3.9 Circulating DNA for molecular diagnostics 299

3.9 Circulating DNA for molecular diagnostics 299

ESSENTIALS Short fragments of cell-​free DNA are released into the plasma when cells die. In patients with cancer some of this circulating DNA is re- leased by tumour cells; in pregnant women some is derived from the fetus; and increased amounts are found in many pathological conditions associated with cell death. In each of these circumstances, analysis of cell-​free DNA can provide useful clinical information (e.g. detection or monitoring of cancer, determination of mutation status of a fetus). With further improvement in analytical technologies and developments of new markers, it is likely that the application of cir- culating cell-​free DNA and cell-​free RNA species in molecular diag- nostics will increase in the future. Introduction Cell-​free DNA is present in the plasma of human subjects. These DNA molecules are short fragments that are released when cells die (Fig. 3.9.1). In cancer patients a proportion of such circulating cell-​ free DNA is released by tumour cells and thus carries molecular signatures of cancer. Such signatures include oncogene mutations, copy number aberrations, DNA methylation changes, and viral sequences in cancer associated with virus infections. By detecting such signatures in plasma, it is possible to detect, monitor and prog- nosticate cancer, and obtain information guiding targeted therapy (Table 3.9.1). In pregnant women, fetal DNA is found circulating in maternal plasma. Such fetal DNA is released by the placenta and carries gen- etic and DNA methylation signatures of the fetus (Fig. 3.9.1). Hence, analysing the plasma DNA of a pregnant woman allows determin- ation of certain genetic characteristics of a fetus (e.g. sex, blood group type, mutation status, and chromosomal constitution; see Table 3.9.1). Such an approach has been referred to as non​invasive pre- natal testing and is now used worldwide, particularly for screening for fetal chromosomal disorders such as Down’s syndrome. Apart from oncological and prenatal applications, cell-​free DNA analysis has also been explored in several emerging areas. Such ap- plications are built on the concept that circulating DNA is a marker of cell death and hence is released in increased amounts in many pathological conditions associated with cell death (Fig. 3.9.1). Examples include the monitoring of rejection episodes following transplantation, trauma, and stroke. With further improvement in analytical technologies and developments of new markers, it is likely that the application of circulating cell-​free DNA in molecular diag- nostics will increase in the future. For many years, nucleic acids extracted from cellular materials (e.g. blood cells and buccal cells) are the predominant materials used for molecular analysis. However, over the last few years, there has been increased interest in the use of extracellular nucleic acids for a variety of molecular diagnostics. This chapter provides an overview of this emerging area (Fig. 3.9.1 and Table 3.9.1). History The existence of cell-​free DNA in plasma has been attributed to Mendel and Metais in 1948. This work is remarkable as it predated the discovery of the double helical structure of DNA by Watson and Crick in 1953, and—​perhaps because it was so far ahead of its time—​ its significance remained unrecognized for many years. In the 1970s, researchers showed that the concentrations of circulating DNA in cancer patients were higher than those without cancer. However, the origin of such excess circulating DNA remained uncertain for many years due to the limitations in technology at that time. With the advent of the polymerase chain reaction (PCR), it was shown in 1994 by Vasioukhin et al. and Sorenson et al. that circulating DNA in cancer patients carried mutations present in the tumour cells, thus demonstrating that a proportion of such circulating DNA molecules is released by the tumour cells. This realization laid the foundation for performing ‘liquid biopsies’ of tumours, in which the sampling of bodily fluids, most typically blood, allows genomic information regarding cancer to be obtained in a non​invasive manner. The presence of circulating tumour DNA in plasma prompted other researchers to look for other types of circulating DNA. In 1997, Lo et al. demonstrated that cell-​free fetal DNA was present in the plasma and serum of pregnant women. This discovery laid the foundation for the development of non​invasive prenatal testing (NIPT). In 1998, the same group of researchers showed that DNA 3.9 Circulating DNA for molecular diagnostics Y.M. Dennis Lo and Rossa W.K. Chiu

300 SECTION 3  Cell biology was released by a transplanted organ (e.g. kidney and liver) into the plasma of a transplant recipient, which opened up a new approach for monitoring rejection following transplantation. In 1999, two independent groups of researchers demonstrated that mRNA released by tumours could be detected in the plasma and serum of cancer patients. In 2000, it was shown that fetal mRNA could also be detected in the plasma of pregnant women. This discovery of circulating mRNA has opened up the possibility of performing gene expression profiling of a tumour or fetus in a non​invasive manner. Developments since then have provided Cancer Other pathologies Organ transplantation Prenatal DNA DNA methylation polymorphisms Viral sequences miRNA mRNA DNA mutations Fig. 3.9.1  Circulating nucleic acid molecules are released from dying cells into plasma, either as a result of normal cell turnover or pathologies. The detection of circulating nucleic acids have been applied as non​invasive means for cancer assessment, prenatal assessment, transplantation monitoring, and the assessment of other pathologies, such as those associated with inflammatory, ischaemic, and immunological cellular damages. Circulating nucleic acid species that have been detected in human plasma include DNA, messenger RNA (mRNA), micro RNA (miRNA), DNA mutations, DNA methylation signatures, DNA polymorphic sequences, and viral nucleic acid sequences. Table 3.9.1  Clinical applications of circulating nucleic acid analysis Applications Clinical utilities Cancer assessment Diagnosis Prognostication To inform choice of therapy Treatment monitoring Screening Prenatal assessment Fetal sex determination (sex-​linked diseases, congenital adrenal hyperplasia) Blood group determination (rhesus D blood group incompatibility) Chromosomal aneuploidies (e.g. trisomy 21, trisomy 18, trisomy 13) Subchromosomal aneuploidies (microdeletions, microduplications) Single gene disease diagnosis Transplantation Graft rejection Monitoring Other pathologies (inflammatory, ischaemia, immunological) Detection Prognostication Identify organs involved (tissue mapping)

3.9  Circulating DNA for molecular diagnostics 301 many powerful methods for analysing such circulating DNA and RNA species. Circulating nucleic acids for cancer detection The first species of circulating tumour-​derived DNA detected in the plasma of cancer patients consisted of oncogene mutations. Since then, many other types of tumour-​derived DNA have been detected in plasma or serum. The main difference between plasma and serum in the context of cell-​free DNA is that DNA is released from the white blood cells during the blood clotting process through which serum is formed. Hence, the fractional concentration of tumour DNA in serum is typically lower than that in plasma. For this reason, most workers in the field prefer to use plasma. In addition to oncogene mutations, microsatellite alterations, fusion genes, DNA methyla- tion changes, and viral sequences associated with cancer have been detected in plasma or serum. In general, a cancer-​associated DNA sequence that is easily dif- ferentiated from any sequence that is present in the human genome represents a good marker for detection. Thus, it was perhaps not sur- prising that viral sequences which were not present in the human genome were used in some of the earliest work elucidating the mo- lecular characteristics and clinical applications of circulating DNA in cancer. One such example is the measurement of Epstein–​Barr virus (EBV) DNA sequences in the plasma of patients with nasopha- ryngeal carcinoma, which is a cancer that is particularly common in south China where it is associated with EBV infection, and EBV DNA is found in the tumour tissues of virtually all cases. In the late 1990s, it was shown that high concentrations of EBV DNA could be found in the plasma and serum of nasopharyngeal carcinoma pa- tients, and the concentrations of such circulating EBV DNA were found to increase with the stage of disease, thus suggesting that they are related to tumour load. Following treatment, the concentrations of circulating EBV DNA would typically decrease, and upon relapse or progression of disease the concentrations of circulating EBV DNA would increase. It has been demonstrated that such circulating EBV DNA consists of short fragments of DNA, rather than intact virions. All of the aforementioned characteristics of circulating EBV DNA are shared by many other species of tumour-​derived DNA in plasma. One powerful application of tumour DNA in plasma is for guiding treatment using targeted therapy. Hence, the presence in the plasma of cancer-​associated mutations which are the targets for specific agents (e.g. epidermal growth factor receptor gene mutations that respond to tyrosine kinase inhibitors) can be used as a predictor that a patient would likely respond to particular therapy. Following initi- ation of effective therapy, DNA fragments carrying the targeted mu- tations would reduce in concentration in plasma. Upon emergence of a tumour cell clone that was resistant to the targeted therapy, DNA fragments carrying the originally targeted mutations would typically increase in plasma, together with those carrying the genetic signa- ture of resistance. With the advent of massively parallel sequencing, it is now possible to detect and measure the concentration of mul- tiple mutations in plasma accurately and sensitively. Apart from mutations that alter the sequence of a cancer genome, cancer cells also exhibit numerous epigenetic changes, which are biochemical modifications of the genome that do not involve a change in the DNA sequence. One of the best-​studied epigenetic changes is DNA methylation. There are numerous changes in DNA methylation in a cancer genome when compared with the genome of non​malignant cells, and such changes have been used as targets for detecting circulating DNA in the plasma of cancer patients. Examples of such markers include the p16 and RASSF1A genes which are hypermethylated in multiple cancer types. Another ex- ample is the hypermethylation of the septin 9 gene that has been used for the screening of colorectal carcinoma. In addition to circulating DNA-​based markers, several mRNA and miRNA species that are preferentially expressed in tumour cells, when compared with non​malignant cells, have been detected in plasma. However, most of the efforts in the use of circulating nu- cleic acids for cancer detection are based on DNA, rather on RNA markers. Reasons for such a course of development include the rela- tive stability of DNA over RNA in general, the ease of extraction and analysis, and the specificity of the resulting tests. Circulating fetal DNA and non​invasive prenatal testing Cell-​free fetal DNA was first demonstrated in maternal plasma in 1997 through the detection of Y-​chromosomal sequences that male fetuses released into their pregnant mother’s plasma. While such sequences are generally referred to as ‘fetal’ in origin, they are actually released from the placenta. Such cell-​free fetal DNA sequences are de- tectable from the early first trimester. By the 10th week of pregnancy, cell-​free fetal DNA represents a mean of 15% of the DNA in maternal plasma. The absolute concentration of cell-​free fetal DNA per unit volume of maternal plasma continues to increase during pregnancy. However, following delivery of the fetus, cell-​free fetal DNA is cleared very quickly, with an estimated half-​life of some 16 minutes. Certain pregnancy-​associated disorders, such as pre-​eclampsia and preterm labour, have been associated with an increase in the absolute concen- tration of cell-​free fetal DNA in maternal plasma. Cell-​free fetal DNA in maternal plasma consists of short fragments of DNA, which interestingly have a shorter size distribution than the background maternally-​derived DNA in plasma. This size dif- ference has been exploited as an approach for enriching fetal DNA, either physically (e.g. through electrophoresis) or bioinformatically (e.g. by computationally ‘targeting’ the short DNA fragments that have been sequenced from maternal plasma). It should, however, be noted that the size difference between circulating fetal and maternal DNA does not allow the complete separation of these two species of circulating DNA. The first diagnostic applications of circulating cell-​free DNA in- volve DNA sequences that the fetus has inherited from its father and which are absent in the pregnant mother’s genome. The first example of such sequences are Y-​chromosomal sequences of a male fetus. The detection of fetal Y-​chromosomal sequences in maternal plasma enables a non​invasive approach for determining the sex of a fetus. Such an approach is useful in the prenatal investigation of preg- nant women who are carriers of mutations for sex-​linked disorders (e.g. haemophilia). The second example of such sequences are RHD sequences of a RhD-​positive fetus, coding for the RhD blood group antigen, that are absent in the genome of a RhD-​negative pregnant mother. This approach is useful for investigating RhD blood group incompatibilities between the pregnant mother and her fetus. The

302 SECTION 3  Cell biology third example of a sequence that a fetus has inherited from its father, but absent in the pregnant mother’s genome, is a mutation that the fetus has inherited from its father. When such an approach is used for the detection of an autosomal dominant disorder, the presence of a paternally inherited mutation in the plasma of a pregnant woman who does not have that mutation herself indicates that the fetus has inherited it and thus is at risk of the disorder. The first such mutation detected was a mutation of the fibroblast growth factor receptor 3 gene causing achondroplasia. When such an approach is used for an autosomal recessive dis- order, one method is to focus on genetic disorders which can be caused by multiple mutations and in which the father and the mother of the fetus carry different mutations. One then attempts to detect the paternally inherited mutation in the maternal plasma. Provided that the assay is sensitive enough, the absence of such a mutation in maternal plasma is taken to indicate that the fetus has not inherited the paternal mutation and hence does not suffer from the disorder. Conversely, the detection of the paternally inherited mutation in maternal plasma would indicate that the fetus has in- herited the paternal mutation but does not provide any information regarding its inheritance of the maternal mutation, hence invasive prenatal diagnosis would still be necessary in this circumstance. The development of more precise methods for measuring DNA sequences in plasma, such as single molecule PCR (or ‘digital’ PCR), has allowed the informativeness of plasma DNA-​based non-​ invasive prenatal testing for autosomal recessive disorders to be en- hanced. This approach is based on the concept that in the genome of a pregnant woman who is a carrier for an autosomal recessive dis- order, the ratio of the mutant copy and the normal copy of the gene implicated in the disorder should be 1:1. Hence, in the maternal plasma, the ratio of the pregnant mother’s own mutant and normal copies of the gene should also be 1:1. However, the fetus would be releasing its own DNA into maternal plasma as well. If the fetus had one copy of the mutant gene and one copy of the normal gene, then the ratio of these versions of the gene would remain unchanged from 1:1 (Fig. 3.9.2). On the other hand, if the fetus had two copies of the mutant gene, then the concentration ratio in maternal plasma would be biased in favour of the mutant gene. Finally, if the fetus had two copies of the normal gene, then the concentration ratio in maternal plasma would be biased in favour of the normal gene. This method has been referred to as the relative mutation dosage approach and has been used in autosomal recessive disorders such as β-​thalassaemia and sickle cell anaemia. Apart from autosomal recessive disorders, this approach can also be used for sex-​linked Mother heterozygous Fetus homozygous for mutation Mutation Fetus heterozygous Mother heterozygous Mother heterozygous Fetus homozygous for normal allele Normal allele Linked polymorphic alleles Fig. 3.9.2  Non​invasive fetal genotyping for single gene disease diagnosis are performed for women who are carriers of a disease mutation by comparing the relative abundance of the mutant and normal alleles in maternal plasma. When a fetus is homozygous for the mutation, the plasma DNA sample shows an overrepresentation of molecules carrying the mutation (left panel). When a fetus is homozygous for the normal allele, the plasma DNA sample shows an overrepresentation of molecules carrying the normal allele (right panel). When a fetus is heterozygous for the maternal mutation, there is equal representation between the mutant and normal alleles in maternal plasma (middle panel). The assessment could be performed by directly comparing the amounts of the DNA molecules carrying the normal and mutant alleles, termed relative mutation dosage (RMD). Alternatively, haplotype based quantitative comparison could be made, termed relative haplotype dosage (RHDO) analysis. The accumulative abundance of the polymorphic alleles linked to the mutation would contribute to the measured representation of the mutant allele. Similarly, the accumulative abundance of the polymorphic alleles linked to the normal allele would contribute to the measured representation of the normal allele. Fetal DNA molecules in maternal plasma are shorter than the maternal molecules, hence these are depicted as the smaller molecules in the illustration. The paternally inherited fetal DNA molecules are depicted as the orange molecules.

3.9  Circulating DNA for molecular diagnostics 303 disorders in which the mother is a carrier of the disease gene on the X chromosome (e.g. haemophilia A). In addition to measuring the dosage of the mutant and normal gene, one can also measure the relative dosage of alleles of single nucleotide polymorphisms (SNPs) that are linked to the gene. Such an approach is particularly robust when multiple SNPs, which are grouped together in a haplotype, are analysed. Such an approach is referred to as the relative haplotype dosage (RHDO) method (Fig. 3.9.2), and has been used for the non​invasive prenatal testing of genetic disorders such as congenital adrenal hyperplasia, β-​thalassaemia and haemophilia A. Haplotype information is conventionally constructed by analysing DNA samples from multiple family members. However, the recent availability of methods for direct haplotype determination has sim- plified the generation of haplotype information for RHDO analysis. Perhaps the most important application of NIPT to date is the use of this technology for detecting fetal chromosomal aneuploidies such as Down’s syndrome. The general concept of this approach is the de- tection of the subtle quantitative aberration in the chromosome in- volved in the aneuploidy in maternal plasma. For example, for the detection of fetal Down’s syndrome, there would be a small increase in the concentration of chromosome 21-​derived DNA sequences in maternal plasma compared with the other chromosomes. One widely used method for detecting such a quantitative aberration of sequences in maternal plasma is with the use of massively parallel sequencing. Apart from Down’s syndrome, such an approach has also been used for the NIPT of trisomy 18, trisomy 13, sex chromosome aneuploidies, and even aberrations involving only part of a chromo- some (e.g. a subchromosomal deletion or duplication). While NIPT for chromosomal aberrations such as Down’s syn- drome are highly accurate, its diagnostic accuracy is not 100% and so it is widely regarded as a highly accurate screening test, rather than as a diagnostic test. Hence, an abnormal result would still need to be con- firmed with an invasive method (e.g. amniocentesis). There are multiple reasons why NIPT is not and perhaps cannot be 100% accurate. Firstly, the robustness of NIPT is related to the fractional concentration of fetal DNA in a particular maternal plasma sample. It is known that some 1–​ 2% of maternal plasma samples contain very low amounts of circulating fetal DNA and hence would be regarded as uninformative for NIPT for chromosomal aneuploidies. Secondly, fetal DNA in maternal plasma is derived from the placenta. It is possible that the placenta might contain a clone of cells with a different chromosomal constitution to the cells of the fetus’s body, a phenomenon known as confined placental mo- saicism. Thirdly, in twin pregnancies, DNA is released by both fetuses into the maternal plasma. However, occasionally, if one twin has subse- quently died, in a scenario that has been referred to as a ‘vanishing twin’, its placenta might continue to release DNA into maternal plasma. If the vanished twin has a chromosomal aneuploidy, then such an aberration might be observed in maternal plasma and might be regarded as a ‘false-​ positive’ result for the remaining healthy twin. NIPT for chromosomal aneuploidies is now performed in over 90 countries in millions of preg- nant women per year, as a result of which some centres have reported a reduction in the use of invasive prenatal testing by some 30%. With the increased use of NIPT worldwide, several groups have come across pregnant women who also have cancer during preg- nancy. Such subjects exhibit genomic aberrations that are detectable in their plasma which originate from the cancer, rather than from the fetus. These studies suggest that pregnant women should be in- formed about this possibility during the counselling for NIPT. Several proof-​of-​concept publications have shown the possibility of performing non​invasive prenatal fetal whole genome sequencing from maternal plasma. However, the high costs of such analysis and the difficulty in data interpretation and genetic counselling have re- stricted these to the research domain at the present time. Fetal RNA and miRNA have also been detected in maternal plasma, but due to the relative complexity of sample stabilization, extraction, and analysis, such approaches have not been used in actual clinical use. Other emerging applications In 1998, it was shown that Y-​chromosomal DNA sequences could be found in female subjects who had received a male liver or kidney through transplantation. The presence of donor-​derived DNA in the plasma of transplantation recipients has now been generalized to bone marrow transplantation, heart transplantation, and lung transplant- ation. Furthermore, this approach has now been extended beyond sex-​mismatched transplantation through the use of SNP markers which are able to differentiate a donor’s from a recipient’s DNA. In addition, through the precise measurement of the concentration of the recipient’s DNA in the donor’s plasma, such as single molecule PCR or massively parallel sequencing, it has been found that rejection episodes are associated with an elevation in donor’s DNA in plasma. This observation is consistent with the concept that DNA is released into plasma when cells die. Plasma DNA analysis is thus a non​invasive approach for monitoring rejection following transplantation. The concept that plasma DNA is a marker for cell death phe- nomena is an important one as it suggests that the concentration of plasma DNA would be elevated in many clinical scenarios associ- ated with tissue damage. Indeed, increase in the levels of circulating DNA has been reported in pulmonary embolism, stroke, trauma, autoimmune diseases, and so on. Early work in this area was per- formed using DNA markers that were not tissue specific. However, the recent development of DNA methylation markers that are spe- cific for particular tissues will likely lead to renewed interest in this area of work. If future technologies would allow plasma DNA to be analysed more rapidly and cheaply, it is possible that circulating DNA analysis would eventually be regarded as a type of biochemical body scan, similar to how serum biochemistry is used nowadays. FURTHER READING Bianchi DW, Chiu RWK (2018). Sequencing of Circulating Cell-free DNA during Pregnancy. New England Journal of Medicine, 379, 464–73. Jiang P, Lo YMD (2016). The long and short of circulating cell-​free DNA and the ins and outs of molecular diagnostics. Trends Genet, 32, 360–​71. Lam WKJ, Jiang P, Chan KCA, et al. (2018). Sequencing-based counting and size profiling of plasma Epstein-Barr virus DNA enhance popu- lation screening of nasopharyngeal carcinoma. Proceedings National Academy of Sciences (USA), 115(22), E5115–E5124. Minear MA, et al. (2015). Noninvasive prenatal genetic testing: cur- rent and emerging ethical, legal, and social issues. Ann Rev Genomics Hum Genet, 16, 369–​98. Thierry AR, et al. (2016). Origins, structures, and functions of circu- lating DNA in oncology. Cancer Metastasis Rev, 35, 347–​76. Wong AI, Lo YMD (2015). Noninvasive fetal genomic, methylomic, and transcriptomic analyses using maternal plasma and clinical im- plications. Trends Mol Med, 21, 98–​108.

SECTION 4 Immunological mechanisms Section editors: John D. Firth, Christopher P. Conlon, and Timothy M. Cox 4.1 The innate immune system  307 Paul Bowness 4.2 The complement system  315 Marina Botto and Matthew C. Pickering 4.3 Adaptive immunity  325 Paul Klenerman and Constantino López-Macias 4.4 Immunodeficiency  337 Sophie Hambleton, Sara Marshall,
and Dinakantha S. Kumararatne 4.5 Allergy  368 Pamela Ewan 4.6 Autoimmunity  379 Antony Rosen 4.7 Principles of transplantation immunology  392 Elizabeth Wallin and Kathryn J. Wood