic stimulation. In recent years it has been found that prior raised endogenous levels of insulin-​like growth factor-​1 (IGF-​1) are associated with raised risk of cancers of the breast, colon, and prostate, and there has also been evidence that endogenous and exogenous growth hor- mone may affect the risk of colorectal cancer. Physical inactivity Physical inactivity contributes to the risk of cancer indirectly by increasing the risk of obesity but it may also contribute directly. Associations with colon, breast, and endometrial cancers have fairly consistently been reported, and an association with prostate cancer has been found in most studies. The mechanism of effect is uncertain, but possibilities include the effects of exercise on hormone levels, on immune function, and on intestinal transit time and hence the dur- ation of exposure of the colonic mucosa to faecal carcinogens. Interaction of agents Attribution of the risk of cancer to different causes is complicated by the fact that some agents interact with others to produce effects that are much greater than the sum of the separate effects of each on its own. An example is provided by smoking and asbestos, which multiply each other’s effects so that, compared with non​smokers in general, the incidence of cancer of the lung was increased six- fold among a group of asbestos insulation workers in the United States of America who did not smoke, but were heavily exposed to asbestos dust in the 1940s, 10-​ to 20-​fold among cigarette smokers in general who did not work with asbestos, and nearly 90-​fold among the asbestos workers who also smoked cigarettes regularly. Other examples are provided by smoking and radon (which interact simi- larly, though somewhat less than multiplicatively, to produce cancer of the lung), by smoking and alcohol (which interact to produce cancers of the mouth, pharynx, larynx, and oesophagus), and by in- fection with the hepatitis B virus and aflatoxin (which interact to produce cancer of the liver). Such interactions complicate the attribution of risk, as we may find ourselves appearing to claim that more cancer can be prevented than actually occurs by attributing, say, 80% of lung cancers in men heavily exposed to asbestos to their occupational exposure and 90% of the same cancers to cigarette smoking. Each, separately, is correct, but they cannot be added to show the combined effect. Conclusion Estimates of the proportions of fatal cancers that can be attributed to environmental and behavioural factors, grouped into 11 main categories, are given in Table 5.1.7. The evidence on which these es- timates are based is summarized in this chapter and in greater detail by Doll and Peto (1981), Stewart and Kleihues (2003), IARC (2012) and Thun et al. (2018).

5.1  Epidemiology of cancer 425 The sum of the best estimates in Table 5.1.7 amounts to less than 100%, despite the fact that some of the listed agents interact with one another to augment each other’s effect and that some fatal can- cers are consequently counted twice. The total would be somewhat more than 100%, however, if the true proportions attributable to some of the categories turn out to be nearer the upper end of the acceptable estimates. The estimates in the second and third columns of Table 5.1.7 do not distinguish between factors (such as tobacco) that are suffi- ciently understood to enable specific action to be taken with a guar- antee of success and those (such as diet) that are not. They should not, therefore, be taken as guides to the proportion of cancer deaths that can now be prevented by practicable means. This is illustrated by the fourth column in Table 5.1.7, which shows the proportions of United Kingdom cancer deaths that are reliably known to be avoid- able by practicable means. The percentage attributed to tobacco is more than the sum of the percentages reliably attributable to other specific factors for which practicable preventive measures are avail- able; and tobacco causes about twice as many deaths from other dis- eases as it does from cancer. The position is different in countries such as China, where hepatitis B virus causes are approaching as many cancer deaths as tobacco and the hazard for future generations can be avoided in a cost-​effective way by infant vaccination. Epidemiology of cancer by site of origin In the following account of the epidemiology of cancers arising in specific organs, the description of each type is preceded by notes showing its importance in England. One figure gives the propor- tion of all cancers that arise at the site, from national cancer registra- tions for England in 2014 (Office for National Statistics, 2016) while another gives the proportion of all cancer deaths allocated to the site in the national mortality statistics for England and Wales for 2014 (Office for National Statistics, 2015). A third gives the ratio of the age-​standardized incidence rates in England for each sex in 2014. The way in which the incidence of the disease varies with age is shown for males and females in a series of graphs, using data for England over a five-​year period (2008–​12). Trends in incidence and mortality for each type, along with the trends in possible causative factors, are given by Swerdlow et al. (2001). Trends internationally are commented on in the text, and described more fully in Doll et al. (1994). Comments on the total worldwide frequency of different cancers are based on data from Ferlay et al., (2014); they disregard incidence of non​melanoma skin cancers, for which reliable inter- national statistics are not available. Lip • 0.1% of all cancers and 0.02% of cancer deaths • Sex ratio of rates 1.7:1; age distribution like oesophageal cancer Carcinoma of the lip was one of the first types of cancer to be re- lated to an extrinsic cause when, more than 200 years ago, it was noted to occur characteristically in pipe smokers. Many years later it was realized that the disease could also be produced by smoking cigarettes, although much less readily, so that it must be produced by the chemicals in smoke rather than by the non​specific effect of local heat. It is also much more common in outdoor than in indoor Table 5.1.7  Estimated proportion of United Kingdom cancer deaths in 2005 attributed to previous exposure to different environmental and behavioural factors and proportion of future United Kingdom cancer deaths avoidable by known effects of practicable changes in current exposure levels Factor or class of factors Percentage of UK cancer deaths in 2005 attributed to
previous exposure levels Estimated percentage of future UK cancer deaths avoidable by known effects of practicable changes in current exposure levels Best estimate Range of acceptable estimates Tobacco 28 25–​30 <28a Alcohol 6 4–​8 <6b Ionizing radiation 3c 2–​4 <1 Ultraviolet radiation 1 1 <1 Infection (virus 3%, bacteria 2%) 5 4–​15 <1d Medical drugs <1 0–​1 <1 Occupation 4 3–​5 <1e Pollution 2 1–​5 <1 Diet 25 15–​35 <8f Reproductive and hormonalg 15 10–​20 <1 Other and unknown ? ? ? a The proportion of UK cancer deaths attributed to smoking fell from 34% in 1985 to 28% in 2005 and is still decreasing (as current levels of smoking would cause less than 28% of future UK cancer deaths). b Mostly cancer of the upper aerodigestive tract that could have been avoided by not smoking. c 0.8% diagnostic X-​rays, less than 0.1% all other manmade, 0.7% natural radon in houses, 1.8% other cosmic or terrestrial natural sources. d Cervical cancer currently causes 1.4% of UK female cancer deaths. Without screening it would cause several per cent; with even better screening it would cause a fraction of 1%. e Although asbestos exposure is now strictly controlled, the delayed effects of past exposure probably account for about 3% of current cancer mortality (including more than 1% from mesothelioma), and diesel, coal, and other smoke must also have appreciable effects, especially if they potentiate tobacco smoke. f For those of later middle age living in the United Kingdom, almost half are overweight (BMI 25–​30 kg/​mb) and a quarter are obese (>30 kg/​m2); minimal cancer mortality is at c.25 kg/​m2, and about 8% of current cancer mortality would be avoided if the overweight and obese had a BMI of 25 kg/​m2. (Instead, BMI is increasing by about 1 kg/​m2 per decade.) g Includes other factors affecting the secretion of reproductive hormones.

426 SECTION 5  Principles of clinical oncology workers and is induced by ultraviolet radiation in the same way as other cancers of the exposed skin. Solar ultraviolet radiation and to- bacco account, between them, for the great majority of all cases in the United Kingdom, probably multiplying each other’s effects. The disease is much less common than it used to be, because of the de- crease in both pipe smoking and outdoor work. Oral cavity and pharynx (excluding salivary glands and nasopharynx) • 2.4% of all cancers and 1.4% of cancer deaths • Sex ratio of rates 2.2:1; age distribution like oesophageal cancer Cancers of the tongue, mouth, and pharynx (other than naso- pharynx) are all related to smoking (of pipes, cigars, and cigarettes) and to the consumption of alcohol. The two factors act synergistic- ally and cancers at these sites are extremely rare in non​smokers who do not drink alcohol. There is also fairly consistent evidence of an association of risk with low intake of fruit and vegetables. Cancer of the tongue is much less common in Britain than it was 100 years ago, but the reason for this sharp decline is unknown. One explanation could be the decrease in syphilis, which was commonly believed to be a predisposing factor because of the clinical associ- ation with syphilitic leucoplakia. Recent increases in oral and pha- ryngeal cancer in the United Kingdom but not the United States are partly due to increased consumption of alcohol and possibly, in the case of pharyngeal cancer, due to human papillomavirus infection. Cancers that occur low in the hypopharynx are distinguished by a tendency to affect women who have suffered from iron-​deficiency anaemia and dysphagia. Cancers of the mouth and pharynx (excluding nasopharynx) are particularly common in Southeast and Central Asia where tobacco smoking was largely replaced by chewing tobacco, betel nut or leaf, and lime (calcium hydroxide). A close association with such chewing habits has been established by studies that have shown that the can- cers tend to originate in the part of the mouth in which the quid is usually held—​a characteristic that varies both between individuals and between areas. The materials chewed differ in different places and, al- though the disease is commonly described as ‘betel chewer’s cancer’, betel is not invariably a component of the quid and the most character- istic constituent seems to be a small amount of lime and, in most cases, some form of tobacco. In parts of Asia, the disease is so common that it accounts for 20% of all cancers and in those populations the abandon- ment of chewing would be the single most effective means of reducing the total incidence of cancer—​so long as the habit was not replaced by an increase in tobacco smoking. Among habitual quid chewers, the risks are particularly elevated in those who both chew and smoke—​ indeed, in parts of India most deaths from betel chewer’s cancer could have been avoided if those affected had not also smoked. The incidence might also be reduced by improved nutrition, as the disease in southern Asia tends to be associated with vitamin A deficiency. In parts of India where women tend to smoke local cigars and cig- arettes with the burning end inside the mouth to prevent them going out, the habit is associated with cancer of the palate. Salivary glands • 0.2% of all cancers and 0.1% of cancer deaths • Sex ratio of rates 1.4:1; age distribution, see Fig. 5.1.3 The salivary glands are not common sites for cancer anywhere. They are, however, relatively more common in circumpolar Inuits than others. No causative factors are known other than ionizing radiation exposure, and no notable changes in incidence over time have been reported. Nasopharynx • 0.1% of all cancers and of cancer deaths • Sex ratio of rates 3.0:1; age distribution, see Fig. 5.1.4 Cancers of the nasopharynx, unlike those in other parts of the pharynx, are not related to alcohol and are only weakly related to tobacco. They are rare in most populations but are common in Southern China, especially so in Cantonese originating from parts of Guangdong, where the disease is among the most common types 0 1 2 3 4 5 6 7 8 9 0– 5– 10– 15– 20– 25– 30– 35– 40– Age group Rate (per 100000) 45– 50– 55– 60– 65– 70– 75– 80– 85+ 10 11 Female Male Fig. 5.1.3  Annual incidence of cancers of the salivary glands, by age and sex. 0– 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rate (per 100 000) 1.0 1.1 1.2 1.3 1.4 5– 10– 15– 20– 25– 30– 35– 40– Age group 45– 50– 55– 60– 65– 70– 75– 80– 85+ Female Male Fig. 5.1.4  Annual incidence of cancer of the nasopharynx, by age and sex.

5.1  Epidemiology of cancer 427 of cancer. A weak relationship with human leukocyte antigen type has been reported. Moderately high rates have been observed in Eskimos, American Indians, Malays, Filipinos, Thais, and in parts of North Africa. Rates decrease over succeeding generations in Chinese migrants to (low-​risk) Western countries. DNA characteristic of the Epstein–​Barr virus (EBV) has been detected in the nuclei of nasopharyngeal cancer cells and patients with the disease tend to have unusually high antibodies against EBV-​ related antigens. Among adults, sudden increases in certain EBV antigens in the blood often precede the appearance of a cancer by a few years. Infection with the EBV is, however, almost universal and can be only one of several agents that act in combination to produce the disease. One such agent in Southern China occurs in the ‘salted fish’ on which children are commonly weaned. This strongly fla- voured delicacy bears little relation to the salted fish eaten elsewhere, and might better be described as decomposing fish:  it contains various mutagens, and exposure to it in childhood when infection with EBV first occurs may alter the usual lifelong balance between host and virus in some hazardous way. Oesophagus • 2.5% of all cancers and 4.7% of cancer deaths • Sex ratio of rates 2.5:1; age distribution, see Fig. 5.1.5 Cancer of the oesophagus is the eighth most common cancer in the world. It is exceptional among malignancies in the extent of geographical variation in incidence, both internationally and often also over relatively small distances. Like other cancers of the upper respiratory and digestive tracts, cancer of the oesophagus is closely related to prolonged smoking and the consumption of alcohol. All types of smoking have comparable effects as, so it appears, do all alcoholic drinks, although spirits may be slightly more effective per gram of ethyl alcohol than other alcoholic drinks. Alcohol and tobacco act synergistically and, in the absence of either, the incidence of the disease in Western countries would be greatly reduced. Smoking raises the risk of both squamous cell and adeno- carcinoma of the oesophagus, whereas alcohol affects largely or solely the former. A few cases originate from the scars produced by poisoning with corrosive substances and a very few in conjunction with a particular hereditary form of tylosis (presenting with kera- toses of the palms and soles). The relatively small excess in men probably reflects the existence of other unknown causes in women, possibly nutritional in origin and similar to those responsible for cancers of the hypopharynx. Mortality (which, because of the high fatality rate, approaches incidence) fell progressively in men in Britain from the 1920s to the 1960s, in line with the fall in the con- sumption of alcohol, and rose again after 1960 when the trend in the consumption of alcohol reversed, but has stabilized since 2000. Since pipe smoking affects oesophageal cancer risks at least as strongly as cigarette smoking, no large effects on male oesophageal cancer trends could be predicted from the male switch from pipes to cigarettes, although the switch by females from non​smoking to cigarettes should, other things being equal, have produced a large upward trend. It appears, however, that other things were not equal and some other, possibly nutritional, cause of oesophageal cancer seems to have decreased, for the upward trend in oesopha- geal cancer in women has been moderate. Oesophageal cancer is associated in several studies with low fruit and vegetable consump- tion. In men, by contrast, the rates have increased since 1960 when based on smoking they might have been expected to decrease. To some extent this can be accounted for by the increased consump- tion of alcohol and possibly by an increase in the nitrosamine con- tent of tobacco smoke, which has resulted from changes in the method of curing tobacco and which could have a specific effect on the oesophagus. A part of the increase is due to an increased risk of adenocarcinoma at the lower end of the oesophagus, which may be associated with a decreased prevalence of Helicobacter pylori and gastritis, and an increase in gastro-​oesophageal reflux and Barrett’s oesophagus, which is a common precursor of the tumour. Obesity is associated with risk of adenocarcinoma of the oesophagus, per- haps via an effect on reflux. The balance of adenocarcinoma and squamous cell cancer of the oesophagus has altered greatly over time in Western countries, such that the former, which has been increasing, now generally predominates in men in the United Kingdom and United States, although not in much of Continental Europe. (In China, Japan, and Korea, however, the tumours are overwhelmingly squamous cell.) In Africa and Asia, the epidemiological features are quite dif- ferent and present some of the most striking, unsolved problems in the field of cancer epidemiology. In parts of China (particularly in north Henan but also elsewhere), and on the east coast of the Caspian Sea in Turkmenistan and Iran, oesophageal cancer is the most common type of cancer, with incidence rates in both sexes that are equal to the highest rates observed for lung cancer in men in European cities. Within China, the disease varies more than 10-​fold from one county to another. In parts of Africa, particu- larly in the Transkei region of South Africa and on the east coast of Lake Victoria in Kenya, extremely high rates are also observed, sometimes equally in both sexes and sometimes only in men. In these and several other areas, the high-​incidence zones are strictly localized and the incidence tails off rapidly over distances of 200 or 300 miles (c.300–​500 km). 15– 0 10 20 30 40 50 60 70 80 90 100 110 120 Rate (per 100 000) 20– 25– 30– 35– 40– 45– Age group 50– 55– 60– 65– 70– 75– 80– 85+ Female Male Fig. 5.1.5  Annual incidence of cancer of the oesophagus, by age and sex.

428 SECTION 5  Principles of clinical oncology When tobacco and alcohol are used they increase the hazard, but they are not the principal agents in these high-​incidence areas. Many dietary causes have been proposed, including micronutrient deficiencies, contamination of food and pickled vegetables by fungi (particularly by species of fusaria) with the production of carcino- genic metabolites, an agent associated with the production of beer from maize, drinking very hot beverages, and the residues left be- hind in pipes from smoking opium (which are commonly swal- lowed). None, however, is supported by any impressive, consistent epidemiological data. The high-​incidence area in Iran, which has been intensively investigated, is characterized by extreme poverty and a restricted diet consisting chiefly of home-​made bread and tea, with some sheep’s milk and milk products, and very little meat, vegetables, or fruit. In this area the disease has been common for centuries. In southern Africa, however, it seems to have become common only since the First World War. In China, where cancer of the oesophagus was the second most important neoplastic cause of death in the 1970s, incidence is still high, and it currently accounts for 13% of all cancer deaths. Stomach • 1.8% of all cancers and 2.7% of cancer deaths • Sex ratio of rates 2.4:1; age distribution like oesophageal cancer Until about 1980, gastric cancer was responsible for more deaths from malignant disease worldwide than any other; it is now third to lung and liver cancers with over 700 000 deaths per year, mainly in developing countries. Over the last 50 years, the incidence has declined in Western countries (see Fig. 5.1.6), and recently it has begun to do so in South America and Japan. The highest rates now are in parts of Japan, China, and Korea, with moderately high rates in countries in the ex-​Soviet Union and Eastern Europe and in parts of South America, while low rates are found both in North America and Australasia, and in some of the least developed parts of Africa. This contrasts with the strong socioeconomic gradient in incidence of the tumour seen within Western countries. Irrespective of whether the incidence in a country is high or low, the sex ratio is generally between 1.5 and 3 to 1. In migrants from high-​risk to low-​risk countries, for instance, from Japan to the United States of America, risk decreases with longer time since migration, but can take two or more generations to reach local levels. Risk of gastric cancer is raised in relation to gastritis associated with chronic infection by H. pylori (sometimes leading to atrophic gastritis), a diet deficient in fruit and green and yellow vegetables, and a poor diet with large amounts of salt and salt-​preserved food. Chronic infection with H. pylori, which is very common, is a major cause of peptic ulcer, a finding that is of consid- erable practical value in patients with ulcers, because the infection can generally be eliminated from the stomach by a short course of appropriate antibiotic therapy and this provides long-​term protec- tion against recurrence. Whether such treatments will have any ma- terial effect on the incidence of stomach cancer remains, however, to be shown. How these various factors influence the production of the disease is unclear. One possibility is that they encourage or dis- courage the formation of carcinogens in vivo, particularly perhaps the production of nitrosamines; but if they do, the intake of nitrates (which can be converted into nitrites by bacterial enzymes) is not a rate-​limiting factor. Changes in the prevalence of the aforemen- tioned three factors could have contributed to the decline in the in- cidence of the disease, but they could not have brought about such a large and widespread reduction in risk, and it seems probable that the better preservation of food, resulting from the extensive use of refrigeration, has played the major part. No risk has been detected from the consumption of mutagens produced by the different methods of cooking meat and fish, nor from food additives or pesticide residues. Some food additives may, on the contrary, have served to reduce risk (by avoiding food spoilage and hence improving nutrition, by avoiding contamination by carcinogen-​producing microorganisms, or by some antioxidant or other protective effect on the gastric epithelium). Risk of gastric cancer is also raised, moderately, by smoking, and raised by exposure to ionizing radiation. Large bowel • 11.5% of all cancers and 9.9% of cancer deaths • Sex ratio of rates 1.5:1; age distribution like oesophageal cancer Cancers of the colon and rectum ought to be considered separately, as their causes are not identical. Cancer of the colon, for example, tends to occur more often in women than in men, particularly when it occurs on the right side, while cancer of the rectum is nearly twice as common in men. The geographical distribution also differs slightly, colonic cancer varying in incidence more than rectal cancer. Separate consideration may, however, sometimes be misleading as cancers commonly occur at the rectosigmoid junction and the site of origin of these cases is not recorded consistently. Moreover, there is a growing tendency to describe both diseases merely as ‘cancers of the large bowel’, which, according to the internationally agreed coding rules, are classed as cancers of the colon. The two diseases will, there- fore, be considered together. Cancers of the colon and rectum are the third most commonly in- cident cancer, and the fourth most common cause of cancer death, in the world. Over 1.3 million cases occur per year worldwide, mainly in developed countries. The disease is most common in Western countries, but incidence has generally stabilized or decreased in these countries in recent years, especially at younger ages. In Japan, where incidence used to be very low, rates have risen to be similar to those in the United States of America and Western Europe, and indeed in parts of Japan are now almost the highest in the world. Rates in migrants from low-​risk to high-​risk countries, for instance, in previous times from Japan to the United States, tend to gain much or all of the host population risks within the first generation. 1860 0 10 20 30 Age-standardized rate per 100 000 population 40 50 60 70 1880 1900 1920 1940 Year of death 1960 1980 2000 Males Females 80 Fig. 5.1.6  Mortality from cancer of the stomach, England and Wales, 1868–​1997, ages 35 and older, by sex.

5.1  Epidemiology of cancer 429 In most parts of Asia, and in Africa and Eastern Europe, large bowel cancer has been relatively uncommon (except in areas where chronic schistosomal infestation of the large intestine is prevalent; for example, high rectal cancer rates are found in Chinese counties in which Schistosoma japonicum was, until recently, a major cause of death). Rates tend to rise markedly, however, with the introduction of a Western lifestyle. Incidence rates in different countries correlate closely with the per caput consumption of fat and meat and crudely with the con- sumption of processed foods from which the natural fibre has been removed. Ways in which these and other dietary constituents might influence the development of the disease have been discussed under diet. Other factors associated with increased risk are obesity and physical inactivity. A  weak but reasonably consistent association with smoking has been observed. There has been an association with consumption of alcohol in most studies and with a high-​fat diet. Cases in childhood or early adult life occur as a complication of familial adenomatous polyposis and of hereditary non​polyposis colorectal cancer (HNPCC) syndrome. These conditions are de- termined by dominant genes, which increase the susceptibility to the disease so much that, unless prophylactic measures such as colec- tomy are undertaken, cancer is highly likely to develop at or before middle age. Many other cases develop from adenomatous polyps and a few occur as a complication of long-​standing ulcerative col- itis and Crohn’s disease. There is substantial evidence of reduced colorectal cancer risk among long-​term users of non​steroidal anti-​inflammatory drugs. There is also increasing evidence for an association of colorectal cancer risk with hormones of the growth hormone/​IGF-​1 axis:  colorectal cancer risks are increased in pa- tients with acromegaly; raised risks have been found in the general population in relation to prior greater levels of IGF-​1; and raised risks have been found in patients treated with growth hormone. Anal intercourse causing infection with types 16, 18, or some other specific types of the human papillomavirus is a probable cause of some anal carcinomas in both sexes, but patients who have sexu- ally transmitted anal warts that are due to other types of human papillomavirus are not for this reason at special risk of anal cancer. Liver • 1.5% of all cancers and 3.1% of cancer deaths • Sex ratio of rates 2.3:1; age distribution, see Fig. 5.1.7 Liver cancer is the second most common cause of cancer death in the world, with four-​fifths of cases occurring in developing coun- tries, and one-​half occurring in China alone. Incidence is consid- erably greater in men than women almost everywhere. Incidence rates have tended to be overestimated in developed countries be- cause the primary condition is often confused with metastases to the liver from cancer in various other organs, particularly at older ages. In recent decades, however, there has been a large increase in the United Kingdom and the United States from the very low level that existed previously, perhaps due in part to an increased prevalence of infection with hepatitis C. The disease is common in Southeast Asia (the highest recorded rates are in parts of China and Thailand), Korea, and Japan, and rates are also high in tropical Africa. In China it accounts for 15% of all cancer deaths and in sub-​Saharan Africa it is the second most common cancer in men. Most cases derive from the main cells of the organ (hepatocellular carcinomas) and are attributable pri- marily to chronic active infection, established early in life, with the hepatitis B virus, exacerbated by consumption of some spe- cific metabolite (e.g. aflatoxins) of particular types of fungi that contaminate stored foods. Neonatal vaccination against the virus produces a marked decrease in the proportion of children who be- come chronically infected. This has begun in many countries, now including the whole of China and parts of tropical Africa, and has already produced a decreased risk of hepatocarcinoma at young ages. Some cases, however, are caused by chronic infection with hepatitis C (a blood-​borne RNA virus that cannot be avoided by immunization; see Chapter 8.5.22). In developed countries, although some cases are also due to in- fection with hepatitis B and C viruses, more arise as complications of cirrhosis of the liver attributable to heavy and prolonged con- sumption of alcohol or, rarely, to haemochromatosis, certain types of porphyria, α1-​antitrypsin deficiency, and hereditary tyrosinaemia type 1. Occasionally, liver cancer is produced by drugs. A few cases have occurred in young men who have taken androgenic anabolic steroids to increase their muscular strength and a few from the use of steroid contraceptives, either arising de novo or from benign adenomas, which are themselves rare complications of the use of steroid contraceptives. Some can be attributed to smoking, for an association has been observed in parts of China where little alcohol is drunk and case–​control studies in Europe have shown an associ- ation after alcohol consumption has been taken into account. A second histological type (cholangiosarcoma) arises from the intrahepatic bile ducts, tends to occur at a somewhat later age than hepatocellular carcinoma, and, although generally less common than hepatocellular carcinoma, nevertheless accounts for an ap- preciable proportion of cases. In parts of China, Thailand, and elsewhere in south or east Asia it can be produced by chronic in- fection with liver flukes (Clonorchis sinensis or Opisthorchis viver- rini). In northeastern Thailand the latter fluke causes one of the highest rates of liver cancer in the world. In developed countries, primary sclerosing cholangitis is the main known risk factor for cholangiosarcoma. 0– 0 10 20 30 40 50 60 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.7  Annual incidence of cancer of the liver, by age and sex.

430 SECTION 5  Principles of clinical oncology A third histological type that is extremely uncommon every- where has been variously described as reticuloendothelioma or angiosarcoma. It was first recognized as a complication of the use of Thorotrast as a contrast agent in neuroradiology, a long-​abandoned practice that led to chronic retention of insoluble thorium radio- nuclides in the marrow, spleen, and liver. In 1973, the disease was found to be an occupational hazard for men exposed to vinyl chloride monomer. A few hundred cases have occurred throughout the world in men who were heavily exposed in the manufacture of vinyl chloride polymer, and linear extrapolation suggests that the minute amounts that have leached out of plastic consumer products might have caused only a dozen or so cases altogether in the general public, if indeed they have produced any. A third, and even rarer, cause is prolonged exposure to inorganic arsenic, such as used to result from the medical prescription of Fowler’s solution. Despite these multiple causes only one case of hepatic angiosarcoma nor- mally occurs annually per 10 million people, which is why the rec- ognition of new causes has been easy. The relative rarity of cancer of the liver in most developed coun- tries is intriguing, since most of the carcinogens thus far discovered in experimental animals induce, perhaps with other cancers, tu- mours of the liver. Gallbladder and extrahepatic bile ducts • 0.6% of all cancers and of cancer deaths • Sex ratio of rates 0.9:1; age distribution like oesophageal cancer Cancers of the gallbladder and extrahepatic bile ducts are nearly always classed together, which is unfortunate as the causes differ. The former is more than twice as common in women as in men, is probably associated with obesity, and is usually preceded by (and probably caused by) cholelithiasis. The latter is slightly more common in men and is increased in incidence by liver fluke infection, primary sclerosing cholangitis, and long-​standing ul- cerative colitis. Both types are uncommon, and their aggregate varies only moderately from one population to another. The highest rates are recorded in Korea and Chile, and in women also New Delhi, India. The incidence of cancer of the gallbladder has fallen sharply in women in the United States in recent decades, but levelled off after falling in men. The decreases may be partly due to an increase in the rate of cholecystectomy in people who, having gallstones, are at greatest risk of cancer of the gallbladder. Pancreas • 2.7% of all cancers and 5.5% of cancer deaths • Sex ratio of rates 1.2:1; age distribution like oesophageal cancer Cancer of the pancreas is two to three times more common in regular cigarette smokers than in lifelong non​smokers. The chem- icals in cigarette smoke that specifically cause pancreatic cancer have not been identified, but the volatile nitrosamines in smoke that are absorbed from the alveoli and carried to the pancreas in the blood- stream are likely candidates. The disease is twice as common in dia- betics as in the population as a whole and risk is raised in patients with chronic pancreatitis. Cancer of the pancreas is generally regarded as a disease of the developed world, but the diagnosis is difficult in the absence of a well-​developed medical service and some of the relatively small geographical and temporal variations may be due to variation in diagnostic standards. The greatest reported rates are in US blacks. Mortality rates in the United States have been stable for the past 40 years, and in Britain stable apart from a decrease in men in the 1980s and early 1990s. Nose and nasal sinuses • 0.9% of all cancers and 0.02% of cancer deaths • Sex ratio of rates 1.7:1; age distribution like oesophageal cancer Surprisingly, in view of the widespread exposure of the human nose to tobacco smoke and other airborne toxins, cancers of the nasal cavity itself are extremely rare. Most arise from the paranasal sinuses. Several occupational hazards have been recognized, including the refining of nickel, processes giving rise to exposure to strong sulphuric acid mists, and the manufacture of hardwood fur- niture and leather goods. It would be wrong, however, to conclude that all contact with nickel, hardwood dust, and leather creates a hazard. The hazards have been observed in special occupational situations in which exposure has been intensive and prolonged. The nickel-​refining hazard was first observed in South Wales where the nickel carbonyl process was used, but similar hazards were subse- quently observed with other refining processes in Canada, Norway, and the Soviet Union. In the Welsh refinery the workplace expos- ures were much heavier before the Second World War, and (des- pite the continued use of the nickel carbonyl process in Wales) no hazard of nasal sinus cancer has been observed among men first employed there since 1950. The hazard in furniture workers was first observed in High Wycombe (southern England) and appears to have followed the introduction of high-​speed woodworking ma- chinery early in the 20th century. A hazard certainly affects some other groups of woodworkers, but should not be assumed to affect furniture workers in general. Most nasal and nasal sinus cancers are squamous carcinomas, but the hazard from hardwood dust characteristically produced adenocarcinomas. In some of the groups exposed to this hazard, as many as 5% of the men developed the disease. This meant that the risk of adenocarcinoma was increased 1500 times (as this histo- logical type of the disease is normally very rare) and the hazard was, in consequence, easy to confirm once suspicion had been aroused. Chromate workers are sometimes said to experience a hazard of nasal cancer, but this may be an error due to confusion with the char- acteristic ‘chrome ulcer’ of the nasal septum. Such ulcers have not generally been found to become malignant. A causal excess of nasal sinus cancer has been seen, however, in women employed in the United States in the early 20th century to apply radium-​containing luminescent paint to dials and clocks, who ingested the radium when they licked the brushes to shape their tips. Risk of nasal cancer is also modestly related to smoking. Larynx • 0.6% of all cancers and 0.5% of cancer deaths • Sex ratio of rates 5.6:1; age distribution, see Fig. 5.1.8 Cancers of the larynx, like cancers of the oesophagus and buccal cavity, are closely associated with tobacco smoking and with the

5.1  Epidemiology of cancer 431 consumption of alcohol. The two agents act synergistically and in the absence of either, the disease is rare. The different parts of this small organ are, however, related to the two agents differently. Cancers of the glottis are strongly related to smoking, particularly to cigarette smoking, and only weakly to alcohol, while cancers of the epilarynx resemble cancers of the neighbouring hypopharynx and are strongly related to both agents and to pipe and cigar smoking equally with cigarette smoking. The highest reported incidence of laryngeal cancer in men is in the Azores and in parts of Spain and Cuba. Rates in women are relatively low everywhere and generally a small proportion of those are in men in the same place. Trends with time vary consid- erably between countries and between the sexes, reflecting trends in smoking and alcohol consumption, and probably some other aetiological factor, perhaps nutritional in character. There has been fairly consistent evidence for an inverse association of risk with fruit and vegetable consumption. That there are other causal factors is evident from the relatively high incidence rates in parts of India, Turkey, North Africa, and Brazil, which cannot be ac- counted for by tobacco and alcohol. The disease has also occurred as an occupational risk in the manu- facture of mustard gas and in processes that cause exposure to strong sulphuric acid mists. Lung • 12.6% of all cancers and 21.5% of cancer deaths • Sex ratio of rates 1.4:1; age distribution, see Fig. 5.1.9 Nearly all lung cancers are bronchial carcinomas and should prop- erly be so described. The term ‘lung cancer’ is, however, in such common use that it is used here as synonymous with bronchial carcinoma, although it actually includes a very small proportion of alveolar cell carcinomas and other rare types of cancer with dif- ferent characteristics. Lung cancer is the most common cancer in the world, with over 1.8 million new cases per year, and the most common cause of cancer death. Until the 1920s, lung cancer was uniformly rare (except in the Hartz mountains, see ‘Occupation’, next). In the next two dec- ades, German and then British pathologists began to comment on an apparent increase, but this tended to be dismissed as an artefact of the greatly improving methods of diagnosis and the establishment of special centres for thoracic disease. Gradually, however, the increase became so pronounced and the change in the sex ratio so marked that the increase could no longer be dismissed as wholly artefactual and by the late 1940s, when the age-​standardized mortality rate in men in the United Kingdom had increased 20 times, it was clear that the developed world had begun to see an epidemic of lung cancer comparable in severity to the epidemics of infectious disease of the past, though with a longer time scale. Until the 1940s, the increase among British women was largely a diagnostic artefact. Since 1950, however, diagnostic standards in middle age have changed very little, the increase in British men has been replaced by a decrease, while the increase among middle-​aged women has continued for longer, be- fore flattening (Fig. 5.1.10). As a result, the sex ratio (male rate divided by female rate), at for example 50–​54 years of age, which 0 10 20 30 Rate (per 100000) 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.8  Annual incidence of cancer of the larynx, by age and sex. 0– 0 100 200 300 400 500 600 Rate (per 100000) 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.9  Annual incidence of cancer of the lung, by age and sex. 6000 5000 4000 3000 2000 1000 0 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 Percentage of 1911–15 rate Males Females Fig. 5.1.10  Mortality from lung and pleural cancers, England and Wales, 1911–​2014, ages 0–​84, by sex. (Lung and pleural cancers aggregated because the data do not allow their separation before 1960. The trends are virtually for lung cancer, however, as pleural cancer has been so much less common.)

432 SECTION 5  Principles of clinical oncology rose from 1.8 after the First World War to 8.9 after the Second World War, was reduced to 1.1 in 2014. Changes in treatment have had little effect on the fatality rate, which remains extremely high, and real changes in mortality closely reflect real changes in incidence. Smoking These time trends can be explained almost entirely by the effect of smoking tobacco, particularly in the form of cigarettes, which caused more than 90% of all lung cancers in the United Kingdom in the early 1990s. Evidence of this effect was first obtained in the middle of the last century by comparing the smoking histories of patients with different diseases (case–​control studies). It was found that the proportion of patients who had never smoked was much smaller if they had lung cancer (the ‘cases’) than if they had some other disease (the ‘controls’), and the proportion who had smoked heavily was correspondingly greater. Further evidence was obtained by asking large numbers of ap- parently healthy men and women what they smoked and then fol- lowing them up to determine the causes of death of those who had died. Cohort studies of this type, in the United States, in doc- tors in the United Kingdom, and in other groups, have all shown similar results, the risk increasing with the amount smoked, and varying with the length of time cigarettes had been smoked. If attention is restricted to populations in which most cigarette smokers had been smoking cigarettes regularly since early adult life, lung cancer is about 20 times more common in regular cig- arette smokers than in lifelong non​smokers and up to 40 times more common in very heavy smokers. At first the relationship was less marked in women than in men, but this was because female smokers who were old enough to have a high risk of cancer ei- ther had not begun smoking cigarettes so early in adult life or had smoked them less intensively when they began, and the sex differ- ences in behaviour and risk have both been progressively elimin- ated with the passage of time. Further studies have found that the relative risk of lung cancer has increased with decreasing age of starting to smoke and de- creased with the number of years that smoking has been stopped (detectable at 5 years after stopping, but never reaching the risk of a lifelong non​smoker); that the national increases in incidence have appeared at appropriate times after the increase in cigarette sales (after due allowance is made for a spurious increase due to improved diagnosis and appropriate differences in consumption by men and women); and that there is a general parallelism between the incidence of the disease in different countries and social and religious groups and their prolonged consumption of cigarettes. Finally, and most encouragingly, the trend in mortality has reversed following reduction in smoking. By 2014, the mortality from lung cancer among men in their thirties in Britain was only one-​eighth of that of men of the same ages 60 years earlier, corresponding to the earlier changes in the prevalence of smoking. The reduction in tar delivery between 1939 and 1965 contributed to the reduction in lung cancer in young men after the war, but the later reduction had little effect because of changes in the way cigarettes were manufac- tured and in the way they were smoked to ensure an adequate intake of nicotine. At older ages the decreases are less striking, but they are now seen at all ages in British men. In British women, however, although peaks were followed by decreases at ages under 75 some years ago, there has been a stabilization or increase in rates in the last 10–​15 years. In recent years, it has been shown that indoor air pollution with tobacco smoke—​‘passive smoking’—​increases lung cancer risk, by about 15–​30% from long-​term adult exposure as a never-​smoking spouse of a smoker. Occupation Several other causes of lung cancer have been discovered as a result of observations in industry. Many thousands of men and women have experienced significant hazards from exposure to asbestos or to polycyclic hydrocarbons (from the combustion of fossil fuel). The former has given rise to hazards in asbestos mines, asbestos textile works, and insulation work in the shipbuilding and con- struction industries and the latter to specific hazards in the manu- facture of coal gas in coking ovens, in steel works, in aluminium foundries, and wherever substantial amounts of incompletely com- busted fumes were released into the working environment. Much smaller numbers of men have experienced substantial hazards from radon in the air of mines (not only when mining radioactive materials, but also when mining haematite and fluorspar under conditions in which radon seeped into the mine air from streams and the surrounding rock); from the manufacture of chromates and chrome pigments; from the refining of nickel, beryllium, and cadmium; from arsenic (in the manufacture of arsenical pesticides and in the refining of copper, which is always contaminated with arsenic); from exposure to bischloromethyl ether in the chemical industry; from exposure to vinyl chloride; from the manufacture of mustard gas, to a small extent from exposure to silica if sufficient to cause silicosis; and from painting work and in rubber manufac- ture. In one extreme situation (in the cobalt mines of the Hartz mountains in central Europe, which were subsequently mined for radium and uranium), the absolute risk of contracting lung cancer due to the occupational hazard of radon was so large that more than half the workers contracted the disease. In several other situ- ations with heavy exposure to asbestos or the early stages of nickel refining, the occupational hazard has affected as many as 20–​30% of the exposed men. Atmospheric pollution Some of the materials responsible for these occupational hazards—​ particularly the combustion products of fossil fuels—​are or have been widely distributed in the air of towns and it is still uncertain how far they have, in this way, contributed to the production of the disease in the general population. That lung cancer was more common in big towns than in small towns and rural areas is cer- tain, but this held as strongly for Oslo and Helsinki, two relatively unpolluted cities, as for more polluted ones. Differences between the largest towns and the least populated areas have seldom been more than threefold and much of the difference can be accounted for by past differences in cigarette smoking. Attempts to ‘allow for’ cigarette smoking have usually been inadequate, as it is impos- sible to take full account of such factors as the age of starting to smoke cigarettes, the amount smoked daily at different periods, and the method of smoking (number of puffs, depth of inhaling, and so on).

5.1  Epidemiology of cancer 433 It is clear, however, that in the absence of cigarette smoking any effect of urban pollution in developed countries is relatively small. Estimates, based on extrapolation from the heavy pollution with coal smoke that used to occur in large towns, suggest that in such towns it may have contributed, in synergism with smoking, to as much as 10% of the risk of lung cancer, but would have caused very little risk in non​smokers. On this basis, the present levels of pollution with benzo[a]pyrene and the other known lung carcino- gens in town air can be only very small. Modern pollution with ultrafine particles (<10 µm diameter) may, however, be more haz- ardous. Study of residents in six contrasting cities in the United States in which information about personal smoking habits had been obtained suggests that the risk in the most polluted city com- pared with that in the least polluted could be increased by about one-​quarter in both smokers and non​smokers. The position in some developing countries is different: notably in parts of China, where intense indoor pollution with smoke and fumes from heating and cooking more than doubles the risk of lung cancer in non​smokers. Radon The effect of another form of pollution—​that of indoor air with radon arising from naturally occurring radium in rock and soil—​ has been estimated by extrapolation from the effects of the much larger doses to which some groups of underground miners have been exposed, and by direct observation in studies of people with and without lung cancer. These studies suggest that indoor radon may contribute to about 3% of lung cancers in the United Kingdom and about twice as much in the United States. The absolute effects are far greater in smokers than in lifelong non​smokers, so that in the absence of smoking few cases would be produced. Geographical differences The development of the male lung cancer epidemic and the early signs of its departure have been most prominent in the United Kingdom and Finland, since the switch of young men to cigar- ettes was largely complete in these countries by the 1920s. In the United States, where cigarette consumption doubled during the Second World War, the peak of mortality occurred a little later. In some other developed countries, the development of the epi- demic is still further behind and it is only just beginning to ap- pear in many developing countries. For example, Chinese males, who now consume about a third of the world’s cigarettes, experi- enced a 10-​fold increase in cigarette consumption per head be- tween the 1950s and 1990s that may well eventually cause almost a million cancer deaths a year when the young men of today reach middle age. In women, development of the epidemic has generally been later than in men. (Only in the Maori population of New Zealand did it occur at the same time.) In the United Kingdom, the United States, and a few other developed countries, the female lung cancer rates from smoking are already substantial, but in others, such as Spain and France, the epidemic in women has scarcely begun. The greatest recorded incidence rates in men world- wide are in parts of Turkey, and in blacks in parts of the United States, and the greatest in women in American Indians in parts of the United States. A relatively high risk has long been noted in Chinese women who are non​smokers, irrespective of their country of residence, which is probably due to their exposure to mutagens in the fumes from oils used in cooking with a wok and from the coal smoke with which many Chinese homes have been heavily polluted. Mesothelioma (of the pleura and peritoneum) • 0.8% of all cancers and 1.6% of cancer deaths • Sex ratio of rates 6.0. Age distribution like laryngeal cancer The existence of a specific type of tumour arising from the pleura, or less commonly the peritoneum, was debated by pathologists until 1960 when Wagner and his colleagues reported that six African patients with a similar type of ‘peripheral lung cancer’ had all lived in villages that were heavily polluted with dust produced by the mining of blue asbestos (i.e. crocidolite). Since then, occu- pational asbestos exposure (in asbestos mines, shipyards, building construction, asbestos product manufacture, and other work) has been shown to be responsible for the great majority of mesotheli- omas, which are the predominant cancers of the pleura and peri- toneum. They are much less likely to be produced by white asbestos (chrysotile) than by brown asbestos (amosite) or blue, as the two latter persist for longer in the lungs. A few cases arise from neigh- bourhood pollution with asbestos or secondary contamination (e.g. from household contact with asbestos workers) and some in Turkish villages are due to the weathering into the general at- mosphere of erionite fibres in local rock and houses; these fibres are physically similar to asbestos although chemically different. A few cases have been caused by radiotherapy, and natural ion- izing radiations may be responsible for most of those that are not associated with asbestos. An SV40-​like virus has been found in some tumours, but it is uncertain whether it plays a part in causing the disease. Mesotheliomas seldom occur less than 15  years after first ex- posure to asbestos, commonly occur 25–​30 years afterwards, and may be delayed for 50 years or more. Hence cessation of use of as- bestos (peak imports to the United Kingdom, and peak production worldwide, were in the 1970s) will only lead to decreasing meso- thelioma rates several decades later. In the last two decades, the recorded mortality in men under age 65 in Britain has begun to decrease. Almost all mesotheliomas are fatal. Due to confusion with lung or other types of cancer, it is still uncertain how many cases have occurred each year and some of the large increase in Western coun- tries since 1960 may be artefactual. The highest recorded rates of mesothelioma incidence now are in men in Bremen, Germany and Genoa, Italy and rates are unusually high in the United Kingdom compared with other countries. Rates in women in Western coun- tries tend to be much lower than in men. Pleural mesothelioma is not related to cigarette smoking and the occupational hazard affects smokers and non​smokers alike. Bone • 0.2% of all cancers and of cancer deaths • Sex ratio of rates 1.2:1; age distribution, see Fig. 5.1.11 Sarcomas can affect any bone, but characteristically affect the long bones in adolescence. After 45 years of age they occur

434 SECTION 5  Principles of clinical oncology most commonly in bones affected by Paget’s disease (osteitis deformans), which predisposes to sarcoma so strongly that as many as 1% of all people affected by the disease eventually develop a bone tumour. Many different histological varieties occur, some of which appear to have different causes. Osteogenic sarcomas and chondrosarcomas are the most common, the former account­ ing for nearly all the adolescent peak. One rare type (Ewing’s tu- mour) occurs only in children and young adults and is almost unknown in black people, irrespective of the society in which they live. Ionizing radiation is the main known extrinsic cause. Cases have been produced after high-​dose radiotherapy, especially such radio- therapy to childhood cancer patients, and after internal radiation from radionuclides including thorium in Thorotrast, an erstwhile contrast medium, radium in ‘luminizers’, once used to paint clocks and dials, and radium-​224 therapy. Bone cancer risk has also been found raised after alkylating agent treatment of childhood cancers, and osteosarcoma risk is raised in several rare cancer syndromes, for example, Li–​Fraumeni syndrome (OMIM 151623), and in retino- blastoma patients. National statistics in Britain record no substantial change in inci- dence over the last 30 years. Internationally, bone cancer is relatively rare everywhere, with less geographical variation than for most cancers. Connective tissues • 0.6% of all cancers and of cancer deaths • Sex ratio of rates 1.5:1; age distribution, see Fig. 5.1.12 Sarcomas of the soft tissues include a variety of different diseases, all of which are rare everywhere. Some occur in genetic syndromes, for example, Li–​Fraumeni syndrome and neurofibromatosis type 1 (OMIM 162200), and others are caused by ionizing radiation. A  few might be caused by intensive immunosuppression or ex- posure to chlorophenols and related compounds, but the evidence is inconclusive. Melanoma of the skin • 4.4% of all cancers and 1.6% of cancer deaths • Sex ratio of rates 1.1:1; age distribution, see Fig. 5.1.13 Melanoma accounts for a small proportion of incident skin cancers but for most skin cancer deaths. Incidence, and to a lesser extent mortality, rates have been increasing in white populations as far back as data are available. In recently born generations in several popula- tions, however, this trend has stabilized or reversed. The incidence of the disease varies inversely with the amount of skin pigmentation, both comparing whites with non​whites, and when comparing within whites, in whom skin sensitivity to sun- shine (ease of burning and tanning) and fair or redhead complexion 0– 0 1 2 3 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.11  Annual incidence of cancer of bone, by age and sex. 0– 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.12  Annual incidence of cancer of connective and other soft tissue, by age and sex. 0– 0 10 20 30 40 50 60 70 80 90 100 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.13  Annual incidence of melanoma of the skin, by age and sex.

5.1  Epidemiology of cancer 435 predict risk. Risk is also related strongly to numbers of benign moles and atypical moles on the skin, and less strongly to markers of cu- taneous ultraviolet damage such as solar keratoses. There is a par- ticularly great risk for patients with giant congenital naevi and those with xeroderma pigmentosum. In white people the tumour occurs most commonly on the legs in women and the trunk in men, and is least common on the buttocks and soles of the feet (areas not ex- posed to the sun). In blacks, in whom melanoma is rare, a high pro- portion occur on the soles of the feet. Incidence rates in white people vary roughly in proportion to the flux of sunshine (ultraviolet radiation) in the countries in which they live, although the reverse is true across Europe, probably reflecting darker complexions as one goes south. Risks rise in white migrants from countries with low insolation to those with higher insolation, especially if migration is at a young age. The greatest recorded inci- dence is in Queensland, Australia, and in whites in Hawaii, where melanoma is the second (Queensland) or third (Hawaii) most common cancer (disregarding non​melanoma skin cancer). For all skin sites combined, the incidence is not, however, greater in out- door than indoor workers (rather the reverse, in fact, perhaps due to the protective effects of a semi-​permanent suntan). The totality of the evidence suggests that recreational intermittent exposure of un- tanned skin to solar ultraviolet radiation, such as when sunbathing, is the principal cause of melanoma and the reason for the rising rates. The relationship is not simple, however, and indeed melanomas of the head and neck occur typically in elderly outdoor workers and appear to relate to chronic ultraviolet exposure. There is suggestive, but not decisive, evidence that use of sunbeds increases melanoma risk, and inconsistent evidence that PUVA (methoxypsoralen UVA) treatment can do so. Skin (non​melanoma) • 38.0% of all cancers and 0.5% of cancer deaths • Sex ratio of rates 1.6:1; age distribution like oesophageal cancer Non​melanoma skin cancers are the most common cancers in fair-​skinned populations, although rarely fatal. The predominant cause is sunshine (ultraviolet) exposure, and correspondingly the highest reported rates of incidence are in Australia. Rates have been rising in white populations across the world for many years, and tend to be greater in men than in women. The tumours are of two main types: basal cell and squamous cell carcinomas. The former, also known as rodent ulcers, have a causation that appears to relate to both cumulative ultraviolet exposure and intermittent intense exposures such as sunbathing. They occur mainly on parts of the body that are regularly exposed to the sun and, in particular, on the face, head, and neck. They are more common in outdoor workers, such as seamen and farmers, than in indoor workers; more common in fair-​skinned (and blond and red-​haired) than in dark-​skinned (and dark-​haired) people; and are almost un- known in blacks (except those who suffer from albinism). Some few cases have been produced by exposure to X-​rays, but the risk is very small unless the dose is very large and they seldom occur after normal courses of radiotherapy. People who suffer from xeroderma pigmentosum, a hereditary condition in which there is a defect in the enzyme responsible for the repair of the damage done to DNA by ultraviolet radiation, develop large numbers of skin tumours at an early age in response to even quite mild sun exposure (see Chapter 23.9). Squamous cell carcinoma is also produced by ultraviolet radi- ation, risk being proportional to cumulative sun exposure, and in PUVA-​treated patients proportional to cumulative PUVA dose. It accounts for about 20% of cancers on ultraviolet-​exposed skin. It is, however, the principal type of skin cancer produced by various carcinogenic chemicals, and particularly by polycyclic hydrocar- bons in the combustion products of coal. These chemicals have been responsible for the scrotal cancers of chimney sweeps, who accu- mulated soot in the folds of the scrotum; of mule spinners, whose clothes were saturated with carcinogenic oils; and of various other groups of workers whose clothes were contaminated with tar. They have caused (and still do cause) cancers of the forearm in industrial workers whose arms are regularly splashed with tar or carcinogenic oils, cancers of the groin in India, localized by the continued friction of the dhoti cloth, and cancers of the abdomen in Kashmir associated with the habit of carrying a kangri, or small stove, inside the clothes in winter to keep warm. Squamous cell carcinoma has also been due to prolonged ex- posure to arsenic, which is excreted by the skin and in the hair, when it may be accompanied by arsenical pigmentation and keratoses. All these conditions have been produced by prolonged medical treat- ment with inorganic arsenic, which used to be prescribed for a var- iety of chronic conditions, by the consumption of well water from arsenic-​rich soils, and by occupational exposure in the smelting of copper and cobalt (the ores of which often contain arsenic) and in the manufacture of arsenical pesticides. How large a part human papillomaviruses play in the devel- opment of squamous carcinoma of the skin is unclear. The type 5 virus is responsible for the warty lesions of epidermodysplasia verruciformis, some of which progress to cancer, and other types of the virus may contribute to the greatly increased risk that follows the intensive immunosuppression given to enable the survival of organ transplants. A third type is Kaposi’s sarcoma. It is associated with AIDS when AIDS results from homosexual intercourse. Frequent at first, par- ticularly in the United States, the association has become progres- sively less common. Before the advent of AIDS, Kaposi’s sarcoma was common in some parts of Central Africa, where it occasionally affected children, progressed rapidly, and could account for as many as 10% of all hospital patients with cancer. Elsewhere it was rare, but indolent cases occurred occasionally in developed countries, princi- pally on the legs of middle-​aged and elderly men. The disease is ini- tiated by infection with the human herpesvirus type 8, but cofactors are required for tumour development. Breast • 15.6% of all cancers and 7.1% of cancer deaths • Sex ratio of rates 0.01:1; age distribution, see Fig. 5.1.14 Cancer of the breast is the second most common cancer in the world and the most common in women, with over 1.6 million cases occurring per year. Incidence rates in women are greatest in Western countries (greatest in Belgium), somewhat lower in Eastern Europe, and lower again in Asia, Africa, and parts of South America. The geographical differences are unlikely to be chiefly due to genetic

436 SECTION 5  Principles of clinical oncology factors, as rates in migrants from low-​ to high-​incidence countries rise considerably, to levels intermediate between the two, and there is a further rise in succeeding generations. In many countries in- cidence rates have tended to rise slowly over several decades, but mortality rates have started to decrease in recent years in Western countries because of more effective treatments and probably the ef- fect of screening (Fig. 5.1.15). Hormonal factors, particularly oestrogens, are important in the production of the disease. The duration of ovarian activity is rele- vant, as the disease is particularly common in women who have an early menarche and a late menopause (the former being more important than the latter). Pregnancy produces a short-​term in- crease in risk, followed after several years by a lifelong decrease, particularly after teenage or early adult pregnancies. The incidence in later life increases progressively with a woman’s age at the time of her first full-​term pregnancy, being about three times greater when the first birth occurs after 35 years of age than when it occurs before 18 years. Full-​term pregnancies after the first have an add- itional protective effect. Pregnancies that end in abortion have little or no effect, however. The duration of lactation has an additional protective effect but is not marked unless it continues for a year or more. Risk of breast cancer is raised in women with benign breast dis- ease, the degree of risk varying according to the type of disease. Risk is also raised by alcohol consumption, by lack of physical exercise, and by ionizing radiation exposure at young ages, with particularly high risks in women given high-​dose mantle radiation for Hodgkin’s disease. There is not, however, good evidence for causation by any form of environmental pollution. Parity and menstrual differences are insufficient to account for the large variations in the incidence of the disease between different countries, which seem to be correlated with a high standard of living (i.e. with life in a developed country). Diet might play a part, but the evidence is complex and inconclusive. Obesity is associated with a reduced risk before the menopause, for uncertain reasons, and with a raised risk after the menopause. Height is associated with increased incidence both for pre-​ and postmenopausal women. Oestrogens prescribed medically, as HRT after the menopause, increase the risk by about 2% for each year of use; combined with progestogens in the contraceptive pill they increase it by about 25% during use, but the increased risk gradually disappears over 10 years, when use is stopped, as it does after HRT is stopped. Diethylstilboestrol, pre- scribed to pregnant women from the 1940s to the 1970s, increased breast cancer risk in these women as well as causing clear cell adeno- carcinoma of the vagina and cervix in daughters exposed in utero. Tamoxifen, an antioestrogen, reduces the incidence of the disease in the unaffected breast when prescribed for the treatment of breast cancer, and reduces incidence in high-​risk women. As well as the re- lation to sex hormone levels, there is evidence that risk of premeno- pausal breast cancer relates to prior endogenous levels of insulin-​like growth factor-​1 (IGF-​1). Breast cancer has been a particularly fertile area of genetic epi- demiology in recent years, with the identification of several high-​ risk genes, some related to clinical syndromes (e.g. Cowden’s disease, and carriage of an ataxia telangiectasia mutation) but most related only to cancer risk, and with the discovery of more than 150 single nucleotide polymorphisms associated with susceptibility. Uterine cervix • 0.9% of all cancers and 0.5% of cancer deaths • Confined to women; age distribution, see Fig. 5.1.16 Carcinoma of the cervix is the fourth most common cancer in women worldwide, and the most common in parts of Africa; it used also to be common in Europe and North America. It has always been rare in Jewish women and has tended to be less common in Muslim women than in women of other faiths living in the same country (e.g. Hindus in India). Changes in incidence over time have been difficult to assess, partly because mortality data have not always distinguished be- tween deaths due to cancer of the cervix and those due to cancer of the corpus (or endometrium), partly because the introduction of screening programmes has made it possible to diagnose and treat premalignant lesions (see next), and partly because hysterectomy for benign conditions has become progressively more common, with a corresponding reduction in the number of uteri in which the disease could occur. Despite these complications there can be no doubt that the disease has become substantially less common in Europe and North America than it was before the Second World War. 5– 0 100 200 300 400 500 10– 15– 20– 25– 30– 35– 40– Age group Rate (per 100000) 45– 50– 55– 60– 65– 70– 75– 80– 85+ Female Male Fig. 5.1.14  Annual incidence of breast cancer, by age and sex. 80 60 40 20 0 1860 1880 1900 1920 1940 Year of death Age-standardized rate per 100000 population 1960 1980 2000 Fig. 5.1.15  Mortality from breast cancer in women, England and Wales, 1868–​1997, ages 35 and older.

5.1  Epidemiology of cancer 437 The rarity of the disease in Jewish women and its relative rarity in Muslim women suggest that male circumcision may reduce the risk of its development, but this is unlikely as the state of circum- cision of her husband has no substantial effect on a woman’s risk of developing the disease in communities in which only some men are circumcised. Cleanliness is likely to be protective, as the disease is relatively uncommon in communities that practise ritual ablution before and after intercourse and, within each community, it becomes less common with rising socioeconomic status. Squamous carcinoma, which constitutes the vast majority of all cases, is intimately connected with sexual activity. It almost never oc- curs in virgins and increases in frequency with the number of sexual partners that a woman or her partner has had and with younger age at first sexual intercourse. Almost all cases are attributable in part to infection with certain types of the human papillomavirus, most notably types 16 and 18. A vaccine effective against these two types has been developed, and vaccination should greatly reduce future incidence. The development of squamous carcinoma is preceded by patho- logical changes limited to the epithelium, known as cervical intraepithelial neoplasia (CIN) types I, II, and III. CIN III is associ- ated with the same types of virus as squamous carcinoma, but CIN I and CIN II generally are not. The changes may progress from one to another, finally leading to carcinoma, but the early lesions (CIN I and II) commonly regress and even CIN III (previously known as carcinoma in situ) may do so occasionally. The lesions can be recog- nized in cervical smears and destroyed by lasers or extensive biopsy and the occurrence of clinical disease can be greatly reduced by the examination of all sexually active women every two or three years and the treatment of advanced CIN lesions. Other factors associated with the production of the disease are high parity, the use of oral contraceptives, and cigarette smoking. Both of the latter tend to be associated with behaviour conducive to venereal infection, but it appears that this tendency cannot wholly account for their association with the disease. That smoking may be responsible for some cases is suggested by the presence of mutagens in the cervical mucus of smokers that are not present in the secre- tions of non​smokers. Adenocarcinoma of the uterine cervix is generally uncommon, but has become somewhat more common recently in several coun- tries. It is related to human papillomavirus infection, but also appears to relate to factors similar to those for endometrial adenocarcinoma. Endometrium (corpus uteri) • 2.6% of all cancers and 1.3% of cancer deaths • Confined to women; age distribution like cancer of ovary The epidemiological features of endometrial cancer are in many re- spects the opposite of those of cervical cancer. Histologically, it is nearly always an adenocarcinoma. It is common in developed coun- tries, especially parts of the United States of America, and rare in poor populations. It is inversely related to parity, but not otherwise related to coitus, and is unaffected by the number of sexual partners. Like cancer of the breast, it is positively associated with late meno- pause, and perhaps with early menarche. Incidence in the United Kingdom and most Western countries has increased markedly in the last 20 years. In the United States there was a peak in the 1970s followed by a decline, as discussed next, but rates have been fairly stable since 1980. The one factor known to produce the disease is regular exposure to oestrogens, unopposed by progestogens. This leads to endometrial hyperplasia and eventually, in some cases, to cancer. Known causes include oestrogen-​secreting tumours of the ovary, the use of oral contraceptives in which oestrogens and progestogens are prescribed sequentially (types that have now been abandoned), the use of un- opposed oestrogens to relieve menopausal and postmenopausal symptoms, and adiposity. The last causes the disease because oes- trogens are produced in the body after the menopause in adipose tissue from the adrenal hormone, androstenedione. Tamoxifen, an analogue of natural oestrogens, which blocks oestrogen receptors in the breast and hence acts as an antioestrogen, can, due to dif- ferences between the hormone receptors in different tissues, have a pro-​oestrogenic effect in some other organs, and increases the in- cidence of endometrial cancer in proportion to the length of treat- ment. Endometrial cancer risks are reduced in users of combined (concurrent oestrogen and progestogen) oral contraceptives. It is improbable that oestrogens are initiating agents. They are not mutagens in vitro and the changes that took place in the incidence of the disease in the United States following the increase and sub- sequent reduction in the use of unopposed Premarin (a conjugated oestrogen) for the treatment of menopausal symptoms occurred so quickly that they make sense only if oestrogens act on some late stage(s) of the carcinogenic process. Endometrial cancer risk is raised in women who are physically inactive and in women with dia- betes, in each instance with evidence that the relation may be more than just a consequence of obesity. Endometrial cancer risk, at least postmenopausally, appears to be reduced among smokers. Ovary • 2.0% of all cancers and 2.5% of cancer deaths • Confined to women; age distribution, see Fig. 5.1.17 About 90% of ovarian cancers are of the surface epithelium, and the causes of the tumour that have been recognized may refer only 15– 20– 25– 30– 35– 40– Age group Rate (per 100000) 45– 50– 55– 60– 65– 75– 70– 85+ 80– 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Female Fig. 5.1.16  Annual incidence of cancer of the uterine cervix, by age.

438 SECTION 5  Principles of clinical oncology to these. International variation in ovarian cancer incidence is less than for most cancers, with high rates generally in Western coun- tries, but also, for instance, in the Philippines. The highest recorded rates are in Latvia and parts of England. Incidence decreases pro- gressively with increasing number of children. There is no strong relation to age at menarche or age at menopause, however. Risk of the disease is reduced by the use of oral contraceptives, more greatly with longer use, and seems to depend on the lifetime number of ovulations. Risk is decreased by tubal ligation and perhaps by hys- terectomy, and increased by mutations in the BRCA1 and BRCA2 genes. There is also an association of mucinous ovarian cancers with smoking. Prostate • 13.4% of all cancers and 7.1% of cancer deaths • Confined to men; age distribution, see Fig. 5.1.18 Cancer of the prostate is the second most common cancer in men worldwide and is found mainly in Western countries. Rates are par- ticularly low in much of Asia and parts of North Africa. It is more characteristically a disease of old age than any other cancer, so that it comes to play a much larger part in clinical experience as the pro- portion of old people in the population increases. It is unusual in that foci of cells resembling cancer can be found in a high propor- tion of clinically normal prostates, so that the recorded incidence is drastically increased by increasing the number of prostatic biopsies. Increases in incidence have been recorded in many Western coun- tries. The introduction of prostate-​specific antigen (PSA) testing has given rise to considerable artefacts in recorded rates in the United States. Some increase in mortality had been recorded in Britain and the United States, up to the early 1990s but in the last 10 years there has been a decrease by a half in the United States. The weight of evi- dence suggests that the disease is principally due to factors that have affected society for many years, but what these factors are remains obscure. Associations have been reported with both increased and decreased sexual activity and there is some evidence, not conclusive, for a reduced risk in men who eat more tomatoes and tomato prod- ucts, the main source of lycopene. There is also some evidence for reduced risk in men who take considerable physical exercise. On general grounds it seems likely that the disease is dependent on sex hormone imbalance (particularly as castration or oestrogen admin- istration slows the progression of clinical disease) but the nature of the imbalance is unknown. Decreased risks of prostate cancer have been found in patients with Klinefelter’s syndrome and those with diabetes; the former, and there is some evidence that also the latter, have reduced androgen levels. An association of prostate cancer risk has been found with prior raised circulating levels of IGF-​1. Vasectomy was thought to increase the incidence of the disease, but probably does not. Two epidemiological observations stand out:  the exceptionally high incidence in black populations in the United States (the highest recorded), and the low (although increasing) incidence in parts of Japan in contrast with other developed countries. Both may be partly due to genetic factors, but they are not wholly so, as Japanese and blacks have much higher rates in the United States than they have in Japan and Africa, respectively. Testis • 0.7% of all cancers and 0.03% of cancer deaths • Confined to men; age distribution, see Fig. 5.1.19 Testicular cancers are of two main types. Seminomas, which are the more common, have a peak incidence at about 35 years of age and teratomas, commonly called embryonal carcinomas in the United States, have a peak incidence about 10 years younger. Testicular cancer is the most common cancer in young white men in many countries, but much less common in non​white groups living in the same areas, except Polynesians, and is comparatively rare in most of Asia. Tumours after 50 years of age are mostly lymphomas and are now classed as such. Both genetic and environmental factors are important. On the one hand, the disease is uniformly rare in black populations, whether in Africa or in the United States. On 0– 5– 10–15–20–25–30–35– Age group Rate (per 100 000) 40–45–50–55–60– 75–80– 65–70– 85+ 0 10 20 30 40 50 60 70 Female Fig. 5.1.17  Annual incidence of cancer of the ovary, by age. 0– 10–15–20–25–30–35–40– Age group Rate (per 100 000) 45–50–55–60–65– 80– 70–75– 85+ 0 100 200 300 400 500 600 700 800 900 Male Fig. 5.1.18  Annual incidence of cancer of the prostate, by age.

5.1  Epidemiology of cancer 439 the other, it has increased in incidence over many decades in white populations around the world. In Britain, the increase began in the 1920s and affected first the higher socioeconomic groups. The in- crease trebled the mortality at 15–​34 years of age and produced a sharp peak in young adult life that had not previously been pre- sent. Mortality has greatly decreased in recent decades, however, as treatment has improved (Fig. 5.1.20), although in the United States the decrease has stalled in the last 15 years. The disease is generally more common in more prosperous populations. Greatest incidence rates are in parts of Chile and of Switzerland, and in New Zealand Maoris. Testicular cancer risk is greatly raised in men with XY go- nadal dysgenesis, and in brothers, and to a lesser extent fathers, of cases. The cancer is much more likely to occur in an undescended than in a normal testis (c.10% of cases in whites are in men who have had maldescent), and in a testis opposite one that has been cancerous, and is also associated with prior inguinal hernia, but otherwise its causes are unknown. The leading hypothesis, in part because of the age distribution and association with cryptorchidism, has been that the aetiology is prenatal, due to exposure in utero to raised maternal oestrogen levels during the first trimester of preg- nancy. Potential prenatal factors have been extensively investigated, with the strongest evidence for reduced risk with late birth order and raised risk for dizygous twins and boys born prematurely, but none are established. Penis • 0.2% of all cancers and 0.1% of cancer deaths • Confined to men; age distribution like cancer of the salivary glands Carcinoma of the penis has not been common in the past few dec- ades except in some parts of tropical Africa and Brazil, where it has accounted for 10% of all cancers in men. Recorded rates now are low everywhere, although greatest still in tropical Africa and Brazil. It is avoided almost entirely by circumcision at birth and is very rare if circumcision is carried out in boyhood. Phimosis is a risk factor. In developed countries penile cancer is rare even in the absence of cir- cumcision if the glans, coronary sulcus, and foreskin are kept clean. The oncogenic types of the human papillomavirus (principally types 16 and 18) can usually be identified in the malignant cells and are important causes of the disease. Kidney • 3.5% of all cancers and 2.6% of cancer deaths • Sex ratio of rates 1.9:1; age distribution like liver cancer Cancers of the kidney are of three main types: nephroblastomas (or Wilms’ tumours), adenocarcinomas (or hypernephromas) of the renal parenchyma, and transitional and squamous cell carcinomas of the renal pelvis. The first are limited to childhood, occur with al- most equal frequency everywhere, and apart from a few of genetic origin, are of unknown aetiology. The second constitute by far the majority of all cases, are more common in Europe (greatest in the Czech Republic) and North America (greatest in some American Indians) than in Africa and Asia, and have been increasing in inci- dence in many Western countries. Cigarette smoking is one cause, but the association is weak and it does not account for more than about one-​quarter of the cases. Obesity is also a risk factor, and phenacetin-​containing analgesics may have been, but the evidence is less clear than it is for renal pelvis cancers. The third type of renal cancer (carcinoma of the pelvis) constitutes some 10% of all cases. Three established causes are occupational exposure to the chemicals that cause cancer of the bladder, cigar- ette smoking, and the consumption of phenacetin in large enough amounts to produce analgesic nephropathy. In all three cases, the hazards are relatively small (two-​ to threefold). Cancer of the renal pelvis is also caused by ingestion of plants containing aristolochic 0– 10– 5– 15–20–25–30–35–40– Age group Rate (per 100 000) 45–50–55–60–65– 80– 70–75– 85+ 0 2 3 5 7 9 11 13 16 15 18 1 4 6 8 10 12 14 17 19 Male Fig. 5.1.19  Annual incidence of cancer of the testis, by age. 300 250 200 150 100 50 0 1910 1920 1930 1940 1950 1960 1970 1980 15–49 50–84 1990 2000 2010 2020 Percentage of 1911–15 rate Year of death Fig. 5.1.20  Mortality from cancer of the testis, England and Wales, 1911–​2014, by age.

440 SECTION 5  Principles of clinical oncology acid, which are used in Chinese herbal remedies and is responsible for Balkan nephropathy (Chapter  21.9.2), which increases renal pelvis cancer risk several hundredfold. Bladder • 2.9% of all cancers and 3.4% of cancer deaths • Sex ratio of rates 3.6:1; age distribution like oesophageal cancer Cancer of the bladder is almost universally several times more common in men than women. Its greatest recorded incidence in men is in parts of Europe, with low rates in much of Africa, Asia, and South America, and in non​whites compared with whites in the United States of America. The tumour can be produced by cigarette smoking, occupational exposure to aromatic amines, infection of the bladder with Schistosoma haematobium, the use of phenacetin-​ containing analgesics, the medical prescription of chlornaphazine (N,N′-​bis(2-​chloroethyl)-​2-​naphthylamine) and cyclophospha- mide, and ionizing radiation. There has been evidence for a rela- tion to chronic consumption of inorganic arsenic, a contaminant of the water supply in parts of Taiwan and other countries. There has also been some evidence for an association with urinary tract in- fection, supported by raised risks in paraplegics, who tend to have frequent urinary tract infections. Most bladder cancers are transi- tional cell carcinomas, but those associated with schistosomiasis are characteristically squamous carcinomas. It is not surprising that the bladder should be affected by many chemicals, as any noxious small molecules in the blood will tend to be found at greatly increased concentration in the urinary tract. Cigarette smoke contains sev- eral mutagenic chemicals that enter the bloodstream and thence the bladder, so that when tested in vitro on bacterial DNA the urine of cigarette smokers is found to be mutagenic, while that of non-​ smokers is barely active. Occupation An occupational cause was first suspected in 1895 in Germany, when Rehn commented on a cluster of cases in men using aniline for the manufacture of dyes. Aniline, however, is not carcinogenic in experimental animals; more recent studies have failed to in- criminate it epidemiologically, and it seems likely that other car- cinogenic chemicals were present as impurities. Four aromatic amines that are carcinogenic in experimental animals have been shown to cause bladder cancer in humans:  2-​naphthylamine, benzidine, 3,3′-​dichlorobenzidine, and 4-​aminobiphenyl. The first is one of the most powerful human carcinogens yet known and was responsible for the development of bladder cancer in all the 19 men who were employed in distilling it in a British fac- tory. Its manufacture in Britain was stopped in 1949, but small amounts continued to be imported until the 1960s. Other aro- matic amines that may cause bladder cancer include auramine, magenta, and, perhaps, 1-​naphthylamine. The last is dubiously carcinogenic in experimental animals and it seems probable that the cases associated with its use have been due to a small pro- portion of 2-​naphthylamine present as an impurity in the com- mercial material. These chemicals were used in the manufacture of dyes, in the rubber industry as antioxidants (1-​naphthylamine and 4-​aminobiphenyl) and hardeners (benzidine), and in labora- tories as a reagent (benzidine). 2-​Naphthylamine is also found in the combustion products of coal and may have been responsible for the risk of bladder cancer in men who made coal gas. Various other occupational associations, including leatherworking and aluminium work, have been reported, but are less clearly aetio- logical. As many as 10% of cases were, at one time, attributable to occupational causes in Britain and North America, but the pro- portion should now be much less. Smoking The most important cause numerically is cigarette smoking, which probably accounts for about half the total number of cases in Britain and North America. 2-​Naphthylamine and 4-​aminobiphenyl are present in cigarette smoke, but whether the amounts are sufficient to account for the carcinogenic effect is uncertain. Medicines The medicinal causes have, by contrast, been responsible for rela- tively few cases. Chlornaphazine was used briefly for the treat- ment of myelomatosis, until it was found to be metabolized into 2-​naphthylamine. Cyclophosphamide is used primarily for the treatment of malignant disease, but it is also used as an immuno- suppressant. In large doses it may cause sloughing of the bladder mucosa and, occasionally, cancer. High levels of consumption of phenacetin-​containing analgesics led to bladder cancer as well as renal cancer risk, but these drugs have been banned in Western countries for the last 30 years. Parasitic infection Heavy infection of the bladder with Schistosoma haematobium has been found to be a cause of the disease, most notably in Egypt and Tanzania. Artificial sweeteners Artificial sweeteners came under suspicion as potential bladder carcinogens because of the results of animal experiments in which, first, mixtures of cyclamates and saccharin and then saccharin alone were shown to cause bladder cancer in rats. The human use of cyc- lamates was banned before saccharin came under suspicion and it now appears that the ‘positive’ results of animal experiments with cyclamates alone were due to impurities. Saccharin has been shown to cause bladder cancer in rats but the quantities that had to be given were large, constituting a few per cent of the feed. The human evi- dence is extensive and could hardly be more negative, except that it does not cover lifelong use. Brain and other central nervous system • 1.5% of all cancers and 2.6% of cancer deaths • Sex ratio of rates 1.5:1; age distribution, see Fig. 5.1.21 Tumours of the brain and nervous system are of several different histological types, some of which may not be clearly either be- nign or malignant. One type occurs characteristically in child- hood (medulloblastoma), another in adult life (glioblastoma), and a third (astrocytoma) at all ages. Despite the overall male pre- dominance, one type (meningioma) is more common in women. Recorded incidence of brain and other central nervous system tumours tends to be greatest in white populations in Western countries, but geographical variations are less marked than for most tumours.

5.1  Epidemiology of cancer 441 A moderately large secular increase in incidence in old age has been recorded in many countries, which might be attributable to improved diagnosis with CT scans and MRI. Little or no increase in mortality has been reported in or before middle age and the re- corded increases in incidence are certainly largely, and possibly wholly, artefactual. The only established external cause is ionizing radiation. No new environmental cause has been established, but many have been suspected without convincing evidence, including electromagnetic fields associated with the use of electri- city (50–​60 Hz) and mobile telephones (cell phones). A small pro- portion of brain tumours are attributable to high-​risk hereditary syndromes, most commonly neurofibromatosis. The presence of allergy is associated with reduced glioma risk, but the reason is unknown. Thyroid • 1.0% of all cancers and 0.2% of cancer deaths • Sex ratio of rates 0.4:1; age distribution, see Fig. 5.1.22 The thyroid is particularly sensitive to ionizing radiation in child- hood, but risks after adult exposures are relatively small. Substantial numbers of cases have occurred among the survivors of the atomic explosions in Hiroshima and Nagasaki, children who were exposed to large amounts of radioactive iodine following the Chernobyl ac- cident, and young people whose necks were irradiated in infancy for the treatment of an enlarged thymus (a condition now con- sidered to be perfectly normal, but at one time thought to be a cause of sudden death). Fortunately, the thyroid tumours produced by ionizing radiation are nearly all of the papillary and follicular types, which respond well to treatment. No external causes are known of the medullary and anaplastic types, which have a high fatality and occur only in adult life. Many medullary thyroid cancers, however, are inherited as an autosomal dominant, alone or as part of the multiple endocrine neoplasia type 2 (MEN2) syndrome. Papillary and follicular thyroid cancers are less often genetic. Associations of thyroid cancer risk have been found with various benign thyroid conditions, but causality remains unclear. The disease is most common in women in South Korea. Increases in recorded incidence have been seen in recent decades in the United States and several other countries; it is unclear how much of this is due to changes in diagnostic completeness and criteria. Hodgkin’s disease (Hodgkin’s lymphoma) • 0.6% of all cancers and 0.2% of cancer deaths • Sex ratio of rates 1.4:1; age distribution, see Fig. 5.1.23 Hodgkin’s disease is best thought of as at least two diseases, one affecting primarily youths and young adults, the other primarily middle-​aged and older people. This division is suggested partly by the existence of two peaks in the age-​specific incidence rates, partly by the histological appearances (younger patients tending to have the nodular sclerotic form of the disease and older patients the mixed cellular form), and partly by the clinical distinction that young patients show mediastinal involvement in more than 50% of cases and infradiaphragmatic involvement in less than 5%, while the reverse tends to be true in older people. There are several reasons for thinking that the characteristic type in young people is infective in origin. In developing countries, Hodgkin’s disease occurs in childhood, but as the standard of living rises, the childhood cases disappear and are replaced by a larger number, and a peak of incidence, in young adults. This is reminis- cent of what happened to poliomyelitis in the first half of the 20th century and suggests that the disease may be due to a ubiquitous infective agent that tends to be contracted at older ages as hygiene improves, rarely causes Hodgkin’s disease, but is more likely to do so if infection is at an older age. Risk in young adults decreases with factors likely to facilitate early infection (e.g. late birth order). That the infectious agent is likely to be the Epstein–​Barr virus (EBV, human herpesvirus type 4) is suggested by the findings that the incidence is increased 5–​20 years after a clinical attack of in- fectious mononucleosis, that abnormal EBV antibody profiles can 0– 0 10 20 30 40 5– 10–15–20–25–30–35–40–45– Age group Rate (per 100 000) 50–55–60–65–70–75–80–85+ Female Male Fig. 5.1.21  Annual incidence of cancer of the brain and other central nervous system (CNS), by age and sex. 0– 0 1 2 3 4 5 6 7 8 9 10 5– 10–15–20–25–30–35–40–45– Age group Rate (per 100 000) 50–55–60–65–70–75–80–85+ Female Male Fig. 5.1.22  Annual incidence of cancer of the thyroid, by age and sex.