22.3.5 The polycythaemias 5227 Daniel Aruch and Ro

22.3.5 The polycythaemias 5227 Daniel Aruch and Ronald Hoffman

22.3.5  The polycythaemias 5227 Latif A-​L, et al. (2013). Allogeneic stem cell transplantation for Chronic Myeloid Leukemia is safe and effective in high risk patients fol- lowing second generation tyrosine kinase inhibitors: a single centre experience. Leuk Res Rep, 2, 47–​50. Mahon FX, et al. (2010). Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete mo- lecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol, 11, 1029–​35. Marin D, et al. (2010). Adherence is the critical factor for achieving molecular responses in patients with chronic myeloid leukemia who achieve complete cytogenetic responses on imatinib. J Clin Oncol, 28, 2381–​8. Marin D, et al. (2011). Assessment of BCR-​ABL1 transcript levels at 3 months is the only requirement for predicting outcome for pa- tients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. J Clin Oncol, 30, 232–​8. Nowell PC, Hungerford DA (1960). A minute chromosome in human chronic granulocytic leukemia. Science, 132, 1497. Palani R, et al. (2015). Managing pregnancy in chronic myeloid leu- kaemia. Ann Hematol, 94 Suppl 2, S167–​76. Pavlu J, et al. (2011). Three decades of transplantation for chronic mye- loid leukemia: what have we learned? Blood, 117, 755–​63. Pinilla-​Ibarz J, et  al. (2000). Vaccination of patients with chronic myelogenous leukemia with BCR-​ABL oncogene breakpoint fusion peptides generates specific immune responses. Blood, 95, 1781–​7. Preudhomme C, et al. (2010). Imatinib plus peginterferon alfa-​2a in chronic myeloid leukemia. N Eng J Med, 363, 2511–​21. Rea D (2015). Management of adverse events associated with tyrosine kinase inhibitors in chronic myeloid leukemia. Ann Hematol, 94 Suppl 2, S149–​58. Ross DM, et al. (2013). Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: re- sults from the TWISTER study. Blood, 122, 515–​22. Sasaki K, et al. (2016). Conditional survival in patients with chronic myeloid leukemia in chronic phase in the era of tyrosine kinase in- hibitors. Cancer, 122, 238–​48. Shah NP, et al. (2004). Overriding imatinib resistance with a novel ABL kinase inhibitor. Science, 305, 399–​401. Soverini S, et al. (2011). BCR-​ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors:  recommendations from an expert panel on behalf of European LeukemiaNet. Blood, 118, 1208–​15. Vardiman, JW (2008). Chronic myelogenous leukaemia, BCR-ABL1 positive. WHO classification of tumours of haematopoietic and lymphoid tissues, 32–7. 22.3.5  The polycythaemias Daniel Aruch and Ronald Hoffman ESSENTIALS Polycythaemia or erythrocytosis is characterized by an abnormal in- crease in the numbers of red blood cells, leading to an elevation in the haemoglobin concentration and haematocrit (>49% in men and

48% in women). The cause may be either (1) primary—​due to an intrinsic defect of haematopoietic stem cells; or (2) secondary—​due to extrinsic stimulation of progenitor erythroid cells by circulating growth factors; and the condition needs to be distinguished from (3) pseudopolycythaemia—​in which haematocrit is raised because the plasma volume is decreased. Normal erythropoiesis The primary controlling factor for erythropoiesis is the glycoprotein hormone erythropoietin. This is produced by the kidney in response to hypoxia, which leads to the accumulation of a transcriptional factor, hypoxia-​inducible factor-​1 (HIF-​1), the principal regulator of numerous genes that participate in the hypoxic response. Mutation in genes encoding for proteins involved in the oxygen-​sensing mech- anism, in the erythropoietin receptor, or in pathways downstream of the receptor can all (rarely) lead to polycythaemia. Secondary polycythaemias Associated with appropriate erythropoietin secretion—​conditions that are ultimately the result of tissue hypoxia and subsequent exces- sive erythropoietin production include (1)  living at high altitude, (2) chronic lung disease, (3) cyanotic congenital heart disease with right-​to-​left shunting, (4) carbon monoxide intoxication—​as occurs in heavy smokers, (5) haemoglobin variants with increased oxygen affinity, and (6) mutations in genes involved in the oxygen sensing pathway—​e.g. von Hippel–​Lindau gene (Chuvash polycythaemia) and prolyl hydroxylases. Associated with inappropriate erythropoietin secretion—​in the absence of tissue hypoxia, inappropriate erythropoietin production commonly originates from the kidney and many renal disorders are associated with erythrocytosis (e.g. renal artery stenosis, polycystic kidney disease, and tumours). Tumour-​associated polycythaemia may also result from cerebellar haemangioblastoma, hepatocellular carcinoma, phaeo- chromocytoma, and other adrenal tumours. Primary polycythaemia—​polycythaemia vera This is a clonal, chronic progressive haematological malignancy characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic elements in the bone marrow. Aetiology—​up to 95% of cases are caused by somatic mutations in the pluripotent haemopoietic stem cells leading to replacement of a key valine residue by phenylalanine at position 617 of the JAK2 kinase (V617F), which releases it from autoinhibition. Less common mutations have been described recently, primarily JAK2 exon 12 and LNK mutations. Clinical features—​may be detected on a full blood count in asymp- tomatic patients, or may present with a wide range of nonspecific symptoms (notably pruritus). Signs include those directly related to polycythaemia (e.g. ruddy complexion), also splenomegaly and hep- atomegaly. Complications of particular note include (1) a thrombotic tendency—​deep venous thrombosis/​pulmonary embolism, hepatic or portal venous thrombosis, venous thrombosis in unusual sites, or transient ischaemic attack/​stroke; (2) other neurological syndromes—​ a wide variety are described; (3) a haemorrhagic tendency—​due to abnormalities of platelet function; and (4)  gout—​associated with hyperuricaemia. Myelofibrosis with marrow failure develops in about half of patients with polycythaemia vera at 20 years. Diagnosis—​using the World Health Organization 2016 criteria, the major criteria are (1) haemoglobin concentration greater than

SECTION 22  Haematological disorders 5228 165 g/​litre in men or greater than 160 g/​litre in women, haemato- crit concentration greater than 49% in men or greater than 48% in women, or increased red cell mass; (2) bone marrow biopsy showing hypercellularity for age with trilineage growth (panmyelosis) including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size); and (3) presence of JAK2 (V617F) or JAK2 exon 12 mutation. Minor cri- teria are (1) trilineage myeloproliferation in the bone marrow, (2) a low serum erythropoietin level, and (3) abnormal marrow prolifera- tive capacity as manifested by the formation of erythroid colonies in the absence of exogenous erythropoietin. Diagnosis requires all three major criteria, or the first two major criteria and the minor cri- terion. Major criterion 2, bone marrow biopsy, may not be required with sustained absolute erythrocytosis: haemoglobin levels greater than 185 g/​litre in men (haematocrit 55.5%) or greater than 165 g/​ litre in women (haematocrit 49.5%) if major criterion 3 and the minor criterion are present. Treatment—​patients should be strongly advised to stop smoking. Phlebotomy should be initiated as soon as the diagnosis is es- tablished to reduce and maintain the haematocrit level at less than 45% in men and less than 42% in women. Low-​dose aspirin should be given. Myelosuppressive therapy with hydroxycarbamide (hydroxyurea) or other agents should be considered in older pa- tients intolerant of phlebotomies and in those with repeated throm- botic episodes and/​or high platelet counts. Those intolerant of hydroxycarbamide may be treated with interferon or ruxolitinib. The optimal myelosuppressive therapy for polycythaemia vera (hydroxycarbamide, pegylated interferon-​α, or ruxolitinib) remains an area of controversy. The resolution of this controversy will require the completion of carefully performed phase III trials. In lieu of such trials, it remains the physician’s choice when to initiate therapy and with which agent. Such therapy should be reserved for patients with high-​risk disease who have repeated thrombotic events in the face of strict haematocrit control. Although interferon has been reported to transiently eliminate cells with the JAK2 V16F mutation, the effects of this on the incidence of thrombotic episodes and disease evolution remain the subject of speculation. Haematopoietic stem cell trans- plantation is a potentially curative option for myelofibrosis but is not indicated in polycythaemia vera. Prognosis—​survival is about 18  months in untreated patients whereas with appropriate management, survival of over 10 years is now common. Patients previously treated with alkylating agents and/​or radioactive phosphorous have an increased long-​term risk of leukaemia. Rare causes of primary polycythaemia These include primary familial and congenital polycythaemia—​ caused by germ-​line mutations in the erythropoietin receptor gene and genes encoding components of the JAK2–​STAT pathway; may be suggested by family history (autosomal dominant). Introduction Erythropoiesis is the process responsible for maintaining a normal red blood cell mass. This is a tightly regulated process, which maintains a balance between the production and destruction of erythrocytes. Polycythaemia or erythrocytosis is a distinct group of disorders characterized by an abnormal increase in the numbers of red blood cells, leading to an elevation in the haemoglobin concen- tration and haematocrit. Absolute polycythaemias (increased red cell mass) can be attributed to either an intrinsic defect of haem- atopoietic stem cells (primary) or to the stimulation of progenitor cells by excessive levels of circulating growth factors (secondary). A pathophysiological classification of polycythaemia is provided in Box 22.3.5.1. Patients with absolute polycythaemias should be dis- tinguished from individuals in whom a minimally elevated haem- atocrit is not accompanied by a corresponding absolute increase in the red cell mass (spurious polycythaemia, stress erythrocytosis, Gaisbock’s syndrome), but rather by a contraction of plasma volume. Haematocrit levels above 49% in men and 48% in women are ab- normal and require further evaluation to determine the cause of the polycythaemia. Erythropoietin (EPO), a 34.4-​kDa glycoprotein hormone, is the primary humoral regulator of erythropoiesis. Alterations in its pro- duction are accompanied by adjustments in the rate of red cell pro- duction. Production of EPO is controlled by the relative supply of oxygen to the kidney and can increase by 1000-​fold in states of se- vere hypoxia. Under normal conditions, EPO production is medi- ated by decreased oxygen content of haemoglobin within red cells, termed hypoxaemia, which leads to decreased oxygen delivery to tissues. Box 22.3.5.1  Classification of polycythaemia Relative polycythaemias Associated with volume loss or contraction: • Gastrointestinal losses: diarrhoea, vomiting, ileostomy • Renal losses: osmotic diuresis, therapeutic diuresis, Addison’s disease, hypercalcaemia • Insensible losses: profuse sweating, fever • Stress or Gaisbock’s polycythaemia Absolute polycythaemias Primary polycythaemias: • Primary familial and congenital polycythaemia • Polycythaemia vera Secondary polycythaemias associated with appropriate secretion of EPO: • Smokers’/​hookah polycythaemia • Hypobaric hypoxia (high altitude) • Chronic pulmonary disease • Alveolar hypoventilation (Pickwickian syndrome) • Congenital heart diseases associated with right-​to-​left shunts • High-​affinity haemoglobins • 2,3-​DPG deficiency • Methaemoglobinaemias • Chuvash polycythaemia (VHL mutations) • HIF-​1α mutations • Prolyl hydroxylase mutations Secondary polycythaemias associated with inappropriate secretion of EPO: • Polycythaemia of renal disease • Tumour-​associated polycythaemia • Endocrine disorders—​phaeochromocytomas, aldosterone-​producing adenomas, Cushing’s syndrome, iatrogenic androgen administration, acromegaly

22.3.5  The polycythaemias 5229 Decreased tissue oxygenation (partial pressure of oxygen (Po2) <60 mmHg) is associated with accumulation of hypoxia-​inducible factor-​1 (HIF-​1), the major transcriptional factor responsible for activation of the EPO gene. The HIF transcriptional system is a master regulator of the hypoxic response controlling a large number of genes including phosphoglycerate kinase, glucose transporter-​1, vascular endothelial growth factor, and EPO. HIF-​1α is continu- ously synthesized irrespective of oxygen availability. It is barely de- tected in steady-​state cells because of its rapid degradation by the ubiquitin proteasome pathway. During hypoxic conditions, a rapid accumulation of HIF-​1α occurs due to a blockade of its degradation. This pathway permits a rapid response to hypoxia without activating transcriptional/​translational machinery. Increased HIF-​1α mRNA and protein levels are induced by hypoxia while protein levels rap- idly decay during normoxia. The degradation of HIF-​1α requires the von Hippel–​Lindau (VHL) protein, oxygen, and three different iron requiring proline hydroxylase (PH) enzymes. The VHL gene is a tu- mour suppressor gene, which participates in the hypoxia-​sensing pathway by binding HIF-​1α facilitating its degradation by the pro- teasome. The PH enzymes, which hydroxylate HIF, are required for HIF proteolytic degradation by promoting the interaction between HIF and VHL. As oxygen levels decrease, hydroxylation of HIF de- creases and HIF-​1α is no longer able to bind VHL and becomes stabilized. In normal individuals, VHL protein binds to hydroxyl- ated HIF-​1α and causes its ubiquitination and proteasomal degrad- ation. Genetic mutations in VHL or PH are frequently capable of leading to inherited forms of erythrocytosis by causing alterations in the binding of the VHL to HIF-​1α, leading to its accumulation and activation of hypoxia-​related genes including EPO and vascular endothelial growth factor (VEGF). EPO binds to its receptor, and ini- tiates downstream effects via the Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription (STAT) intracellular signalling pathways. Binding of EPO causes dimerization of the EPO receptor, phosphorylation of intracytoplasmic residues, and activa- tion of STAT, which shuttles from the cytoplasm into the nucleus and initiates protein transcription. JAK2–​STAT activation promotes erythroid cell division, differentiation, proliferation, and preven- tion of precursor cell apoptosis. Mutations in the EPO receptor gene leading to its constitutive activation have been observed in some pa- tients with familial and congenital forms of polycythaemia. Oxygen transport is a complex process dependent on a number of variables, including ambient oxygen levels, minute ventilation rates, lung diffusion capacity, cardiac output, red cell mass, regional blood flow, tissue capillary density, and haemoglobin–​oxygen af- finity. Acute changes in tissue oxygen demands or in environmental oxygen levels are compensated not only by increased EPO produc- tion but also increased ventilation rates, cardiac output, blood flow distribution, and haemoglobin–​oxygen affinity (through the modu- lation of 2,3-​disphosphoglycerate (2,3-​DPG) production). Sustained hypoxia is required for polycythaemia to occur as a compensatory mechanism. Relative polycythaemias These disorders are characterized by an elevated haemoglobin or haematocrit level, which occurs as the result of contraction in plasma volume. The red cell mass remains normal. There are two major groups of patients with relative forms of polycythaemia. The first includes patients with more acute conditions associated with significant degrees of dehydration, with a consequential decrease in plasma volume, for example, gastrointestinal fluid losses, thera- peutic diuresis, endocrine disorders such as Addison’s disease, and hypercalcaemia. In most cases, the consequences of volume contrac- tion are clinically obvious. The aetiology of the increase in haemato- crit does not usually present a diagnostic challenge. The second group is associated with a slight but sustained increase in the haematocrit. These patients are frequently active, middle-​aged, mildly hypertensive, obese men subjected to considerable stress who present with persistent polycythaemia. Characteristically, they ap- pear plethoric but without any of the other typical features of poly- cythaemia vera. The cause for the contraction in the plasma volume is poorly understood, but autonomic dysregulation with changes in venous capacitance may be responsible. These patients also have a normal red cell mass. The usual range of haemoglobin levels in these individuals is between 160 and 180 g/​litre with haematocrits ranging from 48 to 55%. Most of these patients seek medical evaluation for an unrelated condition, and are incidentally found to have increased haemo- globin and haematocrit values. Suitable advice regarding weight re- duction, control of hypertension, and smoking cessation is usually provided to these patients. The optimal therapy is unknown but is generally directed to correcting the patient’s underlying cardiovas- cular risk factors. Overfilling blood collection tubes can cause artefact, or pseudo­ polycythaemia, due to inadequate sample collection. Attention to this error can frequently avoid unnecessary investigation. Absolute polycythaemia Absolute polycythaemias may be classified as being primarily due to autonomous cell growth or to an enhanced response to growth factors that promotes the proliferation of developing erythroid cells, or secondary, due to excessive production of EPO in response to a variety of stimuli. Primary polycythaemia, caused by defects in haematopoietic stem cells, is accompanied, in general, by low levels of circulating EPO. Germ-​line mutations of the EPO receptor that lead to enhanced erythropoiesis cause primary familial congenital polycythaemias. Polycythaemia vera, the most common primary polycythaemia, is caused by an acquired defect in haematopoietic stem cells resulting in an excessive proliferation of myeloid cells. A mutation in the autoinhibitory domain of JAK2 tyrosine kinase (V617F) has been associated with polycythaemia vera in up to 95% of the cases; JAK2 exon 12 mutations make up the majority of the remaining cases. Most recently LNK mutations have been described. By contrast, secondary polycythaemia is generally caused by ele- vated EPO production and is not associated with mutation in the JAK2 tyrosine kinase. Elevated levels of plasma EPO can accompany systemic hypoxaemia, certain neoplasms, and disorders that impair oxygen delivery to tissues (Box 22.3.5.1). Absolute polycythaemias are accompanied by an increased red cell mass. Documentation of such an increased red cell mass may require a blood volume study with direct determination of both the red cell mass and plasma volume if available. This test is presently available in select referral centres and the need for its performance

SECTION 22  Haematological disorders 5230 is limited to special situations due to the availability of JAK2 mu- tation analysis. A haematocrit value greater than 60% in men and greater than 55% in women, however, is almost always associated with absolute erythrocytosis. In such cases, it is usually unnecessary to perform blood volume studies to be assured that the patient has an absolute polycythaemia. The presence of a JAK2 mutation with intermediate elevations of haemoglobin or haematocrit elevations is diagnostic of polycythaemia vera. Secondary polycythaemias associated with appropriate EPO secretion This group of polycythaemias encompasses a number of conditions that are ultimately the result of tissue hypoxia and subsequent exces- sive EPO production leading to erythrocytosis. These disorders are collectively regarded as hypoxic erythrocytoses. Hypobaric hypoxia At high altitudes, the barometric pressure and, consequently, the ambient oxygen tension are reduced, resulting in alveolar and ar- terial hypoxia. Natives of the Andes (South America) who live above 4200 m have been reported to have haematocrit values 30% higher than individuals who live at sea level. Acutely, changes in minute ventilation, heart rate, blood flow, and haemoglobin–​oxygen affinity occur as an individual reaches a high altitude. Serum EPO is ele- vated initially, but eventually returns to the normal range in the ab- sence of extreme hypoxia. This decline will not prevent the increase in red cell mass, which will be sustained, because early unsustained elevations of EPO promote expansion of the erythroid progenitor pool. Only very small quantities of the hormone are subsequently required to sustain the red cell mass under normal circumstances. Healthy highlanders develop pulmonary hypertension, right ven- tricular hypertrophy, and increased amounts of smooth muscle cells in distal pulmonary arterial branches, which leads to increased pulmonary vascular resistance and pulmonary artery pressures as compared to individuals who live at sea level. Due to these adap- tive changes, healthy highlanders are able to perform physical ac- tivities similar to or often even more strenuous than those living at sea level. On the other hand, many individuals living at high alti- tude do not demonstrate an increased haemoglobin concentration. A  high-​frequency missense mutation in the EGLN1 gene, which encodes prolyl hydroxylase domain 2 (PHD2), has been described in Tibetans. This mutation reduces hypoxia-​induced erythropoiesis secondary to HIFs that is otherwise the cause of erythrocytosis at altitude elevations. Chronic mountain sickness is a pathological loss of adaptation to high altitude by highlanders that occurs in native or lifelong resi- dents living above 2500 m. They suffer from headaches, fatigue, impaired exercise tolerance, cyanosis, clubbing, right heart failure, and absolute polycythaemia. These symptoms frequently resolve with descent to lower altitudes. The prevalence of chronic moun- tain sickness is higher in men than in women and increases with altitude, ageing, associated lung disease, history of smoking, and air pollution. The chronic response to high altitudes is probably deter- mined by poorly defined genetic factors, which likely contribute to the development of chronic mountain sickness. The major mech- anism underlying chronic mountain sickness is relative hypoventi- lation, since healthy highlanders characteristically hyperventilate. Chronic mountain sickness is a common problem, affecting 6 to 8% of males in La Paz (Peru), for instance. The definitive treatment of chronic mountain sickness is descent to lower altitudes or sea level. Phlebotomy and acetazolamide therapy are recommended for af- fected individuals who must remain at high altitudes. Chronic pulmonary disease Pulmonary diseases are a common cause of secondary polycy- thaemia. Defects in gas exchange result in hypoxia, with conse- quent increases in EPO and red cell mass. Not every patient with hypoxia secondary to respiratory disease develops polycythaemia; however, the presence of concurrent inflammation or infection may blunt the marrow response to hypoxia. It is important to be aware that smoking itself may also contribute significantly to the polycy- thaemia associated with chronic respiratory disease. Phlebotomy may be indicated in patients with relatively high haematocrit levels (55–​60%), given the known deleterious effects of hyperviscosity. Chronic oxygen therapy in patients with severe chronic obstructive pulmonary disease has resulted in relief of hypoxia and modest re- ductions of haematocrit levels. Smokers’/​hookah polycythaemia Smoking even in the absence of chronic lung disease is associated with secondary erythrocytosis. Increasingly recognized as well is the association with chronic hookah use. These disorders are associated with elevated carboxyhaemoglobin levels. Smoking cessation is both diagnostic and therapeutic as the erythrocytosis will resolve. Alveolar hypoventilation Hypoventilation may lead to hypoxia and an EPO-​mediated in- crease in red cell mass. These disorders include the sleep apnoea syndrome and supine hypoventilation. Up to 25% of patients with unexplained polycythaemia are subsequently found to have sleep apnoea. Common symptoms include loud snoring, breathing pauses, feelings of nonrefreshing sleep, and excessive daytime sleeping. In these conditions, significant degrees of hypoxia may occur without evident parenchymal pulmonary disease. Decreases in blood oxygen content may occur intermittently; consequently, EPO levels and arterial blood gas values may be normal. Diseases affecting the central nervous system may also impair respiratory centre function and trigger hypoventilation. These defects have been described in association with encephalitis, cerebrovascular accidents, and drug intoxication (e.g. barbiturates). Impaired skel- etal muscle function of the chest wall or diaphragm may also suffi- ciently compromise alveolar ventilation to trigger polycythaemia. In these cases, correction of hypoxia is warranted. The role of phle- botomy is unclear, but not unreasonable in patients with significant elevations in haematocrit and associated cardiovascular or cere- brovascular disease. The obesity–​hypoventilation syndrome seen in morbidly obese individuals is characterized by chronic hypoxia and hypercapnia due to alveolar hypoventilation with a resultant increase in EPO production, polycythaemia, and cor pulmonale. Effective treatment includes surgically induced weight loss, nasal continuous positive airway pressure ventilation, and, occasionally, the use of respiratory stimulants. Cardiovascular disease Cyanotic congenital heart diseases with an associated right-​to-​left shunt result in oxygen desaturation and an elevation of EPO, causing

22.3.5  The polycythaemias 5231 secondary polycythaemia. After compensatory erythrocytosis in re- sponse to oxygen desaturation occurs, serum EPO levels may return to normal levels. An extremely elevated haematocrit may be detri- mental to optimal oxygen delivery. Some children with congenital heart disease may develop extreme haematocrit values (≥80%), which leads to a significant risk of a thrombotic event, especially during periods of dehydration due to hyperviscosity and sludging within the microcirculation. The treatment of erythrocytosis in pa- tients with cyanotic congenital heart disease is controversial and should be individualized. Phlebotomy may be indicated in some instances where it has been shown to improve cerebral blood flow and neurological symptoms and to increase exercise capacity. To date, there is no consensus defining the precise target haematocrit values for therapeutic phlebotomy in the management of patients with these disorders. It is also important to note that serial phle- botomy will cause iron deficiency and subsequent microcytosis. Microcytosis may actually worsen the symptoms of hyperviscosity and a trial of iron replacement may be warranted. Carbon monoxide intoxication Chronic carbon monoxide intoxication most commonly occurs as a consequence of smoking. Elevated haematocrits have been re- ported in 3% of all smokers. Other less common causes include work-​related exposures such as those seen in caisson workers, truck drivers, or tunnel toll-​collectors. Carbon monoxide has a much higher affinity for haemoglobin than oxygen does, thereby reducing the amount of oxygen that can be bound and transported by haemo- globin. It also shifts the oxygen–​haemoglobin dissociation curve to the left, decreasing the ability of haemoglobin to release oxygen to peripheral tissues. Furthermore, carbon monoxide impairs normal compensatory mechanisms: carboxyhaemoglobin is known to de- crease 2,3-​DPG production by red cells and to reduce the affinity of haemoglobin for 2,3-​DPG. Polycythaemia due to chronic carbon monoxide intoxication may be associated with an increased risk of thromboembolic phenomena. Phlebotomy may be indicated in pa- tients with very high haematocrit values (>55–​60%). The decreased oxygen-​carrying capacity associated with carbon monoxide intoxication is not detected by standard blood gas meas- urements; therefore, a direct measure of carboxyhaemoglobin levels is required. Morning carboxyhaemoglobin levels ranging from 4 to 20% have been reported. Individuals with chronic carbon monoxide poisoning may experience neuropsychiatric and cardiac abnormal- ities. The treatment is smoking cessation or removal of the patient from the source of carbon monoxide. High-​affinity haemoglobins At least 50 haemoglobin variants exhibit increased avidity for oxygen. These mutations are inherited in an autosomal dominant manner. Oxygen transport by haemoglobin occurs as a function of the oxygen–​haemoglobin affinity curve. This function is represented by a sigmoid curve and is a reflection of the initial binding of oxygen by deoxygenated haemoglobin occurring with significant difficulty. As oxygen molecules are bound to normal haemoglobin, further binding is facilitated by structural changes that occur in the haemo- globin molecule. High-​affinity haemoglobin variants arise when mutations alter key amino acid residues in regions of haemoglobin that affect these rearrangements, or at the interface between α and β chains. Another group of mutations induces changes in oxygen affinity indirectly, by causing structural changes in haemoglobin re- gions that are critical for the binding of 2,3-​DPG. Increases in oxygen affinity result in a shift of the oxygen dissoci- ation curve to the left. Consequently, haemoglobin binds oxygen more readily and retains more oxygen at lower Po2 levels. This ultim- ately results in decreased delivery of oxygen to tissues where capil- lary Po2 is low (35–​45 mmHg). Mild tissue hypoxia then triggers an increase in the production of EPO with consequent polycythaemia. Oxygen affinity by variant haemoglobin is usually measured as the P50o2, which represents the partial oxygen pressure at which 50% of haemoglobin is saturated with oxygen. This analysis is necessary for the identification of patients with high-​affinity haemoglobins. High-​ affinity haemoglobins are associated with lower than normal values of P50o2; values below 17 mmHg are usually diagnostic of such an abnormal haemoglobin. Haemoglobin electrophoresis or high- performance liquid chromatography may, on occasion, aid in the recognition of an abnormal haemoglobin, but many high-​affinity haemoglobins display normal electrophoretic mobility or retention times. Conversely, the presence of an abnormal band per se does not provide information regarding oxygen affinity. A study of family members is important, but a negative family history does not negate the diagnosis since there is a high rate of spontaneous mutations. Most patients with high-​affinity haemoglobins have mild poly- cythaemia and are asymptomatic since the compensatory polycy- thaemia results in normal oxygen delivery to tissues. Phlebotomy therapy of such patients has been reported to be of no value and has been shown to reduce exercise tolerance. Methaemoglobinaemia Hereditary methaemoglobinaemia may be associated with a mild polycythaemia. Methaemoglobin results from the oxidation of ferrous ions (Fe2+) to the ferric state (Fe3+). Oxygen does not bind reversibly to methaemoglobin, resulting in a left shift of the oxygen dissociation curve, impaired oxygen delivery, and chronic tissue hypoxia. 2,3-​DPG deficiency This rare familial form of polycythaemia is due to a deficiency of the enzyme biphosphoglycerate mutase. Deficiency leads to a decrease in 2,3-​DPG, resulting in the increased affinity of oxygen to haemo- globin, peripheral tissue hypoxia, and hypoxic erythrocytosis. This disorder should be suspected in patients with familial polycy- thaemia with a low P50o2 in the absence of a mutant haemoglobin. Measurements of 2,3-​DPG in fresh red cell reveals reduced levels. Chuvash polycythaemia Chuvash polycythaemia is a recognized form of congenital and fa- milial polycythaemia endemic to the Chuvash population of the Russian Federation, which has also been reported to occur in a var- iety of other racial and ethnic groups. The extreme elevations of haemoglobin in this autosomal recessive disorder are accompanied by increased EPO levels. Chuvash polycythaemia is caused by germ-​ line mutations in the von Hippel–​Lindau gene, most common being the 598 C→T mutation. Stimulated EPO production is caused by the inability of the mutated von Hippel–​Lindau gene to bind to HIF-​ 1α, which escapes degradation and accumulates. These patients present with isolated erythrocytosis without elevations of white cell or platelet counts, have low blood pressure, varicose veins, and vertebral haemangiomas probably due to increased levels of VEGF.

SECTION 22  Haematological disorders 5232 Mortality, often attributable to cerebral infarction or haemorrhage, is higher than 25% by age 40. The roles of both aspirin and venesec- tion in the treatment of Chuvash polycythaemia are unclear. There is no effective therapy for reducing symptoms, complications, or mor- tality. JAK2 inhibition has been investigated as a possible therapy and is currently the subject of a clinical trial (NCT01730755). Prolyl hydroxylase mutations Mutations in the prolyl hydroxylases are inherited as an autosomal dominant condition and are associated with failure of hydroxylation of prolyl residues of HIF-​1α, increased HIF levels, and increased EPO production leading to the development of erythrocytosis. In contrast to the patients with Chuvash polycythaemia, patients with mutations of prolyl hydroxylases do not have the clinical conse- quences described previously. Secondary polycythaemias associated with the inappropriate secretion of EPO Enhanced EPO levels and secretion occur in the absence of tissue hypoxia in this group of disorders. The EPO response is therefore inappropriate to systemic oxygen requirements. Polycythaemia of renal disease As the kidney is the principal site of EPO production, it is not sur- prising that renal disorders may be associated with erythrocytosis or anaemia. Patients with hypertension and renal artery stenosis have a higher incidence of erythrocytosis than similarly hypertensive pa- tients without renal artery disease. Other benign kidney diseases associated with an increase in EPO production and erythrocytosis include polycystic kidney disease (acquired or familial) and renal cysts. Unusual patients with glomerulonephritis may also occasion- ally present with an elevated haematocrit. An uncommon cause of polycythaemia is Bartter’s syndrome, a hereditary tubular disorder characterized by hypokalaemia secondary to renal potassium loss in association with elevated plasma renin activity and aldosterone secretion. Post-renal transplant polycythaemia is defined as a per- sistently elevated haematocrit higher than 51% after renal trans- plantation without an elevation of the white blood cell or platelet count. Between 5 and 13% of patients have been reported to de- velop erythrocytosis 8 to 24 months after renal transplantation. It has been postulated that the excessive response to EPO from the donor kidney in a patient with previously low EPO levels could cause this elevation in haematocrit in these patients. Approximately 60% of patients with post-​transplant erythrocytosis experience headaches, plethora, lethargy, and dizziness and approximately 10 to 20% develop thromboembolic complications. Retention of the native kidney is essential for the development of post-​transplant erythrocytosis with the native kidney overproducing EPO leading to the development of erythrocytosis. Frequently the erythrocytosis resolves with the removal of the kidney. Angiotensin-​converting en- zyme inhibitors have proved useful in controlling post-​transplant polycythaemia, but phlebotomy may still be required in patients with haematocrit levels over 55 to 60% to rapidly decrease the risk of thrombotic complications. Tumour-​associated polycythaemia A number of tumours are associated with an inappropriately in- creased production of EPO, including benign and malignant tumours of the kidney, hepatomas, cerebellar haemangioblastomas, parathyroid adenomas, Leydig tumours, paraganglioma, men- ingioma, and phaeochromocytomas. Polycythaemia occurs in 1% of patients with renal carcinomas, 9 to 20% of patients with cere- bellar haemangioblastomas, and 10% of patients with hepatomas. Resection of the tumour, if feasible, may be associated with re- gression of the polycythaemia. Therapeutic phlebotomy is recom- mended in patients with extreme increases in the haematocrit. Two plasma cell dyscrasias associated with erythrocytosis are POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes) syndrome and TEMPI (telangiectasias, erythrocytosis with elevated EPO levels, mono- clonal gammopathy of unknown significance, perinephric fluid collections, and intrapulmonary shunting) syndrome. Treatment is directed at the underlying disease. In TEMPI syndrome, the most effective treatment has been bortezomib which suggests that the TEMPI syndrome is a consequence of the abnormal plasma cell clone. Endocrine disorders Phaeochromocytomas and aldosterone-​producing adenomas have been associated with increased levels of EPO. Mild forms of poly- cythaemia have also been observed in some patients with Cushing’s syndrome, probably related to marrow stimulation by steroid hor- mones. Recombinant human EPO has been abused by athletes and its use can lead to erythrocytosis. The recombinant form of EPO can be distinguished from native EPO by its electrophoretic mobility. Drug associated In addition to exogenous administration of EPO-​stimulating agents, androgen administration, either illicit or for hypogonadism, can be associated with erythrocytosis. Cessation of the offending agent will resolve the erythrocytosis. The development of tyrosine kinase inhibitors in cancer, particularly renal cell carcinoma, has resulted in the observation of erythrocytosis associated with sunitinib, sorafenib, and axitinib, usually with normal or high EPO levels. While the erythrocytosis associated with these agents may resolve with time, in other cases, phlebotomy may be needed if the agents require continuation. Primary polycythaemia Primary familial and congenital polycythaemia Primary familial and congenital polycythaemia is an inherited form of polycythaemia caused by mutations in the EPO receptor, thereby resulting in the hypersensitivity of erythroid progenitor cells to EPO and low serum EPO levels. Most of the disease-​causing mutations result in truncation of the cytoplasmic C-​terminal portion of the EPO receptor, which leads to constitutive activation of the receptor due to loss of its negative regulatory domain. In the autosomal dom- inant form of the disease, family members have plethora, headaches, dizziness, nosebleeds, and exertional dyspnoea. These symptoms resolve with phlebotomy and reduction of the haematocrit. Unlike those with polycythaemia vera, primary familial and congenital polycythaemia patients have normal platelet counts, are JAK2 V617F negative, lack splenomegaly, and do not progress to acute leukaemia. Not all cases of primary familial and congenital polycythaemia can be attributed to the mutations of the EPO receptor (which occur in

22.3.5  The polycythaemias 5233 c.10–​20% of cases), suggesting that other genetic defects can lead to a similar phenotype. An up-​to-​date database with primary and sec- ondary causes of congenital erythrocytosis can be found at http://​ www.erythrocytosis.org as well as information regarding reference laboratories that perform mutational analyses, which can be used to establish these diagnoses. Polycythaemia vera Polycythaemia vera is a clonal, chronic progressive haematological malignancy characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic elements in the bone marrow. The other myeloproliferative neoplasms include essential thrombocyth- aemia and primary myelofibrosis. The hallmark of polycythaemia vera is an absolute form of erythrocytosis usually associated with leucocytosis, thrombocytosis, splenomegaly, hypersensitivity of haematopoietic progenitor cells, and the JAK2 V617F mutation. This mutation accounts for the cytokine hypersensitivity, which charac- terizes haematopoietic progenitors from patients with this disorder. The use of recently developed molecular tools to detect JAK2 V617F has already revolutionized the diagnosis of polycythaemia vera (Table 22.3.5.1). This mutation is a recognized molecular target for therapy. In contrast to those with other haematological malig- nancies, patients suffering from polycythaemia vera may enjoy pro- longed survival, provided that the excessive production of red cells and platelets is controlled. The development of myelofibrosis and/​or acute leukaemia occurs occasionally. Epidemiology Polycythaemia vera was thought to be a rare disorder, with an es- timated yearly incidence in the Western world between 5 and 17 cases per million population. Recent data suggest that its preva- lence might be higher than previously expected with rates of at least 300 cases per million population being reported. The very high association between JAK2 V617F and polycythaemia vera will probably have an impact on future studies of the incidence and prevalence of this disease, and could change our knowledge of the average age of diagnosis. A very low incidence of two cases per year per million population has been reported in Japan. These differences suggest that environmental as well as genetic factors might be important. Polycythaemia vera is slightly more common in men than in women, with a male/​female ratio of 1.2:1. The average age at diagnosis is 60 years; it is very rare in individuals younger than 30 years of age. Only a handful of cases have been reported during childhood. Biological and molecular aspects The exaggerated production of red cells, granulocytes, and platelets in polycythaemia vera suggests that the fundamental defect occurs at the level of the pluripotent haematopoietic stem cell. The clonal, and thereby malignant, nature of polycythaemia vera was first es- tablished by the cellular analysis of blood cell production in African American women heterozygous for X-​linked glucose-​6-​phosphate dehydrogenase isoenzymes. These results have been confirmed using restriction fragment length polymorphisms of the active X chromosomes. In patients with polycythaemia vera, EPO concentrations often fall below the levels observed in normal individuals. These low levels persist even after repeated phlebotomies, suggesting that excessive production of EPO is not a critical component in the pathogenesis of this disorder. Using in vitro cell-​culture systems, polycythaemia vera bone marrow cells can form erythroid col- onies in the absence of EPO and are hypersensitive to EPO (en- dogenous colony formation). Polycythaemia vera progenitor cells are also hypersensitive to other cytokines such as stem-​cell factor, interleukin-​3, and granulocyte–​macrophage colony-​stimulating factor. The presence of JAK2 V617F mutations can now explain both the hypersensitivity to EPO and to multiple other growth factors which use the JAK2–​STAT5 signalling pathway. A valine to phenylalanine substitution occurs within exon 14 of the JH2 domain of the JAK2 tyrosine kinase gene. In the normal kinase, this domain has an inhibitory effect over the catalytic domain, JH1. A mutation in the JH2 domain disrupts this autoinhibitory effect and renders the kinase constitutively active, leading to a constant downstream phosphorylation and substrate activation. STATs are intracytoplasmic molecules that initiate gene tran- scription. One of the target genes of STATs is BCL2L1, which ex- presses an antiapoptotic protein, overexpressed in polycythaemia vera and believed to play an important role in increased survival of erythroid precursors and megakaryocytes. Overexpression of JAK2 V617F in cell lines is associated with increased cellular pro- liferation and cytokine hypersensitivity and its transfection into marrow cells which are then transplanted into mice is associated with the development of a clinical phenotype similar to polycy- thaemia vera. Progression of the disease has been also shown to Table 22.3.5.1  The 2016 WHO diagnostic criteria for polycythaemia vera Major criteria 1 Evidence of elevated red cell mass: Hgb >165 g/​litre (men), >160 g/​litre (women), or Hct >49% in men, >48% in women, or Increased red cell mass (>25% above mean normal predicted value) 2 Bone marrow biopsy showing hypercellularity for age with trilineage growth (panmyelosis) including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size) 3 Presence of JAK2 V617F or JAK2 exon 12 mutation Minor criterion 1 Subnormal serum erythropoietin level Hct, haematocrit; Hgb, haemoglobin. The diagnosis of polycythaemia vera is made in the presence of all three major criteria or the first two major criteria and the minor criterion. Bone marrow biopsy many not be required in cases with sustained haemoglobin levels >185 g/​litre in men (haematocrit 55.5%) or 165 g/​litre in women (haematocrit 49.5%) if major criterion 3 and the minor criterion are present. Source data from Arber DA, et al. (2016). The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Blood, 127, 2391–​405.

SECTION 22  Haematological disorders 5234 correlate with the level of allele chimerism. Retrospective analyses have revealed that patients with a low burden of the mutated al- lele can evolve over time to a higher burden of the mutated allele. Loss of heterozygosity on the short arm of chromosome 9 (the location of the JAK2 gene) is a consequence not of gene deletion but rather uniparental disomy or mitotic recombination. Even in patients with a low burden of JAK2 V617F, erythroid progenitors that are homozygous for JAK2 V6717F are usually present. This finding is characteristic of polycythemia vera but occurs less fre- quently in primary myelofibrosis and rarely in essential thrombo- cythaemia. The burden of JAK2 V617F is correlated with disease progression and the development of complications in polycy- thaemia vera patients. Additional mutations of JAK2 that are as- sociated with erythrocytosis have also been identified. Somatic gain-​of-​function mutations involving exon 12 rather than exon 14 have been identified in patients with isolated erythrocytosis and low serum EPO levels. All erythroid colonies cloned from the haematopoietic cells of such patients are heterozygous for this mutation. JAK2 exon 12 mutations could therefore identify a distinctive myeloproliferative disorder that affects patients who currently carry a diagnosis of idiopathic erythrocytosis. Finally, mutations in LNK, primarily in exon 2, have been described in patients with polycythaemia vera and other myeloproliferative neoplasms lacking JAK2 mutations. LNK, or lymphocyte-​specific adaptor protein, functions to inhibit wild-​type and mutant JAK2 phosphorylation. The exact mechanism by which mutations in LNK result in polycythaemia vera is under investigation. Pathobiology Patients with polycythaemia vera have an increased thrombotic tendency resulting from the expansion of the red cell mass which represents the main cause of mortality in these patients. There is a direct relationship between the risk of thrombosis and age, with the incidence of cardiovascular complications being higher in patients over 65 years of age. Younger individuals are also at risk for throm- botic episodes, many of them life-​threatening, such as Budd–​Chiari syndrome, cerebrovascular thrombosis, cerebral sinus thrombosis, acute myocardial infarction, and pulmonary embolism. The main rheological abnormality is elevation of the total blood viscosity. Cerebral blood flow is reduced in patients with polycythaemia vera and a haematocrit of 53 to 62%. Reductions in blood flow are cor- rectable by phlebotomy. Even small reductions in the haematocrit result in significant reductions in blood viscosity and increased cerebral blood flow, thereby reducing the likelihood of thrombus development. Thrombocytosis and functional platelet abnormalities and increased white blood cell count (found in 50% of the patients) are frequently present, and may play a role in the development of thrombosis. Patients with polycythaemia vera are also at an increased risk of developing life-​threatening haemorrhagic complications. Abnormalities in platelet function and number have been impli- cated. Qualitative platelet abnormalities include defective platelet aggregation in vitro, acquired storage pool disease, and dysregulated thromboxane A2 metabolism. Acquired von Willebrand’s disease has been described in patients who have very high platelet counts (>1000 × 109/​litre), in association with life-​threatening bleeding episodes. No laboratory test has proven useful for the a priori identification of patients at an increased risk of developing haemor- rhagic or thrombotic events. The progression to a post-​polycythaemic-​related myelofibrosis phase of the disease is a common cause of morbidity. This stage is characterized by cytopenias, marrow fibrosis, and extramedullary haematopoiesis. There are conflicting data on the incidence of this complication. Some studies reported a very low incidence after 10 to 20 years; others reported that up to 25 to 50% of patients with polycy- themia vera may develop polycythemia vera-​related myelofibrosis. The fibroblastic component represents a reactive event, and may be due to the local release of growth factors, particularly transforming growth factor-​β, by haematopoietic cells. The association between the treatment modality and the development of myelofibrosis is as yet unclear. There is, however, an established association between the treatment type (alkylating agents and radioactive phosphorus (32P)), and the development of acute leukaemia. It must be empha- sized, however, that even those patients treated with phlebotomy alone have a leukaemogenic risk significantly higher than that ex- pected in the general population. Clinical manifestations The clinical manifestations of polycythaemia vera are the direct consequence of the excessive production of cellular elements of the various haematopoietic cell lineages. The routine and widespread use of laboratory screening tests during medical evaluations has led to an increased detection of asymptomatic patients. In contrast, symptomatic patients may present to their physician with a large array of nonspecific com- plaints including headache, weakness, pruritus, dizziness, exces- sive sweating, visual disturbances, paraesthesias, joint symptoms, and epigastric distress. Approximately one-​third of patients will have lost 10% of their body weight by the time they come to med- ical attention, presumably due to the associated hypermetabolism. Joint disease is usually the manifestation of gout, due to the in- creased production of uric acid. The most important signs on phys- ical examination include ruddy cyanosis, conjunctival plethora, splenomegaly, hepatomegaly, and hypertension. Patients left without appropriate treatment are at a particu- larly high risk of developing thrombotic or haemorrhagic events. In fact, thrombosis may be the cause of death in up to 30 to 40% of patients. Thrombosis may occur in the deep venous system of the lower extremities, or present as a pulmonary embolism. Cerebrovascular, coronary, and peripheral vascular occlusions are not rare. Thromboses at unusual sites are also characteristic of poly- cythaemia vera. They include occlusion of the splenic, portal, hep- atic, and mesenteric veins. Cardiac valve abnormalities affecting the aortic or the mitral valves are commonly seen, frequently in the form of leaflet thickening or frank vegetations. These lesions are as- sociated with the occurrence of arterial thromboembolism. Hepatic venous or inferior vena caval thrombosis is known as Budd–​Chiari syndrome and is characterized by hepatosplenomegaly, ascites, oe- dema of the peripheral extremities, jaundice, abdominal pain, and distension of superficial abdominal veins as a result of portal hyper- tension. The prevalence of myeloproliferative neoplasms in patients with splanchnic vein thrombosis was estimated to be as high as 49% for hepatic vein thrombosis and 23% for portal vein throm- bosis. Often these patients will present with normal haemoglobin

22.3.5  The polycythaemias 5235 and haematocrit levels. This phenomenon is regarded as ‘inapparent polycythaemia vera’ and requires a full evaluation for the presence of myeloproliferative neoplasms. Detection of the JAK2 V617F muta- tion will help identify and appropriately treat these patients earlier. Up to 34% of patients with portal vein thrombosis and up to 58% of patients with Budd–​Chiari syndrome may have a myeloproliferative neoplasm, identified by the presence of JAK2 V617F. Iron defi- ciency may also mask the expected erythrocytosis in some patients with polycythaemia vera. ‘Masked’ polycythaemia vera may exist even in those without Budd–​Chiari syndrome or iron deficiency and describes those patients with erythrocytosis who do not meet the cut-​offs set forward by diagnostic criteria but who have bone marrow biopsies consistent with polycythaemia vera. They often have thrombocytosis, and discriminating these patients from essen- tial thrombocythemia is important as the thrombotic risks differ be- tween these two diagnoses. The 2016 WHO diagnostic criteria have set lower thresholds for haemoglobin and haematocrit compared to the 2008 WHO diagnostic criteria, which should reduce the number of patients that fall into this category. Leucocytosis, thrombocytosis, and splenomegaly are usually present. Neurological abnormalities occur in up to 60 to 80% of patients. They include transient ischaemic attacks, cerebral infarction, cere- bral haemorrhage, confusional states, fluctuating dementia, and in- voluntary movement syndromes. Dizziness, paraesthesiae, tinnitus, visual problems, and headaches are common symptoms attributed to the hyperviscosity state. Small infarcts in the basal ganglia region, also known as lacunae, may explain some of the transient neuro- logical manifestations. Symptoms of carotid, vertebral, or basilar ar- tery insufficiency occur frequently. Peripheral vascular insufficiency may be manifested by intense redness or cyanosis of the digits, burning, classical erythromelalgia, digital ischaemia with palpable pulses, or thrombophlebitis. Erythromelalgia consists of a burning pain in the digits of either the lower and/​or upper extremities, an ob- jective sensation of increased temperature, and relief by cooling. If left untreated it may evolve into gangrene. Antiplatelet aggregation therapy rapidly reverses the symptoms. Peripheral pulses are usually normal in these patients, as this phenomenon is due to changes in the microcirculation related to arteriolar activation and aggregation of platelets in vivo. Haemorrhagic complications are the cause of death in 2 to 10% of patients with polycythaemia vera; 30 to 40% of patients will ex- perience a haemorrhagic event sometime during the course of their disease. Peptic ulcer disease occurs frequently and contributes to the gastrointestinal tract being the most common source of bleeding. Oesophageal varices are another common site of bleeding in patients with intra-​abdominal thromboses. The bleeding diathesis may relate to abnormalities in platelet function, and thus occurs frequently after the ingestion of anti-​inflammatory agents. Spontaneous bleeding is rare. Recent data suggest that low-​dose aspirin might not increase the frequency of life-​threatening haemorrhages. Generalized pruritus affects 50% of all patients, but its aetiology is unknown. Increased blood and urine histamine levels have been implicated. Pruritus triggered by water contact is characteristic, and very poorly tolerated. There is no relationship between the severity of the disease and the intensity of the pruritus. Up to 20% of pa- tients experience persistent pruritus even after normalization of their counts. The risk of postoperative complications is high in patients with polycythaemia vera. Bleeding, thrombosis, or a combination of both can occur. The risk is higher for those patients who undergo surgery with uncontrolled erythrocytosis. Inadequately controlled disease may be associated with almost an 80% risk of complications. The duration of controlled blood counts is also important: the longer this duration is, the less the risk of complications (as low as 5%). Polycythaemia vera evolves to polycythaemia vera-​related myelofibrosis in up to 50% of the patients 10 to 20 years after the initial diagnosis. It is characterized by increased splenomegaly, tear- drop red cells, a leucoerythroblastic blood picture, marrow fibrosis, and a normal or decreasing red cell mass. Fatigue, dizziness, weight loss, anorexia, progressive anaemia, and thrombocytopenia associ- ated with bleeding are common. Patients with progressive anaemia should be evaluated for folate and iron deficiency. Occasional pa- tients will respond to iron supplementation with resurgence of erythropoiesis. Severe hyperuricaemia may induce gout or uric acid nephropathy. Polycythemia vera-​related myelofibrosis portends a grave prognosis, with over two-​thirds of patients dying within 3 years. In the appropriate setting, strong consideration should be given to allogeneic stem cell transplantation, which offers an oppor- tunity for cure. The evolution to acute leukaemia is probably the natural con- sequence of the malignant nature of polycythaemia vera, which can be accentuated by therapeutic interventions commonly used for its treatment, such as alkylating agent or 32P. In a recent study, older age and prior exposure to 32P and busulfan, but not hydroxycarbamide therapy, was associated with an increased risk of leukaemia. Between 30 and 50% of patients who develop leu- kaemia have previously developed myelofibrosis whereas 50% progress directly from the erythrocytotic phase. A  significant number of patients experience a myelodysplastic interval before transforming to acute leukaemia. Patients should be treated with either decitabine or standard acute myeloid leukaemia induction in preparation for taking these patients rapidly to allogenic stem cell transplantation. Laboratory evaluation Laboratory evaluation of patents with erythrocytosis involves the careful use of a battery of diagnostic tests. The diagnosis of poly- cythaemia vera has been dramatically simplified with the advent of the molecular tests for the JAK2 V617F mutation. The use of analyses for the JAK2 V617F and JAK2 exon 12 mutations has proven particularly useful in diagnostically challenging cases and in patients with inapparent erythrocytosis and a serious thrombotic event occurring especially at a younger age. Allele-​specific poly- merase chain reaction methods can be used to detect JAK2 V617F in at least 95% of patients with polycythemia vera. Those pa- tients who fulfil clinical criteria for polycythemia vera but are JAK2 V617F negative should be evaluated for JAK2 exon 12 mu- tations. Approximately 10% of such patients who are negative for JAK2 V617F harbour an exon 12 JAK2 mutation. Rare mutations in calreticulin (CALR) and LNK have been described. Quantitation of the red cell mass to document absolute erythrocytosis remains a useful diagnostic test in characterizing patients without JAK2 mutations or erythrocytosis, though its availability is limited. Approximately two-​thirds of the patients present with leucocytosis

SECTION 22  Haematological disorders 5236 and approximately 50% have thrombocytosis. Red cell morph- ology usually reflects an underlying iron-​deficiency state pre- sent in the great majority of patients: microcytosis, hypochromia, polychromatophilia, poikilocytosis, and anisocytosis are frequently seen. White blood cell morphology is usually normal. Increased numbers of basophils, eosinophils, and immature myeloid cells are observed. Megathrombocytes are often seen in the peripheral blood smear. Platelet counts are usually less than 1000 × 109/​litre, but higher counts may be seen. The progression to polycythaemia vera-​related myelofibrosis is characterized by the appearance of a leucoerythroblastic blood picture with the presence in the periph- eral blood of teardrop red cells (dacrocytes), immature myeloid cells, megathrombocytes, and nucleated red blood cells. Bleeding time and platelet aggregation studies are frequently, but not always, abnormal. Prolongation of prothrombin and partial thrombo- plastin times are frequently encountered, usually reflecting a la- boratory artefact due to erythrocytosis (the volume of plasma in the collection tube might be too small relative to the amount of citrate anticoagulant present in these tubes). At diagnosis, serum EPO levels are either reduced or within the lower limits of normal. Low levels persist in two-​thirds of patients after normalization of the haematocrit. In patients with extreme thrombocytosis, acquired von Willebrand’s disease occurs, characterized by a significant decrease in large von Willebrand’s factor multimers due to their adsorption to platelets and megakaryocytes. This acquired defect occurs mainly in patients with very high platelet counts (>1000 × 109/​litre) and resembles type 2 von Willebrand’s disease. The defect is corrected by normalization of the thrombocytosis. Elevations in leucocyte alkaline phosphatase (70%), serum vitamin B12 levels (40%), and serum vitamin B12 binding pro- teins (70%) are common, as are hyperuricaemia and increased hista- mine levels. Bone marrow examination reveals a hypercellular marrow with trilineage growth (panmyelosis) with prominent erythroid, granulo- cytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes. Iron stores are usually absent prior to treatment. Reticulin is often seen, but is not predictive of evolution into the myelofibrotic phase. Cytogenetic abnormalities have been observed in 25% of patients, but none is characteristic. A recent study using fluorescence in situ hybridization analyses has shown that abnor- malities involving chromosome 9 rearrangements are common, being present in up to 53% of patients with polycythaemia vera. A gain in 9p is the most frequent genomic abnormality in polycy- thaemia vera. JAK2 is located on 9p and the duplication of 9p in polycythaemia vera is thought to be the consequence of homolo- gous recombination. Other chromosomal abnormalities involving chromosomes 1, 5, 7, 8, 12, and 13 have been associated with disease progression. Diagnostic criteria for polycythaemia vera The diagnosis of polycythaemia vera has been greatly simplified by JAK2 molecular testing. The 2016 World Health Organization (WHO) diagnostic criteria for polycythaemia vera require the presence of all three major criteria, (an increased haemoglobin, haematocrit, or red cell mass; presence of a JAK2 mutation; and consistent bone marrow biopsy findings) or the first two major cri- teria plus a subnormal serum EPO level (Table 22.3.5.1). It is im- portant to note that a bone marrow biopsy remains an important procedure except in cases with a JAK2 mutation and subnormal EPO level as well as significant erythrocytosis (haemoglobin levels

185 g/​litre in men (haematocrit 55.5%) or 165 g/​litre in women (haematocrit 49.5%)). Approach to the patient with polycythaemia It is wise to avoid the temptation of diagnosing polycythaemia on the basis of a single blood count unless extremely high haematocrit levels are observed. A rational diagnostic approach is required to avoid unnecessary emotional distress to the patient as well as ex- pensive and unnecessary evaluations (Fig. 22.3.5.1). Dehydration from any cause can produce a spurious elevation in the blood counts. Heavy smokers with mild polycythaemia should be asked to stop smoking and their counts repeated after a few weeks. Once a genuine elevation of haemoglobin or haematocrit has been established, the next step is to decide whether this represents an ab- solute increase in total red cell mass, or merely a relative phenom- enon. A blood volume study with direct quantitation of both red cell mass and plasma volume can be helpful in making this distinction if available. In patients with extreme degrees of erythrocytosis (haem- atocrit >55% in men and >49.5% in women) one can be assured that the red cell mass is elevated. If absolute polycythaemia is confirmed, it is essential to elucidate whether it is the consequence of a primary myeloproliferative disorder such as polycythaemia vera or a sec- ondary condition. The determination of EPO levels may be useful in differentiating between polycythaemia vera and secondary polycythaemia. An ele- vated serum EPO level is indicative of the presence of a secondary polycythaemia and a low level supports the diagnosis of polycy- thaemia vera, but a normal EPO value does not exclude hypoxia-​ induced causes of erythrocytosis or the autonomous production of EPO leading to erythrocytosis. Normal values may also be encoun- tered in some cases of polycythaemia vera. The presence of leucocytosis, thrombocytosis, or splenomegaly is suggestive of polycythaemia vera as the cause for the elevated red cell mass. Arterial blood gases and the direct determination of oxygen saturation in arterial blood, if decreased, may aid in the recognition of a chronic pulmonary or congenital cardiovas- cular abnormality. If blood oxygen saturation is normal, the quan- tification of haemoglobin’s oxygen affinity (P50o2) may indicate the presence of high-​affinity haemoglobin variant. Otherwise, causes for a physiologically inappropriate polycythaemia should be sought. Molecular methods to detect JAK2 V617F and exon 12 JAK2 mutations provide diagnostic tools for the evaluation of patients suspected of having polycythaemia vera. JAK2 mutation analysis provides a direct means of identifying the overwhelming number of patients with polycythemia vera. There is a small but definite group of patients in whom a specific cause for polycythaemia remains elusive, despite appropriate diag- nostic testing. Examining close relatives might disclose the presence of a familial form of polycythaemia, a rare condition caused by an abnormality in EPO receptor or defects in hypoxia sensing (Chuvash polycythemia). Regular, continued surveillance is recommended for all noncategorized patients, as some of them develop polycythaemia vera in the future.

22.3.5  The polycythaemias 5237 Management of polycythaemia vera The two main goals in the management of patients with polycy- thaemia vera involve the confirmation of the diagnosis and reduc- tion of the red cell mass. The untoward effects of an increased red cell mass on tissue blood flow occur independently from the spe- cific cause of the polycythaemia. It is thus reasonable to recom- mend that all patients with uncorrectable erythrocytosis be offered phlebotomy. The main therapeutic goals are the maintenance of well-​being and the prevention of complications for as long as possible. Several therapeutic strategies have resulted in dramatic increases in the survival of patients. Historical evidence suggests a median survival of approximately 18  months in untreated patients with polycy- thaemia vera whereas with appropriate management, survival of over 10 years is now common. The main therapeutic objective is the reduction of the haematocrit to a normal level. This is usually ac- complished by the implementation of repeated phlebotomies. Every possible effort should be made to discourage patients with polycy- thaemia vera from smoking. A regimen of phlebotomies should be prescribed as soon as the diagnosis has been clearly established. It is often feasible to remove between 350 and 500 ml of blood every other day until the desired haematocrit level is attained. The re- moval of smaller aliquots might be necessary in older patients. In the landmark Cytoreductive Therapy in Polycythemia Vera (CYTO-​ PV) trial, stringent control of the haematocrit at less than 45% versus more permissive control of 45 to 50% was associated with a decreased risk of thrombosis, making lower than 45% the standard of care. Many haematologists still target 42% for women, though this is not based on prospective data. Once the target haematocrit level is achieved, a maintenance regimen should be instituted. Venesection is preferred in those younger individuals without critical elevations in their platelet counts. Myelosuppressive therapy should be considered in elderly patients who are intolerant of phlebotomies, and in younger individ- uals with repeated thrombotic episodes and extremely high platelet counts. There is controversy regarding what represents the optimal myelosuppressive agent. A  major concern has been the possible ­association between exposure to some of these agents and the devel- opment of leukaemia. Hydroxycarbamide is useful for the management of patients with polycythaemia vera and represents the first-​line therapy espe- cially in older patients in whom phlebotomy alone is insufficient or intolerable, due to its minimal leukaemogenic potential. It should, however, be used with great caution in patients formerly treated with radioactive 32P or alkylating agents as the risk of leukaemia is higher. Low-​dose aspirin (81–​100 mg/​day) administered to patients with polycythaemia vera has been shown to decrease the risk of ar- terial and venous events. The European Collaboration on Low dose Aspirin for Polycythaemia Vera (ECLAP) study was a randomized study comparing low-​dose aspirin with placebo in reducing throm- bosis in polycythaemia vera. A clinically significant reduction in rate of thrombosis was seen in favour of low-​dose aspirin compared to Peripheral blood mutation screening for JAK2 V16F and serum erythropoietin measurement; send JAK2 exon 12 if JAK2 V16F is negative JAK2 mutation (+) AND serum EPO level ↓ Hgb 16.5–18.5 g/dL in men, 16– 16.5 g/dL in women OR Hct 49–55.5% in men, 48–49.5% in women Bone marrow biopsy required for definitive PV diagnosis Diagnostic for PV: Bone marrow boipsy not required for diagnosis Bone marrow biopsy required for definitive PV diagnosis If bone marrow not diagnostic, consider congenital polycythaemia with EPOR mutation Consider secondary polycythemia including congenital polycythaemia with VHL mutation Hgb >18.5 g/dL in men, >16.5 g/dL in women OR Hct >55.5% in men, >49.5% in women PV likely PV unlikely Bone marrow biopsy required for definitive PV diagnosis PV possible JAK2 mutation (+) AND serum EPO level normal/↑ JAK2 mutation (–) AND serum EPO level ↓ JAK2 mutation (–) AND serum EPO level ↑ Fig. 22.3.5.1  Diagnostic algorithm for patients with erythrocytosis. EPO, erythropoietin; EPOR, erythropoietin receptor;
Hct, haematocrit; Hgb, haemoglobin; PV, polycythaemia vera; VHL, von Hippel–​Lindau.

SECTION 22  Haematological disorders 5238 placebo. Although there is no overall survival benefit, no significant increased risk of bleeding was reported. In younger patients, given their potential long-​term survival, strong consideration should be given to the use of phlebotomy therapy in combination with low-​dose aspirin, as well as with other apparently nonleukaemogenic interventions such as interferon-​α and anagrelide. In two recent studies, one in polycythaemia vera patients and another in essential thrombocythaemia patients, hydroxyurea has been shown to be nonleukaemogenic, and can be used as an alternative to phlebotomy or in combination with it. Interferon-​α2a and pegylated forms of interferon have been shown to be effective in controlling the blood counts and symp- toms (especially pruritus). The use of pegylated forms of inter- feron has been reported to be associated with a reduction of the percentage of cells bearing the JAK2 V617F mutation, suggesting its activity at the level of an early haematopoietic stem cell/​pro- genitor cell. In patients with a history of thrombosis where un- controlled thrombocytosis is a problem, anagrelide, an inhibitor of megakaryocytic maturation, has proven effective. Ruxolitinib has been evaluated in the phase III RESPONSE trial, which compared best available therapy to ruxolitinib, with primary co-​endpoints of haematocrit control (<45%) and reduction in spleen volume of at least 35% by week 32. Best available therapy included thera- peutic phlebotomy as well as various pharmacological interven- tions including hydroxycarbamide at tolerable doses, interferon-​α, immunomodulators, and anagrelide. In total, 20.9% of patients as- signed to ruxolitinib treatment achieved the composite primary endpoint (38.2% for spleen volume reduction; 60% for haemato- crit control) compared to 0.9% of patients assigned to best avail- able therapy (0.9% for spleen volume reduction and 19.6% for haematocrit control) (P <0.001). Ruxolitinib therapy resulted in a significantly increased proportion of patients with reduction in symptom scores, measured by several validated tools including the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-​ SAF). It is unclear what impact ruxolitinib will have on thrombotic risk and transformation to myelofibrosis or acute leukaemia, which should become clearer with long-​term follow-​up and population-​ based studies. Refractory aquagenic pruritus can be managed in the majority of cases with ruxolitinib. At present, oral melphalan should be used only in patients who are incapable of complying with the other forms of therapy or are unwilling or unable to return for follow-​up. Elective surgery should only be undertaken after adequate and sustained control of the blood counts has been achieved. When emergency surgery is required, the patient should be phlebotom- ized rapidly until a normal haematocrit is achieved, and platelets should be available in case excessive operative bleeding occurs. Patients should be mobilized promptly, and the use of prophy- lactic doses of low molecular weight heparin should be considered unless contraindicated. Dental extractions are associated with an increased bleeding risk and should only be pursued in patients with good haematological control. A particularly high-​risk inter- vention is splenectomy, which has an operative mortality rate of approximately 9%. The haematocrit should be normalized and platelet count maintained below 400 × 109/​litre before splen- ectomy. After splenectomy there is a particular risk of extreme thrombocytosis. Although no prospective studies have been done, prophylactic low molecular weight heparin is probably warranted in all patients undergoing splenectomy during the perioperative period. Due to the high probability of expanding extramedullary haematopoiesis with rapid development of liver enlargement after splenectomy, patients should receive hydroxycarbamide therapy postoperatively. In patients with polycythaemia-​related myelofibrosis, the management is quite similar to that for primary myelofibrosis. Allogeneic stem cell transplantation is now a curative option for patients with myelofibrosis, both primary and secondary to other myeloproliferative neoplasms, and should be considered in appro- priately selected patients. Pregnant patients with polycythaemia vera experience an in- creased incidence of fetal loss, with 30% of pregnancies culminating in spontaneous abortions. Unexpectedly, pregnancy in patients with polycythaemia vera is frequently associated with a gradual normal- ization of blood values, and it is not unusual for a woman who has required extensive therapy for control of her disease to no longer require phlebotomies during pregnancy. The European Leukaemia Net (ELN) has published recommendations about the management of pregnant patients with myeloproliferative neoplasms, which in- cludes risk stratification and treatment considerations. Based on available data, the ELN classifies high-​risk pregnant patients with essential thrombocythaemia/​polycythaemia vera as those with his- tory of thrombosis or myeloproliferative neoplasm-​related haem- orrhage as well as those with previous pregnancy complications, such as intrauterine growth restriction, stillbirth or intrauterine death, peripartum haemorrhage, severe pre-​eclampsia, multiple first-​trimester miscarriages, placental abruption, and platelet counts over 1500 × 109/​litre. Both low-​ and high-​risk essential thrombocythaemia/​polycythaemia vera pregnant patients should be treated with therapeutic phlebotomy to maintain the haemato- crit at less than 45% or midgestation-​specific range, low-​dose as- pirin, and provided with low molecular weight heparin for 6 weeks postpartum. For women with previous thrombosis or pregnancy-​ related complications, low molecular weight heparin should be extended throughout the pregnancy unless there were previous bleeding issues. Additionally, interferon-​α has been considered the most appropriate cytoreductive agent during pregnancy for women with a history of thrombosis. The role of ruxolitinib in pregnancy is not defined. Prognosis The outcome of patients with secondary polycythaemia is usually re- lated to the prognosis of the underlying disorder. In polycythaemia vera, the nature and severity of the complications during the clinical course of the disease are the most important determinants of out- come. Disease duration is also important, as long-​term survival is strongly associated with progression to myelofibrosis or acute leu- kaemia. As previously emphasized, prompt and appropriate therapy results in dramatic improvements in survival. Young patients should be initially managed with phlebotomy and low doses of aspirin. Supplemental therapy with interferon, anagrelide, or hydroxyurea might be required in patients with serious haemorrhagic or throm- botic episodes. The use of either hydroxycarbamide or melphalan appears warranted in the treatment of elderly patients who, because of their age, have a limited survival.