Section 19

Section Editor: Sudhir Mehta Hematology and Oncology

226. Hemolytic Anemias: Diagnostic Approach 231. Immunotherapy in Clinical Practice Shubha Laxmi Margekar, Ashok Kumar, Purnima Margekar, Vineet Talwar, Amrith BP Venu Gopal Margekar 232. PNH—A Great Masquerader: When to Suspect? 227. Thalassemia in Adults—Management Issues Asish Rath, Jasmita Ramesh Aggarwal, Anupam Prakash 233. JAK2 Mutation—What Physicians Should Know? 228. Approach to Thrombocytopenia Tulika Seth Avinash Kumar Singh, Divya Krishna, Gautam Kumar Rakesh, Bhupendra Kumar 234. Approach to a Case of Polycythemia: More Blood May Be Bad…. 229. Management of CML in Resource-limited Settings Tuphan Kanti Dolai, Prakash Singh Shekhawat, Malini Garg Hemant Malhotra, Naveen Gupta, Ajay Yadav 230. Disability-free Hemophilia in India— Myth or Reality Sunita Aggarwal, Ranvijay Singh, Sandeep Garg

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Hemolytic Anemias: 226 Diagnostic Approach

Shubha Laxmi Margekar, Ashok Kumar, Purnima Margekar, Venu Gopal Margekar

Abstract Hemolytic anemias are an important type of anemia in which destruction of RBCs occurs. Manifestations occur in the form of development of anemia along with sign and symptoms of hemolysis. Usually the diagnosis is made by good clinical history relating the cause of anemia and evidence of hemolysis in laboratory evaluation. Therefore, a general approach for making etiological diagnosis of the hemolytic anemia is always needed for classification and management of anemia. In this chapter an effort has been made to provide a relevant approach for the evaluation of hemolytic anemia.

Introduction Causes of anemia that can be treated (anemia due to nutritional deficiencies, gastrointestinal bleed, anemia Anemia is defined as decrease in hemoglobin (Hb) level, of renal origin, and hemolysis) should be looked for according to reference ranges specific for age, gender, and carefully, so that it is not missed. race. For men Hb <13 g/dL and for women Hb <12 g/dL is considered as anemia as per World Health Organization (WHO). Classification The mean life span of Red Blood Cells (RBCs) is 120 „„ On the basis of etiology, hemolytic anemia can be days. Taking a good history followed by proper clinical classified as: examination along with laboratory investigations is still —— Inherited or considered as the best approach for any case of anemia. —— Acquired. In hemolytic anemias, there is premature destruction „„ According to the site of hemolysis (Table 1), it is of RBC, that is, in less than 120 days (which defines the divided into: hemolytic disorder) which in turn to compensate, there —— Intravascular, where RBCs destruction is in the is increase in the production capacity of RBCs by bone circulation or marrow. When the rate of destruction of red blood cell —— Extravascular, where destruction occurs in the exceeds the capacity of producing more red cells by bone spleen or liver (within macrophages). marrow, the hemolytic disorder will manifest as hemolytic „„ From the clinical perspective, hemolytic anemia can be anemias. —— acute or Usually the diagnoses of hemolytic anemia are made —— chronic. through the laboratory investigations but a clinical „„ According to mechanism (location of the abnormality) history along with physical examination is also helpful for (Table 2), there may be providing useful information about presence of hemolysis —— intrinsic (intracorpuscular) defect or and its probable etiology. —— extrinsic (extracorpuscular) defects.

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TABLE 1 Classification according to site of hemolysis

Intracorpuscular factors (Red cell abnormality) Extracorpuscular factors Hereditary Immune hemolytic anemias zz Membrane defect (spherocytosis, elliptocytosis) zz utoimmune hemolytic anemia zz Metabolic defect (Glucose-6-Phosphate-Dehydrogenase (G6PD) —— caused by warm-reactive antibodies deficiency, Pyruvate kinase (PK) deficiency) —— caused by cold-reactive antibodies zz Hemoglobinopathies (unstable hemoglobins, thalassemias, sickle zz Transfusion of incompatible blood Nonimmune hemolytic cell anemia) anemias Acquired zz Chemicals Membrane abnormality-paroxysmal nocturnal hemoglobinuria zz Bacterial infections, parasitic infections (malaria), venons (PNH) zz Hemolysis due to physical trauma —— hemolytic-uremic syndrome (HUS) —— thrombotic thrombocytopenic purpura (TTP) —— prosthetic heart valve —— Hypersplenism

TABLE 2 Classification according to the location of the abnormality

Intracorpuscular defects Extracorpuscular factors Hereditary zz Hemoglobinopathies zz Familial (atypical) hemolytic uremic Syndrome zz Enzymopathies zz Membrane-Cytoskeletal defects Acquired zz Paroxysmal nocturnal hemoglobinuria (PNH) zz Mechanical destruction (microangiopathic) zz Infectious zz Autoimmune zz Toxic agents zz Drugs

Mechanisms involved in anemia include: „„ Hemoglobin disorders: Abnormal hemoglobin is because „„ Inadequate production: Stem cell damage or defective of inherited gene in some people. Abnormal hemoglobin red cell maturation. can lead to easy destruction of red blood cells. Disorder „„ Excessive destruction (hemolysis): Intrinsic defect in red includes sickle cell anemia and the thalassemias. cell leading to shortened lifespan or external factors in „„ Physical damage to RBCs: blood or blood vessels destroy red cells. —— During heart-lung surgery „„ Blood loss (bleeding) —— In patients with artificial heart valves (as blood flows near devices) Causes of hemolytic anemia include: —— In a patients with severe burn (exposure to extreme „„ Inherited red blood cell membrane abnormalities: heat) There is change in the shape of cell due to membrane „„ Physical damage to RBCs: defects and this change in shape is identified as —— During heart-lung surgery abnormal by spleen and destroyed it. —— In patients of artificial heart valves „„ Inherited enzyme deficiencies in red blood cells: —— In patients with severe burn due to exposure to Abnormalities in enzyme levels makes the red blood extreme heat cell fragile, which makes red blood cell prone to get —— Autoimmune hemolytic anemia: It can occur in destroyed easily. autoimmune conditions like lupus, certain types

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of infections, and use of some drugs. This happens Features of Hemolysis when the red blood cells of the body gets destroyed „„ Hemoglobin—Normal to severely reduced by immune mechanism of its own body. „„ MCV, MCH—Usually increased —— Hypersplenism: The spleen is enlarged and „„ Bilirubin is raised (mostly unconjugated) becomes overactive. It traps the circulating red „„ LDH is raised (up to 10 times of normal with intra­ blood cells and destroys it. vascular hemolysis „„ Reticulocytes, n-RBC—Raised General Features of Hemolytic Disorders „„ Haptoglobins are reduced to absent Hemolytic anemias can be differentiated from other „„ Urinary hemosiderin, Urobilinogen are +ve anemias by presence of signs and symptoms arising ↓ because of hemolysis. Blood Smear ↓ General examination: Jaundice, Pallor, Bossing of Skull „„ Spherocytes— —— DCT +ve (Autoimmune hemolysis) Physical findings: —— DCT –ve (H. Sherocytosis, malaria, clostridium) „„ Enlarged spleen (as a preferential site of hemolysis) „„ No spherocytes—Hereditery enzymopathies leading to neutropenia and/or thrombocytopenia „„ Fragmentation—Microangiopathic, Traumatic „„ Enlarged liver (in some cases) „„ Skeletal changes, because of bone marrow over activity (seen in severe congenital forms) Laboratory Evaluation of Hemolysis „„ Hemoglobin—From normal to severely reduced See Table 3. „„ MCV—Usually increased „„ Reticulocytes—Increased (sign of the erythropoietic Metabolic Manifestations of Increased response by the bone marrow) Red Cell Turnover „„ Bilirubin—Increased (mostly unconjugated) There is significant iron loss (requiring treatment), „„ LDH—Increased due to persistent hemoglobinuria because of chronic „„ Haptogloin—Reduced to absent intravascular hemolysis. If frequent blood transfusions are needed or if erythropoiesis is massively increased Two main principles for diagnosis: then iron overload is very common. This iron overload „„ First is to confirm the diagnosis of hemolysis leads to hemochromatosis affecting liver and heart „„ Second is to determine its etiology muscles eventually leading to cirrhosis and heart failure The etiology of hemolytic anemia can be determined respectively. by: „„ A good clinical history Morphological Abnormalities of RBCs in „„ The peripheral blood film Hemoltyic Anemias „„ Schistocytes—thrombotic microangiopathies or Clinical Presentation cardiac prosthetic valves „„ New onset pallor or anemia „„ Spherocytes—hereditary spherocytosis, immune hem. „„ Hemolytic faces—Chipmunk facies anemia, burns, chemical injury to RBC „„ Jaundice „„ Sickled cells—sickle cell disease „„ Splenomegaly, bossing of skull (in severe congenital „„ Elliptocytes—hereditary elliptocytosis cases) „„ Echinocytes—pyruvate kinase deficiency „„ Gall stones „„ Heinz bodies—G6PD deficiency „„ Dark colored urine „„ Basophilic stipplings—thalassemia, Wilson’s disease, „„ Leg ulcers and lead poisoning

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TABLE 3 Laboratory evaluation of hemolysis

Extravascular Intravascular

Hematologic

zz Peripheral blood film zz Polychromatophilia zz Polychromatophilia zz Reticulocyte count zz Raised zz Raised zz Bone marrow examination zz Erythroid hyperplasia zz Erythroid hyperplasia

Plasma or serum

zz Bilirubin zz Unconjugated decreased zz Unconjugated zz Haptoglobin zz Absent zz Absent zz Free hemoglobin zz N/increased (Variable) zz Increased zz Lactate dehydrogenase zz Increased (Variable)

Urine

zz Bilirubin 0 0 zz Hemosiderin 0 + zz Hemoglobin 0 + → severe cases

Flowchart 1: Approach to identify and classify hemolytic anemias

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Red Cell Survival Study —— Schistocytes—Microangiopathic hemolytic To prove that the life span of RBC is reduced than normal anemia PT/PTT, LFT, KFT, Blood Pressure TTP, (about 120 days), then the study labeling the red cells with HUS, DIC, Eclampsia, Preeclampsia, malignant 51Cr can be done and fall in radioactivity over days to hypertension, prosthetic valve. weeks can be measured. Nonradioactive isotope 15N can —— Hypochromic microcytic anemia Thalassemia- also be used now in place of radioactive substance. Hemoglobin electrophoresis. —— Sickle cells-Sickel cell anemia—Hemoglobin Algorithm for the Evaluation and electrophoresis. —— Infection/drug exposure—G6PD activity. Diagnosis of Hemolytic Anemia —— Fever/recent travel—Thick and thin smears, Anemia—present Babesia serology, bacterial cultures. Indirect hyperbilirubinemia—present Reticulocytosis—present Conclusion ↓ Then look for hemolysis: CBC, reticulocyte count, LDH, Hemolytic anemias can be diagnosed on the basis of detailed indirect bilirubin, haptoglobin, and peripheral blood history, general and physical examination. Laboratory smear investigations and peripheral smear are helpful to confirm the hemolysis and to guide further tests to look for etiology. ↓ „„ If negative: Consider alternative diagnosis (other causes of normocytic anemia like chronic kidney Suggested Readings disease, hemorrhage, chronic disease) . 1 Adamson JW, Longo DL. Anemia and polycythemia. Harrisons „„ If positive: Principles of Internal Medicine, 19th edition. pp. 392-400. —— Spherocytes, positive DAT (Flowchart 1)— . 2 Agarwal MB. A Practical Approach to Anaemia. Progress in Immune hemolysis: lymphoproliferative disorder/ Medicine, API. 2017. pp. 334-8. malignancy, autoimmune diseases, drugs, 3. Dhaliwal G, Cornett PA, Tierney LM Jr. Hemolytic anemia. Am Fam Physician. 2004;69:2599-606. infections, transfusion. . 4 Eloy F. Ruiz, Miguel A. Cervantes. Diagnostic approach to —— Spherocytes, negative DAT, family history— hemolytic anemias in the adult. Rev Bras Hematol Hemoter. 2015; Hereditary spherocytosis. 37(6):423-5.

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Thalassemia in Adults— 227 Management Issues

Ramesh Aggarwal, Anupam Prakash

Abstract Beta-Thalassemia can be broadly classified into transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT). The primary management in these patients is regular blood transfusion and adequate iron chelation. Some patients may require splenectomy and hematopoietic stem cell transplantation (HSCT). Majority of the complications arise because of transfusion related iron overload and include cardiac failure, arrhythmias, endocrine complications like hypogonadism, hypoparathyroidism, diabetes, hypothyroidism, delayed growth, osteoporosis, renal complications, and infections. Early initiation of chelation therapy can reverse the process of iron deposition in tissues and salvage or prolong the development of complications. This chapter highlights the challenges which arise in managing patients of beta-thalassemia and summaries the clinical approach in preventing various complications which arise out of iron load in these patients.

Introduction (HSCT) are required. Table 1 summarizes the current therapies available for TDT. The disease β-Thalassemia includes many different conditions like β-thalassemia major, β-thalassemia intermedia, and HbE/β-thalassemia. They can be broadly Complications and Management Issues classified into transfusion-dependent thalassemia (TDT), Majority of the complications in TDT arise because which includes severe forms of HbE/β-thalassemia and of transfusion related iron overload and include β-thalassemia major and non-transfusion-dependent cardiac failure, arrhythmias, endocrine complications thalassemia (NTDT), which includes thalassemia like hypogonadism, hypoparathyroidism, diabetes, intermedia and milder forms of HbE/β-thalassemia hypothyroidism, delayed growth, osteoporosis, renal (Fig. 1). TDT are patients who require regular transfusion complications, and infections. Some complications like of blood to survive whereas NTDT will require blood silent cerebral infarction and pulmonary hypertension are transfusions in certain conditions like pregnancy, surgery, more common in patients with NTDT. and infections.1 Cardiac Complications Management They include cardiomyopathy congestive heart failure, The management of TDT requires regular blood arrhythmias, peripheral vascular disease, and pulmonary transfusion and adequate iron chelation. In some patients, hypertension. Persistent hypoxia, high levels of abnormal splenectomy and hematopoietic stem cell transplantation hemoglobin, and high iron levels in tissues along with

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Fig. 1: Classification of thalassemia based on transfusion dependence

TABLE 1 Treatment options for beta thalassemia2

Treatment modality Merit Demerit

Blood transfusion Helps by suppressing ineffective zz Every 2–4 weeks’ transfusion required for life long erythropoiesis, prevents bone marrow zz Transfusion associated complications like expansion infections, iron overload, and alloimmunization

zz Iron chelation zz Decreases iron overload in myocardium, zz May not be effective in every patient zz Deferoxamine: Parenteral liver, pituitary, etc. zz Side effects are common zz Deferasirox: Oral zz Improves endocrine functions and cardiac zz Parenteral formulation often lead to non- zz Deferiprone: Oral health compliance zz Cost of therapy is high

Hydroxyurea (It is a cytotoxic zz Some hematological parameters may zz Lack of clear evidence of its use in TDT antimetabolite that helps to improve in NTDT patients increase fetal hemoglobin levels) zz Cost of therapy is low

Splenectomy zz The quality of life and growth may improve zz Risk of infection and sepsis increases zz It may improve hemoglobin concentration zz The risk of venous thrombosis and pulmonary zz The frequency of transfusions may decrease hypertension increases zz It also reduces the ability to scavenge toxic-free iron species

HSCT zz Treatment of choice for patients younger zz May not be useful for all patients, only a subset of than 14 years patients qualify for this treatment zz Others may benefit depending on donor zz Young age availability and iron load after this age zz Donor should be a compatible sibling zz The survival rate is up to 90% and the zz Risk of 5–10% mortality disease-free survival rates reaches up to zz Preparation for this treatment requires 80% myeloablative conditioning and can cause zz The quality of life is improved impairment of fertility zz Long-term cost-effective zz Highly trained centers are required zz Very high cost HSCT, hematopoietic stem cell transplantation; NTDT, non-transfusion-dependent thalassemia; TDT, transfusion-dependent thalassemia

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infection with cardiotropic viruses contribute to initiation Endocrine Complications and progression of cardiomyopathy in these patients. They include hypogonadism, growth retardation, diabetes Presenting features are dyspnea, fatigue, or palpitations. 6 mellitus, hypoparathyroidism, and hypothyroidism. Cardiomyopathy in thalassemia presents with two different phenotypes: „„ Dilated cardiomyopathy where the left ventricle is Gonadal Axis dilated and contractility is reduced. This progresses to Hypogonadism can be primary (hypergonadotropic congestive heart failure. hypogonadism) due to iron deposition in gonads or „„ Restrictive cardiomyopathy where the left ventricle secondary (hypogonadotropic hypogonadism) due to filling is restrictive. This phenotype can lead to iron affecting the gonadotrophs in anterior pituitary. pulmonary hypertension, dilatation of right ventricle, Secondary hypogonadism is more common in patients and subsequent heart failure. with TM. Primary amenorrhea precedes secondary Measurement of amino-terminal pro-B-type amenorrhea in most women with thalassaemia major. natriuretic peptide is more reliable in early diagnosis than Screening for hypogonadism should be done annually. Doppler echocardiography.3 Cardiac MRI provides better Careful history should be taken which includes erectile estimate of iron burden than serum ferritin and it has function, libido, spontaneous erections in males been found to be a better predictor of heart failure and and libido, vasomotor symptoms, and menstrual 4 arrhythmia in different studies. history in females. The patient’s genitalia should be examined and loss of secondary sexual characters should Rhythm Disturbances be noted. They are atrial fibrillation, atrial flutter, intra-atrial re- Investigations: entrant tachycardia, ectopic atrial tachycardia, and „„ Serum fasting testosterone, SHBG, LH, FSH, and hCG ventricular arrhythmias. Prolonged QT interval and in males and estradiol, LH, and FSH in females. repolarization abnormalities occur because of iron Management: overload leading to torsades de pointes and sudden cardiac death (SCD).5 „„ Females: Treatment is given to relieve from symptoms of estrogen deficiency like sweats, hot flushes, mood changes and vaginal atrophy. Hormone replacement Management therapy (HRT) is helpful to overcome them and As aggressive iron chelation therapy can restore the prevent osteoporosis. functions of myocardium, it should be the goal: „„ Males: Testosterone replacement in the form of „„ Asymptomatic patients with no cardiac dysfunction transdermal gel or intramuscular injections every 2 and no deposition of iron in heart: healthy lifestyle and weekly helps to improve libido, erectile dysfunction, adequate chelation therapy and may improve bone mineral density and muscle „„ Iron deposition in heart but no cardiac dysfunction: mass. healthy lifestyle and intensification of chelation therapy including use of combination therapy Growth Hormone Axis „„ Cardiac dysfunction with or without symptoms: —— Healthy lifestyle and intensification of chelation Many factors contribute to delayed growth in patients, therapy including use of combination therapy which are iron overload, chronic anemia, hypersplenism, —— Slower transfusion of blood with use of diuretics hypothyroidism, hypogonadism, chronic liver disease, —— Use of ACE inhibitors, or ARII blockers malnutrition, stress, and growth hormone (GH) deficiency. —— Cardioselective beta-blockers like bisoprolol and GH deficiency is common in patients with TM and results carvedilol in short stature and delayed growth. GH deficiency can —— Digitalis if atrial fibrillation is present along with have varied presentations like classic GHD, GH resistance anticoagulation or a combination of both.7 Clinical Assessment should

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be done preferably at regular interval of 6 monthly. It lack of energy, fatigue, decreased appetite, and muscular includes: weakness. Adrenal androgen deficiency can sometimes „„ Patient’s height (standing and sitting) and weight lead to decreased pubic and axillary hairs. „„ Measurement of upper/lower segment ratio and Investigations: calculation of BMI and annual growth velocity (GV) „„ Serum cortisol level, adrenocorticotropic hormone „„ Recording of parental heights at the first visit (calculate (ACTH) stimulation test or rarely insulin stimulation mid-parental height) test (ITT) can be done to assess adrenal insufficiency. „„ Accurate measurement of standing and sitting heights, weight, and head at each visit; measurement of the Treatment: head circumference especially during the first 2 years „„ Acute adrenal crisis is not common in these patients, of life and hence steroids should not be used routinely to „„ Plotting growth data on ethnically adjusted charts or cover asymptomatic individuals. Glucocorticoids can international (WHO) adjusted charts be used in acute stressful conditions.9 „„ Calculating annual growth velocity (GV), body mass index (BMI), and upper/lower segment ratio at every Thyroid visit Primary hypothyroidism is present in 4–29% of patients „„ Assessment of pubertal status as per Tanner stage with TDT and is much common than central or secondary (development of breast in girls and testicular volume hypothyroidism.10 Prevalence of hypothyroidism is in boys) directly proportional to iron load. With the increase in „„ Assessment of bone age serum ferritin the prevalence of hypothyroidism increases. Investigations: Also hypothyroidism is more frequent in splenectomized „„ Serum IGF-1, Serum TSH, and free T4, LH, FSH, and patients than non-splenectomized patients. The reason sex steroids along with X-ray of wrist and hand for bone may be because of the fact that intact spleen acts as age. reservoir of excess iron and functions as a scavenger for Treatment: free iron fraction. „„ Children who are short with low IGF-1 levels and Classification of hypothyroidism: normal GH secretion to stimulation tests may benefit „„ Sub-biochemical hypothyroidism: The response to from IGF-1 or GH-IGF-1 treatment. TRH test is exaggerated and TSH and FT4 are normal. „„ If the bone age is 10 years or greater, then priming with „„ Subclinical hypothyroidism: The TSH is high (>4.2 sex steroids is done before initiating any treatment for mIU/L and <10 mIU/L) and FT4 levels is normal. GH deficiency. „„ Overt (clinical) hypothyroidism: The TSH is high (TSH „„ Treatment of other diseases like diabetes and >10 mIU/L) and FT4 is low. hypothyroidism has to be done simultaneously. Investigations: „„ There must be psychological evaluation and support for conditions that are non-treatable like constitutional „„ Assessment of serum-free T4 and TSH should be done delay of puberty and growth, familial short stature, etc. annually from the age of 9 or earlier if hypothyroidism is suspected. For early diagnosis, TRH stimulation test „„ Treatment with rhGH should start with low doses and then titrated according to IGF-1 levels and growth rate. can be used. In patients with iron overload, there is exaggerated TSH response to TRH, which may evolve Adrenal into subclinical or clinical hypothyroidism. In clinical hypothyroidism, there are the FT4 and basal TSH Patients with thalassemia are often asymptomatic for gradually decrease over time. adrenal insufficiency and may have only biochemical evidence. A study of 56 children suffering from thalassemia Treatment: had shown presence of adrenal insufficiency in about „„ Early detection and adequate chelation therapy may 37.5% asymptomatic patients.8 Acute stress can precipitate reverse the thyroid dysfunction. Patients with overt adrenal insufficiency and symptoms can be feeling of hypothyroidism are treated with L-thyroxine.

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Hypoparathyroidism „„ Bisphosphonates like zoledronic acid have the It is an uncommon and a late complication in TM advantage of intravenous administration and longer patients. Deposition of iron in parathyroid glands results duration of action lasting more than 12 months and in hypoparathyroidism. Hypocalcemia and paraesthesia should be initiated earlier. „„ Other drugs helpful in bone disease include teri­ occur in mild cases and severe cases may present with paratide, denosumab, and zinc. tetany and seizures. Investigations: Glucose Intolerance and Diabetes Mellitus „„ Measurement of calcium, phosphate and parathyroid Patients with thalassemia are susceptible to develop hormone (PTH) levels should be done. In diabetes. The etiology is multifactorial including genetic hypoparathyroidism there is low serum calcium, high predisposition, β-cell destruction because of iron overload, phosphate, and low PTH levels. insulin resistance, insulin deficiency, chronic liver disease, Treatment: and viral infections. Initially patients may present with „„ Supplementation with calcium and vitamin D is done. impaired glucose tolerance because of insulin resistance Calcitriol 0.25 µg twice daily brings the calcium and and later develop insulin deficiency. Some characteristics phosphate levels to normal. In some patients with high of insulin dependent diabetes in thalassemics are: phosphate levels, phosphate binders may be used. „„ Ketoacidosis is an uncommon presentation In severe hypocalcemia, tetany, and cardiac failure, „„ Islet cell antibodies are usually negative intravenous calcium has to be given. „„ HLA haplotypes like DR4, B8-DR3, and BW15 have no association Thalassemia Bone Disease Investigations: Patients with thalassemia can suffer from bone diseases, „„ Fasting blood sugar levels, 2-h oral glucose tolerance which include osteopenia, osteoporosis, bone deformity testing (OGTT) and measurement of insulin levels and fractures.11 Risk factor for bone disease in these should be started at the age of 10 years. HbA1c is patients includes concurrent presence of hypogonadism altered in hemoglobinopathy and cannot be used and deficient growth hormone. Other contributory factors as a reliable marker for monitoring. Instead serum include chronic anemia and marrow expansion, chelator fructosamine levels may be used for monitoring long toxicity, hypercalciuria, and renal dysfunction from term glycemic control. deferasirox, liver disease, and advancing age. Treatment: Investigations: „„ Dietary counseling and weight reduction in meta­ „„ Serum calcium, serum phosphorus, alkaline phos­ bolically obese patients. phatase, and 25-hydroxy vitamin D „„ Chelation therapy should be more intensive. There is „„ PTH, LH, FSH, testosterone levels increase in insulin secretion and decrease in insulin „„ Osteocalcin resistance with intensive chelation therapy. „„ C-terminal telopeptide „„ Data on use of oral antidiabetic drugs is limited. Insulin „„ 24-h urinary calcium is needed in late presentation and complications. „„ X-ray of spine (AP and lateral views) „„ MRI spine: to exclude an intervertebral disc de­ Conclusion generation The management in thalassemia requires a team effort and „„ Dual-energy X-ray absorptiometry (DEXA) scan for a multidisciplinary involvement. Management of TDT mainly assessing bone mineral density (BMD) depends on lifelong transfusion therapy. The complications Treatment: arise because of the iron deposition in various organs and „„ Treatment with calcium and vitamin D can be started infections from frequent blood transfusions. It has been proven although there is not much evidence of improvement in studies that early initiation of chelation therapy can reverse in BMD. Contd...

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Contd... . 5 Aessopos A, Farmakis D, Hatziliami A, et al. Cardiac status in well-treated patients with thalassemia major. Eur J Haematol. the process of iron deposition in tissues and salvage or prolong 2004;73(5):359-66. the development of complications. Pregnancy, fertility, and . 6 De Sanctis V, Soliman AT, Elsedfy H, et al. Growth and endocrine psychosomatic stress are other associated issues in managing disorders in thalassemia: the international network on endocrine patients of thalassemia and require treatment for enhancing complications in thalassemia (I-CET) position statement and quality of life in these patients. guidelines. Indian J Endocr Metab. 2013;17:8-18. . 7 Scacchi M, Danesi L, Cattaneo A, et al. Growth hormone deficiency (GHD) in adult thalassaemic patients. Clin Endocrinol. 2007;67:790-5. References . 8 Poomthavorn P, Isaradisaikul B, Chuansumrit A, et al. High prevalence of “biochemical” adrenal insufficiency in thalassemics: . 1 Musallam KM, Rivella S, Vichinsky E, et al. Non-transfusion- is it a matter of different testings or decreased cortisol binding dependent thalassemias. Haematologica. 2013;98(6):833-44. globulin? J Clin Endocrinol Metab. 2010;95:4609-15. 2. Cappellinia MD, Porterb JB, ViprakasitcV, et al. A paradigm shift on 9. Elsedfy HH, El Kholy M, Tarif R, et al. Adrenal function in thalassemia beta-thalassaemia treatment: how will we manage this old disease major adolescents. Pediatr Endocrinol Rev. 2011;8:295-9. with new therapies? Blood Reviews. 2018;32(4):300-11. 10. De Sanctis V, Soliman AT, Canatan D, et al. Thyroid disorders . 3 Kremastinos DT, Tsiapras DP, Kostopoulou AG, et al. NT-proBNP in homozygous β-thalassemia: current knowledge, emerging levels and diastolic dysfunction in beta-thalassaemia major issues and open problems. Mediterr J Hematol Infect Dis. patients. Eur J Heart Fail. 2007;9:531-6. 2019;11(1):e2019029. 4. Kirk P, Roughton M, Porter JB, et al. Cardiac T2* magnetic resonance 11. Haidar R, Musallam KM, Taher AT. Bone disease and skeletal for prediction of cardiac complications in thalassemia major. complications in patients with β thalassemia major. Bone. Circulation. 2009;120:1961-8. 2011;48:425-32.

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228 Approach to Thrombocytopenia

Avinash Kumar Singh, Divya Krishna, Gautam Kumar Rakesh, Bhupendra Kumar

Abstract Thrombocytopenia is a common medical problem encountered by physicians in their day-to-day OPD as well as IPD practice. Anxiety is at peak for patients as well as young physicians when dealing with case of severe thrombocytopenia. Thorough history, physical examination, and directed investigations lead to correct cause and resolution of thrombocytopenia. Causes range from benign disease like ITP and megaloblastic anemia to serious diseases like leukemia, lymphoma, and TTP. Drugs are culprit in many cases and sometimes misleading drug history leads to unnecessary battery of tests. Once properly diagnosed, correct and timely treatment can resolve thrombocytopenia. Platelet transfusion is not always required and should be reserved for severe thrombocytopenia or with bleeding patients.

Introduction Low Platelets—How Serious It is? By the time you shall be reading this chapter, we hope Platelets play an important role in vessel wall integrity and that world will be on the path of recovery from COVID so low platelet leads to primary hemostatic defects. Its pandemic crisis and message like “stay safe” will be less relevance in an individual patient is variable and depends frequently used. upon the clinical presentation. Thrombocytopenia is a common medical problem Clinically significant bleeding does not occur unless encountered by physicians in day-to-day practice. It platelets are less than 10–20,000 × 109/L, while mild to creates panic among general people as well as practicing moderate thrombocytopenia becomes significant when physicians, which is not always justified. there is additional bleeding risk like surgery, trauma We will describe how to approach a case of thrombo­ or when higher cut-off to be met as when treatment for cytopenia with brief about treatment options of common hepatitis C or chemotherapy is indicated. diseases. In some situation, finding of thrombocytopenia Thrombocytopenia is defined as a platelet count below points toward serious disease like HIV or Myelodysplastic the 2.5th lower percentile of the normal platelet count syndrome and also sometimes it indicates disease activity distribution. Results of the third US National Health and like in thrombotic thrombocytopenic purpura (TTP). Nutrition Examination support the traditional value of 9 150 × 10 /L as the lower limit of normal; however, counts How Important is Setting of between 100 × 109/L and 150 × 109/L do not necessarily indicate disease if they have been stable for more than Thrombocytopenia? 6 months, and the adoption of a cut-off value of 100 × In OPD settings, mostly thrombocytopenia is isolated 109/L may be more appropriate to identify a pathologic and asymptomatic and diagnosis is straight forward condition.1-3 while in inpatient, or in ICU settings, multisystem

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TABLE 1 Clinical scenario and most common causes of thrombocytopenia

Outpatient Inpatient ITP Multisystem illness/ICU Cardiac Pregnancy/Postpartum DITP Infections HIT GT Infections: DITP Cardiac bypass ITP HIV, Hep C, CMV, H. Pylori, Dengue, and other recent viral infection Connective tissue disorder TTP/HUS GP IIB/IIIA inhibitors HELPP syndrome Vaccinations DIC DITP Pre-eclampsia Congenital thrombocytopenia Liver disease/BM disorders Dilutional TTP/HUS Common variable immunodeficiency disease HIT MDS MAS CIT CIT, chemotherapy-induced thrombocytopenia; DITP, drug-induced ITP; MAS, macrophage activation syndrome; MDS, myelodysplastic syndrome.

involvement leads to thrombocytopenia and diagnosis Physical examination should be directed to look for and management is sometimes challenging (Table 1). bleeding, organomegaly, or skeletal abnormality. Pregnancy with thrombocytopenia has to be approached differently as it has significance in the care for mother as Lab Investigations—What Battery of well as the newborn. Tests Required? A structured approach involves clinical details and Even today, initial and basic investigation for support of lab along with other medical disciplines. thrombocytopenia evaluation is complete blood count (CBC) and peripheral smear. Routine tests, like liver Mechanism of Thrombocytopenia function and renal function test, are always required. Major mechanism of thrombocytopenia is reduced Chest X-ray is required to look for focus of infection production as in aplastic anemia, Myelodysplastic and mediastinal mass and USG abdomen is done syndrome (MDS), or chemotherapy induced to look for any evidence of chronic liver disease or thrombocytopenia and platelet destruction as in lymphadenopathy. Autoimmune workup is required in disseminated intravascular coagulation (DIC) or TTP. suspected cases to rule out SLE/APLA syndrome. Infective Less common mechanism is platelet sequestration workup is required in patients who present with classical as in congestive splenomegaly and hemodilution as in features. Immature platelet fraction or reticulated platelets excessive fluid or platelet poor component transfusion. help in differentiation from bone marrow failure (low There are conditions like ITP and hepatitis C, where percentage) and hyper-destructive thrombocytopenia 5,6 multiple mechanisms play role. (higher percentage). Flowchart 1 shows algorithm for thrombocytopenia on the basis of peripheral blood smear 7 History and Physical Examination—How findings. Artefactual thrombocytopenia and conditions does it Help in Evaluation? like Harris platelet syndrome are not so rare problem As for any medical disorder history is very important for encountered in our clinical practice and it has huge thrombocytopenia evaluation and question should be psychological impact on the patient, their immediate care directed toward bleeding history, associated symptoms giver, and physicians.8.They need only counseling that like fever, jaundice, joint pain, medication history, dietary they should not worry and need no intervention. We will habits, addiction and history to rule out autoimmune discuss common conditions leading to thrombocytopenia disease and malignancy. in our practice.

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Flowchart 1: Algorithm for the workup of thrombocytopenia based on peripheral blood smear finding

Isolated Thrombocytopenia List of basic tests required as per international BOX 1 4 It is defined as thrombocytopenia in the absence of RBC consensus report or WBC abnormality and without signs or symptoms of zz Complete blood count systemic illness. Common examples are ITP and D-ITP. zz Peripheral blood smear zz Reticulocyte count Immune Thrombocytopenia (ITP) zz Quantitative Ig level measurement (terms idiopathic and pupura have been removed) zz BM examination (in patients >60 years) Even being the commonest cause of isolated zz Blood group (Rh) thrombocytopenia, there is no single test, which confirms zz Direct antiglobulin test ITP as diagnosis and it remains a diagnosis of exclusion. zz H. pylori Box 1 lists all blood investigations in a suspected case zz HIV of ITP. Antiplatelet antibody assay is not very sensitive, zz HCV but specificity approaches 90%. Table 2 enumerates morphologic features and pointers towards different causes of thrombocytopenia. Bone marrow is not required ITP patients are divided into newly diagnosed unless patients are more than 60 years or with atypical (thrombocytopenia is of less than 3 months duration), features. persistent (thrombocytopenia extends from 3 months to

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TABLE 2 Showing morphological findings on PBS in different causes of thrombocytopenia

Platelets zz Platelet clumping —— Platelet clumping caused by EDTA-dependent platelet autoantibodies is a common cause of artifactual thrombocytopenia. It occurs in about 1 in 1000 nomral adults and is not associated with bleeding or thrombosis zz Platelet size and granularity —— Consistently large platelets suggest hereditary macrothrombocytopenia. Large platelets with a gray color on Wright-Giemsa stain define the gray platelet syndrome, an autosomal-dominant macrothrombocytopenia associated with bleeding tendency due to absent or greatly reduced a-granules —— In thrombocytopenia due to peripheral destruction, large platelets or giant platelets are often seen in addition to platelets of normal size —— When thrombocytopenia is due to reduced platelet production (e.g., after chemotherapy), platelets are of normal size. In myelodysplasitc syndromes, platelets have variable size (giant platelets may be seen) and are frequently hypogranular. In Wiskott-Aldrish syndrome, and X-linked thrombocytopenia, both cuased by mutations of the WAS gene, platelets are smale WBCs zz Leukemic cells —— Malignant hematological disorders (leukemias and lymphomas) are often associated with thrombocytopenia, which is almost never an isolated finding —— Other abnormalities of WBCs, including leukocyte inclusions A constellation of nonspecific abnormalities of WBCs are common to many conditions (e.g., neutrophilia, lymphocytosis, leukopenia, etc.) and may be associated with thrombocytopenia. The presence of hypolobulated neutrophilis (Pelger-Huet anomaly) suggests a myelodysplastic syndrome. Dark coarse granules (toxic granulations) found in neutrophils suggest sepsis. Atypical lymphocytes suggest viral infection. The presence of WBC inclusion (Dohle-like bodies) should be investigated carefully when platelets are mostly large (MYH9-related congenital macrothrombocytopenia) RBCs zz Schistocytes —— The presence of RBC fragments known as schistocytes is indicative of a thrombotic microangiopathy (TTP/HUS) or DIC zz Size and other morphological features. —— Microspherocytes may suggest Evans syndrome, but may also be present along with chistocytes in thrombotic microangiopathies. Macrocytosis (and hypersegmentation of neutrophils) suggest vitamin B12 or folate deficiency. Dacryocytes (teardrop-shaped cells) suggest myelofibrosis. Nucleated RBSc suggest hemolytic anemia, myelofibrosis, or an inflitrative process of the BM zz Parasites —— The presence of intracellular parasites (e.g., in malaria) is diagnostic of infection

12 months), and chronic when thrombocytopenia is for Options in symptomatic patients are steroid (short more than 1 year. course), immunosuppressants (azathioprine, MMF), In ITP patients with emergency (intracranial bleed, Dapsone, MAB (rituximab), Thrombopoietin mimetic massive GI bleed) IVIG, High dose methylprednisolone, (wltrombopag, romiplostim), and splenectomy. Other and anti D (in Rh positive patients) are options. less commonly used options are danogen, vincristine, For asymptomatic or patients with minor bleeding, cyclosporine, and cyclophosphamide. observation and local bleeding control are best options, as most of the morbidity in ITP patients are due to treatment Drug-induced Immune Thrombocytopenia (D-ITP) than due to low platelet count. Good counseling can The list of drugs leading to thrombocytopenia is ever reduce need of drugs and give better quality of life to increasing one. The pathophysiological mechanism of patients. It is important to explain to patients that even D-ITP is due to formation drug dependent antibody with very low platelets, severe or life threatening bleeding against epitope of platelet glycoprotein created by their is rare in ITP. interaction with the drug.

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Sometimes D-ITP can be confused with ITP, and only and in APML. Chronic DIC is seen in solid tumors and good history can help to differentiate. large aortic aneurysm. Table 3 enumerates factors and Diagnosis often is difficult as sometimes not only drug scoring to diagnose overt or non-overt DIC. but even food and beverages can lead to thrombocytopenia. Diagnosis is mostly empirical with recovery of platelet Thrombocytopenia in Cardiac Patients count after discontinuation of drug. Lab diagnosis is by Open heart surgery is an important cause of thrombo­ demonstration of drug dependent antibody by various cytopenia in cardiac patients. Nadir is typically seen on methods. days 2–3 and recovery starts rapidly thereafter. Causes of An overall score of 5 or more is compatible with overt low platelet after surgery are multiple. DIC. A score less than 5 is suggestive of non-overt/low- Severe thrombocytopenia has been observed in grade DIC. 0.1–2% of patients after exposure to gp IIb/IIIa inhibitors (abciximab, tirofiban, eptifibatide). Peculiarity is sudden Thrombocytopenia in Hospitalized Settings onset, usually within hours after surgery, and used to Approximately 1% of admitted patients in acute care resolve by day 10. This has been attributed to presence of hospital are thrombocytopenia but only 30% of them have naturally occurring antibody to neoepitopes exposed by 15 bleeding manifestation.9 As expected it is more common gp IIb/IIIa inhibitors. in ICU, where 8–68% are thrombocytopenic on admission HIT—It occurs in 1–3% of patients receiving heparin and 13–44% during stay in the unit.10 Many potential beyond the first postoperative week and in 10% of 16,17 etiologies like sepsis, DIC, drugs, CABG coexist and it is patients after ventricular assist device implantation. 4T often difficult to elucidate cause of thrombocytopenia described in Table 4 helps in diagnosis of heparin induced 18 ITP should always be considered as it is seen in about thrombocytopenia. 20% of these and antibiotics are the major culprit.11,12 Discontinuation of such drug should always be based on Pregnancy with Thrombocytopenia clinical criteria.13 About 6–15% of women develop thrombocytopenia (<1.5 lakhs/mm3) at the end of pregnancy but platelets Disseminated Intravascular Coagulation less than 1 lakh/mm3 are seen in only 1% of patients. DIC is a consumptive coagulopathy characterized by Most common cause of thrombocytopenia is gestational activation of intravascular coagulation with microvascular thrombocytopenia (GT) (70%), preeclampsia (21%) ITP 19,20 thrombi formation, thrombocytopenia, depletion of (3%). clotting factors, bleeding complications, and end organ damage. It can be acute or chronic. International society Gestational Thrombocytopenia (GT) on thrombosis and hemostasis divides DIC into overt GT is seen in mid second to third trimester of pregnancy (decompensated hemostatic system) or non-overt and it is extreme variation of normal physiological (compensated coagulopathy).14 fall of platelet count. Platelet count usually remains Acute DIC is seen in sepsis, septic shock, after trauma above 70 k/mm3 and if falls to lower than 70 k, then (neurotrauma), after surgery, after obstetric complications alternative diagnosis should be considered. Diagnosis is

TABLE 3 Diagnostic score of DIC

Parameters 0 1 2 3 Platelet count >100 × 109 50–100 ×109 <50 × 109 Elevated fibrin degradation products No increase Moderately increase Strong increase PT more than ULN <3S >3S >6S Fibrinogen >1 gm/L <1 gm/L

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TABLE 4 The 4Ts pretest probability of heparin-induced thrombocytopenia

Points* 4Ts 2 1 0 Thrombocytopenia Platelet count decrease >50% and Platelet count decrease 30–50% or Platelet count decrease <30% or platelet nadir >20 × 109/L platelet nadir >10–19 × 109/L platelet nadir <10–19 × 109/L Timing of platelet Clear onset between d 5 and 10 or Consistent with d 5–10 decrease, but Platelet count decrease <4 d count decrease platelet decreases ≤1 d (prior heparin not clear (e.g., missing platelet counts); without recent exposure exposure within 30 d) onset after d 10; or decreases ≤1 d (prior heparin exposure 30–100 d ago) Thrombosis or other New thrombosis (confirmed); skin Progressive or recurrent thrombosis; non- None sequelae necrosis; acute systemic reaction necrotizing erythematous) skin lesion; post intravenous Suspected thrombosis (not proven) Other causes for None apparent Possible Definite thrombocytopenia Pretest probability socre: 6–8, high; 4–5, intermediate; 0–3 low. *0, 1, or 2 for each category with a maximum possible score of 8.

by excluding other causes and there may similar history stem cell injury, increased autoimmune destruction and of thrombocytopenia in previous pregnancy and fetus/ lung injury.22-25 newborn remains unaffected (normal platelet) and Bleeding due to thrombocytopenia is rarely seen. recovery takes 1–2 months after delivery.

ITP in Pregnancy What are the Indications, Dosing of ITP in pregnancy is seen in about 1–2 of 1,000 pregnancies. Platelet Transfusion? It is the most common cause of thrombocytopenia in „„ Any patient with bleeding due to thrombocytopenia first and early second trimester. One third of patients or severe thrombocytopenia (less than 30 k/mm3 are diagnosed first time during pregnancies and rest has even without bleeding) or patient with moderate history of ITP. thrombocytopenia (30 k to 1 lakh/mm3) and due for Differentiating GT from ITP is difficult and it is more surgery or has other associated bleeding risk need important for neonatal management as 9–15% of neonates platelet transfusion. 3 of ITP mother can have thrombocytopenia and 1–2% can „„ Higher threshold (more than 1 lakh/mm ) is required 21 have intracranial hemorrhage. for patients with life threatening bleeding (ICH) or 3 Whenever platelet is less than 50 k/mm , diagnosis is planned for neurosurgery or ophthalmic surgery. ITP by default, though as in medicine always it should be „„ Exact cut-off depends upon primary disease leading to correlated in clinical context. thrombocytopenia. „„ In aplastic anemia and acute leukemia, platelet COVID-19 and Thrombocytopenia transfusion should be done when platelet count is SARS-CoV-2 leading to present COVID-19 pandemic less than 10 k/mm3 without fever and less than 20 k present commonly with fever, cough, and breathlessness. with fever, for APML threshold is higher due to excess Common abnormality in hematological parameters bleeding risk. includes leukopenia, lymphocytopenia, and „„ Disease like TTP and HIT are conditions where platelet thrombocytopenia. Thrombocytopenia is seen in about transfusion is always avoided and common disease 5% of patients at admission and overall about 36% of like ITP rarely needs platelet transfusion. patients showed thrombocytopenia, most of which are „„ Physicians should think twice before ordering for significant in severe cases. The proposed mechanism is platelet transfusion in asymptomatic thrombo­ multifactorial due to cytokine storm, direct hematopoietic cytopenia patients.

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„„ Dose for adult patient is single donor platelet or 6 . 8 Naina HVK, Harris S. Harris platelet syndrome—underdiagnosed unit random donor platelet (1 RDP per 10 kg). One and unrecognized. Arch Pathol Lab Med. 2008;132(10):1546. 9. Teo CP, Kueh YK. Incidence of thrombocytopenia in an acute care SDP leads to increase of platelet by 40–50 k and RDP hospital. Ann Acad Med Singapore. 1989;18(4):379-81. increase platelet by 5–10 k unless reasons for platelet 10. Hui P, Cook DJ, Lim W, et al. The frequency and clinical significance refractoriness are absent. of thrombocytopenia complicating critical illness: a systematic review. Chest. 2011;139(2):271-8. 11. von Drygalski A, Curtis BR, Bougie DW, et al. Vancomycin-induced Conclusion immune thrombocytopenia. N Engl J Med. 2007;356(9): 904-10. Thrombocytopenia is a common problem encountered 12. Rousan TA, Aldoss IT, Cowley BD Jr, et al. Recurrent acute thrombocytopenia in the hospitalized patient: sepsis, DIC, by physicians in day-to-day in IPD as well as OPD practice. HIT, or antibiotic-induced thrombocytopenia. Am J Hematol. Common causes are ITP, D-ITP, and infections. Good history, 2010;85(1):71-4. physical examination, and directed investigations can lead to 13. George JN, Aster RH. Drug-induced thrombocytopenia: correct diagnosis of thrombocytopenia. pathogenesis, evaluation, and management. Hematology Am Soc Delay in diagnosis and blind treatment leads to unnecessary Hematol Educ Program. 2009;2009:153-8. intervention in many patients, which should always be 14. Taylor FB Jr, Toh CH, Hoots WK, et al. Towards definition, clinical discouraged. Platelet transfusion is required in emergency and laboratory criteria, and a scoring system for disseminated cases and should not be misused, just to overcome fear and intravascular coagulation. Thromb Haemost. 2001;86(5):1327-30. anxiety of low platelet. 15. Bougie DW, Wilker PR, Wuitschick ED, et al. Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated We thank our patients for having faith on us and giving with antibodies specific for ligand-occupied GPIIb/IIIa. Blood. us opportunity to understand “thrombocytopenia” and all 2002;100(6):2071-6. supporting staff including nurses who help us in patient 16. WarkentinTE, GreinacherA. Heparin-induced thrombocytopenia management. and cardiac surgery. Ann Thorac Surg. 2003;76(6):2121-31. 17. Warkentin TE, Greinacher A, Koster A. Heparin-induced thrombocytopenia in patients with ventricular assist devices: References are new prevention strategies required? Ann Thorac Surg. 2009;87(5):1633-40. 1. Cheng CK, Chan J, Cembrowski GS, et al. Complete blood count 18. Lo GK, Juhl D, Warkentin TE, et al. Evaluation of pretest clinical score reference interval diagrams derived from NHANES III: stratification (4 Ts) for the diagnosis of heparin-induced thrombocytopenia in by age, sex, and race. Lab Hematol. 2004;10(1):42-53. two clinical settings. J Thromb Haemost. 2006;4(4):759-65. 2. Stasi R, Amadori S, Osborn J, et al. Long-term outcome of otherwise 19. Boehlen F, Hohlfeld P, Extermann P, et al. Platelet count at term healthy individuals with incidentally discovered borderline pregnancy: a reappraisal of the threshold. Obstet Gynecol. thrombocytopenia. PLoS Med. 2006;3(3):e24. 2000;95(1):29-33. . 3 Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardization 20. Bockenstedt PL. Thrombocytopenia in pregnancy. Hematol Oncol of terminology, definitions and outcome criteria in immune Clin North Am. 2011;25(2):293-310. thrombocytopenic purpura of adults and children: report from an 21. Gill KK, Kelton JG. Management of idiopathic thrombocytopenic international working group. Blood. 2009;113(11):2386-93. purpura in pregnancy. Semin Hematol. 2000;37(3):275-89. . 4 Provan D, Stasi R, Newland AC, et al. International consensus 22. Guan W-j, Ni Z-y, Hu Y, et al. (2020) Clinical characteristics of 2019 report on the investigation and management of primary immune novel coronavirus infection in China. medRxiv. Available from thrombocytopenia. Blood. 2010;115(2):168-86. https://doi.org/10.1101/2020.02.06.20020974 . 5 Pons I, Monteagudo M, Lucchetti G, et al. Correlation between 23. Chang D, Lin M, Wei L, et al. Epidemiologic and clinical characteristics immature platelet fraction and reticulated platelets. Usefulness of novel coronavirus infections involving 13 patients outside in the etiology diagnosis of thrombocytopenia. Eur J Haematol. Wuhan, China. JAMA. Available from https://doi.org/10.1001/ 2010;85(2):158-63. jama.2020.1623 6. Monteagudo M, Amengual MJ, Munoz L, et al. Reticulated platelets 24. Huang C, Wang Y, Li X, et al. Clinical features of patients as a screening test to identify thrombocytopenia aetiology. QJM. infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2008;101(7):549-55. 2020;395(10223):497-506. 7. Stasi R. How to approach thrombocytopenia, Hematology Am Soc 25. Xu P, Zhou Q, Xu J. Mechanism of thrombocytopenia in COVID-19 Hematol Edu Program. 2012;2012(1):191-7. patients. Ann Hematol. 2020;99(6):1205-8.

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Management of CML in 229 Resource-limited Settings

Hemant Malhotra, Naveen Gupta, Ajay Yadav

Abstract After the development, testing and global availability of BCR/ABL targeted tyrosine kinase inhibitors (TKIs), the prognosis of patients with CML in the chronic phase has improved to point that the majority of patients can expect a multi-decade survival which is now close to that of age and sex matched subjects without the disease, at least in the developed world. However, the situation in the low- and middle-income countries (LMIC), like India, may not be as encouraging. Many hematological cancers in developing countries, including CML, have subtle and not so subtle differences in incidence, age of onset, stage at presentation, phenotype, stage-for-stage response rates, and prognosis as compared to their counterparts in the developed world. Several reasons have been postulated for this and these include; socioeconomic factors, genetic differences, environmental factors (infections, particularly, viral infections, exposure to pesticides, etc.), nutritional factors and factors related to availability of drugs, means for monitoring the disease and option for the use of second generation agents. Generic first generation TKIs (imatinib) and also second generation ones are available in many parts of the world but several challenges still remain in providing optimal treatment to the patients with CML in resource-poor countries. These include availability of optimal and high-quality BCR/ABL testing, availability and cost of second and third generation TKIs (nilotinib, dasatinib, bosutinib, and ponatinib) and hematopoietic stem cell transplantation, compliance and toxicities of drugs and ensuring minimal standard-of-care treatment and monitoring for each and every patient diagnosed with CML. Some of these issues will be reviewed and highlighted in this article.

Introduction Our understanding of the pathophysiology of this disease led to the development of targeted therapies Chronic myeloid leukemia (CML) is a clonal hematopoietic in the form of tyrosine kinase inhibitors, which have a disorder of a pluripotent stem cell that leads to increased proliferation of cells of the myeloid lineage. Its hallmark is the specific action to inhibit the BCR-ABL1 fusion oncogene. presence of reciprocal translocation between the long arms The agents were rightly dubbed “magic bullets” and of chromosomes 9 and 22, t(9;22)(q34;q11), which brings revolutionized the practice of oncology. They have led to a the BCR (breakpoint cluster region) gene into proximity paradigm shift in the management of CML, and now most to the ABL1 (Abelson murine leukemia viral oncogene patients with newly diagnosed disease can look forward homolog 1) gene, forming a new fusion chimeric gene— to a multi-decade survival as opposed to the 4–5 years BCR-ABL1—which is the oncogene responsible for the survival in old times. pathogenesis of the disease.1 The truncated chromosome so In this chapter we have discussed the practical aspects formed is termed the Philadelphia chromosome, owing its of usage of these agents with special focus on optimum use name to the city where it was first described.2 in a setting of resource constraints.

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Clinical Features, Diagnosis, and understanding was that treatment has to be continued lifelong for continued suppression of the BCR-ABL1 Risk Stratification transcript. But with tyrosine kinase inhibitors (TKIs), we A significant proportion of patients are nowadays have managed to achieve such deep suppression of the detected in asymptomatic stage with incidentally disease activity that it is possible in a certain cohort of detected leukocytosis. Patients in more advanced stages patients to discontinue TKI after a few years and attain a of the disease present with signs and symptoms of gross state of treatment-free remission (TFR) and in fact, TFR splenomegaly (abdominal fullness and discomfort has now become one of the standard goals of therapy.4,5 with feeling of early satiety), symptomatic anemia, and constitutional symptoms like fever, fatigue, or weight Tyrosine Kinase Inhibitors loss. Hemogram shows leukocytosis, which may be TKIs are orally administered agents, which act by binding accompanied with thrombocytosis and/or anemia. to the ATP binding site in the BCR-ABL1 protein and inhibit The differential leukocyte count shows prominence its kinase activity. These agents are able to provide much of immature precursors of the granulocytic series, like deeper and sustained responses and can provide near myelocytes and metamyelocytes. Circulating blasts are normal life expectancy in a large proportion of patients. also seen. Basophilia is a consistent feature. The absolute Imatinib was the first TKI approved for the use in CML after eosinophil and monocyte counts are usually increased, showing markedly better results than the previous standard although the percentages are not elevated. Small numbers therapy of interferon plus low dose cytarabine in the pivotal of nucleated red blood cells and mild reticulocytosis may IRIS trial.6 Acquired mutations in the kinase domain can be seen. Clinical chemistry may reveal hyperuricemia and render imatinib ineffective in certain proportion of cases. hyperuricosuria, elevated serum lactate dehydrogenase Second generation TKIs, which include nilotinib, dasatinib, (LDH), increased serum vitamin B12-binding capacity, and bosutinib, can be effective in such a setting. These and increased serum B12 levels. Low or absent neutrophil agents are also approved as first line agents where they alkaline phosphatase activity is seen in 90% of patients. have demonstrated superior activity than imatinib with Bone marrow (BM) examination usually shows respect to rapidity and depth of response, although survival marrow hypercellularity up to 75–90% with marked outcomes are similar (ENESTnd,7 DASISION,8 and BFORE9 increase in myeloid precursors. Blasts usually represent trials). The T315I is a unique mutation that renders the less than 5% of cells in CP CML. Presence of more than disease resistant to all four of these TKIs. The 3rd generation 10% blasts indicate transformation to AP. Megakaryocytes TKI ponatinib is effective in such cases.10 These TKIs are may be hypolobated and typically dwarf forms are seen. summarized in Table 1. The definitive diagnosis of CML is established by demonstrating the presence of the characteristic trans­ Monitoring Response to Therapy location and its resultant transcript. This can be done by Initiation of therapy leads to resolution of symptoms, conventional cytogenetics, fluorescence in situ hybridization splenomegaly, and normalization of blood counts. (FISH) or by polymerase chain reaction (PCR). Complete blood count (CBC) needs to be monitored every The disease has a triphasic clinical course through 2 weeks till a state of complete hematological response is chronic phase (CP), accelerated phase (AP), and blast noted. Subsequent depth of response is monitored using phase (BP).1 Without treatment, the disease inevitably techniques that detect the characteristic chromosomal progresses from CP to AP/BP, which have high morbidity translocation using karyotyping or FISH or by detecting and mortality. Risk stratification helps in predicting the BCR-ABL1 transcript using PCR. Monitoring using survival and it can be done using the Sokal score2 or ELTS PCR based assays is the current gold standard as it allows score.3 for detection of minute quantities of the transcript, and hence responses can be monitored to a degree of depth, Treatment which is not possible by any of the other techniques. The standard goals of therapy were relief of symptoms There can be dramatic variations in the absolute and prevention of progression of the disease. The classical transcript value across laboratories and standardization

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TABLE 1 Tyrosine kinase inhibitors for CML

Generation Imatinib Nilotinib Dasatinib Bosutinib Ponatinib* 1st 2nd 2nd 2nd 3rd Dose 400 mg OD 300 mg BD/400 mg BD 100 mg OD/70 mg BD 400 mg OD/500 mg 45 mg OD (May (1st line/ 2nd line) OD initiate at lower dose) Adverse effects zz Myelosuppression zz Myelosuppression zz Myelosuppression zz Diarrhea zz Thrombotic zz GI intolerance zz Hyperglycemia zz Pleural effusion zz Myelosuppression events zz Fatigue zz Hepatotoxicity zz Pulmonary zz Raised ALT zz Hypertension zz Edema zz Cardiotoxicity hypertension zz Arrhythmias zz Skin changes zz Pancreatitis zz Muscle cramps Sensitive kinase None F317L/V/I/C, T315A, Y253H, E255V/K, F317L/V/I/C, T315A, All mutations domain mutations V299L F359V/I/C Y253H, F359V/I/C including T315I *Not available in India at the time of writing.

TABLE 2 Response definitions

Test Frequency of monitoring Definition of response Clinical examination and Every 15 days until CHR, then Complete hematological response: complete blood counts every 3 monthly unless otherwise required zz Platelets ≤450 × 109/L zz WBC count ≤10 × 109/L zz Differential without immature granulocytes and with ≤5% basophils and zz Non-palpable spleen BCR-ABL1 quantitative Three monthly zz Major Molecular Response (MMR): ≤0.1% PCR (ratio of transcript/ zz MR4: ≤0.01% housekeeping gene zz MR4.5: ≤0.0032% expressed on IS) zz MR5: ≤0.001% zz Deep molecular response (DMR)—MR4 or deeper zz Molecularly undetectable leukemia—Undetectable BCR-ABL1 mRNA transcripts Cytogenetics (chromosome Alone not sufficient for monitoring zz Complete (CCyR): No Ph+ chromosome banding analysis of bone To be done in patients with atypical zz Partial (PCyR): 1–35% Ph+ metaphase marrow metaphase cells) translocations, atypical transcripts of BCR-ABL1 zz Minor (mCyR): 36–65% Ph+ metaphase not quantifiable by PCR, at failure or progression to document ACA

techniques are employed in order to homogenize the into IS values.11 The results are defined on a log scale; results. The BCR-ABL1 transcript value is described as a 1%, 0.1%, 0.001%, 0.0032%, and 0.0001% correspond to 2, ratio of the transcript value to that of a housekeeping gene 3, 4, 4.5, and 5 log reductions respectively from the like ABL1 or GUSB. This ratio is further converted to the baseline value of the IRIS trial. BCR-ABL1 quantitative International scale (IS). The baseline 100% was defined PCR is recommended to be performed every 3 months.5 as the median transcript value measured in 30 pooled The standard response definitions are described in samples of patients of newly diagnosed CML CP enrolled Table 2. in the IRIS trial. Reference materials were generated Effective therapy leads to progressive decline in the using this pool of samples and laboratories can use this to BCR-ABL1 IS and target values at different timepoints are generate specific conversion factors to convert their results defined in order to ascertain the efficacy of treatment. The

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Advanced phase CML (AP and BP) can present either Molecular targets of treatment TABLE 3 (BCR-ABL1 IS values) de novo or as progression from CP. TKIs are effective in TKI-naïve patients with de novo AP. Imatinib at higher Optimal Warning Failure dose of 600 mg or any of the 2G TKIs may be considered Baseline NA High risk ACA, high NA in this setting. Patients with AP who have additional risk ELTS score cytogenetic abnormalities or who have progressed after 3 months ≤10% >10% >10% if confirmed TKI failure respond poorly with second line TKIs and within 1–3 months allogeneic stem cell transplant should be considered. 6 months ≤1% >1–10% >10% CML BP can be either myeloid-type or lymphoblastic-type 12 months ≤0.1% >0.1–1% >1% and this should be determined by immunophenotyping. Anytime ≤0.1% >0.1–1% >1%, resistance The outcomes of CML BP remain poor and the only Loss of MMR mutations, high risk modality that can provide long-term cure in setting of BP is ACA allogeneic stem cell transplant. Patients are usually treated with TKI along with acute myeloid leukemia (AML)-like or acute lymphocytic leukemia (ALL)-like chemotherapy molecular timepoints as per the European LeukemiaNet to bring the disease into chronic phase or hematological 2020 guidelines are described in Table 3.5 In case of remission prior to proceeding with transplant. suboptimal response or failure of therapy there is a need When TKIs were first introduced two decades ago it to determine if any mutations are present in the kinase was thought that they have converted CML into a chronic domain. Detection of these mutations serves as a guide disease with near normal life expectancy and lifelong for choosing the most optimum second line therapy. treatment. But long-term experience with TKIs showed that Mutation analysis was conventionally done using Sanger it is possible to go even a step further. These drugs are able sequencing but Next-generation sequencing has proved to achieve sustained deep molecular responses when used to be more sensitive and has become the recommended over a period of few years and it is possible to discontinue 12 technique. the drug in selected patients, providing them with the Imatinib was the first TKI to be approved for use in equivalent of a “cure.” This concept of TFR was initially CML based on the results of the IRIS trial. The starting explored with the use of the older agent interferon.13 dose is 400 mg once a day but higher doses of 600 mg The STIM114 and TWISTER15 trials demonstrated that and 800 mg have also been tried. The second generation TFR can be achieved in patients on long-term imatinib. TKIs were compared with imatinib as the frontline agent Subsequently a large number of trials have been done in newly diagnosed CML CP in various trials and were with both first line and second line TKIs demonstrating found to induce faster and deeper responses as compared the utility and feasibility of TFR.16 Although all studies have to imatinib, but there was no significant superiority in used different criteria for patient selection, a few common progression-free and overall survivals in any of these criteria have emerged. Ideally the patient should be in studies. No head-to-head comparison is available for first CP, on first line TKI for prolonged duration (5 years or any of the 2G-TKIs. Hence, the choice of frontline TKI more), having achieved optimal response at all milestones depends on a combination of various factors including and having achieved a sustained deep molecular response risk stratification, cost, availability, adverse effect profile, (DMR) for 2 years or more. Patients on TFR should be comorbidities, and individual preferences of the physician motivated and have access to regular monitoring (monthly and the patient. The choice of agent for second line use for initial 6 months, 2 monthly for next 6 months, and also depends on similar factors but also take into account subsequently 3 monthly for life).5 The success rates of mutation status. TFR in almost all trials have been in the range of 40–50%.16 Treatment of patients with failure of two previous TKIs Failure of TFR is considered when there is a loss of MMR. is not so straightforward. Allogeneic stem cell transplant Most failures occur in the initial 6 months but thankfully should be considered in this setting. TKIs (2G and 3G) nearly all patient regain MMR following reinitiation of TKI. can be used but responses are seldom durable. If effective, The success of TFR has led to its incorporation as one of the TKIs can be used as an effective bridge to transplant. standard goals of therapy.

MU-229.indd 1468 29-01-2021 15:28:50 Management of CML in Resource-limited Settings CHAPTER 229 1469

Managing CML in Resource Poor Settings peripheral centers. Presentation with a higher risk score leads to inferior survival outcomes. Although major advances have been made in our „„ Age at diagnosis: Patients of CML in Asian countries understanding, treatment, and monitoring of CML, these present a decade earlier than those in the west.20 advances have not universally permeated into clinical This translates to a longer course of the disease and practice, and this is particularly true for low and middle subsequently a longer need for TKIs and monitoring, income countries. Patients and physicians in these regions which adds to the overall financial burden. of the world face unique challenges at every step of disease „„ Investigations: Molecular and cytogenetic methods management. Efforts are needed to ensure that our for diagnosis and monitoring are not readily available patients are able to obtain similar benefits of treatment as in smaller cities. Even with availability, the cost of those in developed nations.17 these investigations ends up posing a hindrance to An algorithm for guidance in resource-limited setting their usage. The standard recommendations on the is provided in Flowchart 1. monitoring frequency (once in 3 months) are seldom, if ever, followed in India. Clinicians have to devise Specific Issues Related to CML Treatment in their own modifications to these guidelines in order to Resource-limited Settings ensure that the patient remains well monitored yet not „„ Delays in diagnosis: A majority of CML patients in India be overburdened by the cost. are diagnosed late and have a higher disease burden „„ Drugs: The cost of imatinib at the time of launching was and higher risk score at the time of diagnosis.18,19 prohibitively expensive, but three major developments This is related to poor awareness of the disease have ensured that today they are within the reach and inadequate access to diagnostic modalities in of nearly every patient who needs it. In this regard,

Flowchart 1: Algorithm for CML-CP treatment in resource-limited settings

Allo SCT, allogenic stem cell transplantation; CP CML, chronic phase CML; TKI, tyrosine kinase inhibitor

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massive strides have been made in the last two References decades. Firstly, the Glivec International Patient . 1 Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Assistance Program (GIPAP), in association with the Tumours of Haematopoietic and Lymphoid Tissues. Vol. 2, 4th Max Foundation, which sought to make imatinib edition. Lyon, France: IARC; 2017. available free of cost to patients particularly in low and . 2 Nowell PC, Hungerford DA. A minute chromosome in human middle income countries.21 India has been the largest chronic granulocytic leukemia. Science. 1960;132:1497. beneficiary of this program, and GIPAP has provided . 3 Sokal JE, Cox EB, Baccarani M, et al. Prognostic discrimination free of cost imatinib to more than 12,000 patients in “good-risk” chronic granulocytic leukemia. Blood. 1984;63(4): (25% of all GIPAP beneficiaries) from 2002 onward. 789-99. The program stopped enrolling new patients in 2016. 4. firrmann M, Baccarani M, Saussele S, et al. Prognosis of long-term survival considering disease-specific death in patients with chronic Secondly, a large number of Indian pharmaceutical myeloid leukemia. Leukemia. 2016;30(1):48-56. companies started production of generic imatinib, . 5 Hochhaus A, Baccarani M, Silver RT, et al. European LeukemiaNet which is available to patients at a much cheaper 2020 recommendations for treating chronic myeloid leukemia. rate. Thirdly, several public funded institutions Leukemia. 2020;34(4):966-84. provide generic imatinib free of cost. These benefits . 6 O’Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with are not restricted to imatinib alone. Nilotinib is interferon and low-dose cytarabine for newly diagnosed chronic- available at a concessional cost as a part of a patient phase chronic myeloid leukemia. New Eng J Med. 2003;348(11): assistance program. Generic formulations of Dasatinib 994-1004. . 7 Saglio G, Kim D-W, Issaragrisil S, et al. Nilotinib versus Imatinib and Bosutinib were launched in early 2020. These for newly diagnosed chronic myeloid leukemia. New Eng J Med. have ensured that even the 2G TKIs are much more 2010;362(24):2251-9. accessible to patients than what they were previously 8. Kantarjian H, Shah NP, Hochhaus A, et al. Dasatinib versus Imatinib and have led to increased use of these agents both as in newly diagnosed chronic-phase chronic myeloid leukemia. New first line and second line. Eng J Med. 2010;362(24):2260-70. „„ Adherence to treatment: Poor adherence to treatment . 9 Cortes JE, Gambacorti-Passerini C, Deininger MW, et al. is a major barrier to obtaining favorable long-term Bosutinib versus Imatinib for newly diagnosed chronic myeloid outcomes.22 Various factors non-adherence relevant leukemia: results from the randomized BFORE trial. JCO. 2017;36(3): to the Indian context are financial constraints, lack of 231-7. 10. Cortes JE, Kim D-W, Pinilla-Ibarz J, et al. A phase 2 trial of ponatinib social support, and poor patient awareness about the in Philadelphia Chromosome–Positive Leukemias. New Eng J Med. disease and treatment. Treating clinicians must pay 2013;369(19):1783-96. adequate attention to addressing these issues. Adequate 11. Branford S. Chronic myeloid leukemia: molecular monitoring in counseling at diagnosis and reinforcement of the same clinical practice. Hematology. 2007;2007(1):376-83. at each hospital visit is essential. Various government 12. Soverini S, Bavaro L, De Benedittis C, et al. Prospective assessment and non-government aid regarding provision of TKIs of NGS-detectable mutations in CML patients with nonoptimal go a long way in ensuring uninterrupted treatment. response: the NEXT-in-CML study. Blood. 2020;135(8):534-41. 13. Mahon FX, Delbrel X, Cony-Makhoul P, et al. Follow-up of complete Conclusion cytogenetic remission in patients with chronic myeloid leukemia after cessation of interferon alfa. J Clin Oncol. 2002;20(1):214-20. CML is a chronic myeloproliferative neoplasm characterized by 14. Mahon FX, R´ea D, Guilhot J, et al. Discontinuation of imatinib in t(9;22)(q34;q11) and the resultant chimeric BCR-ABL1 oncogene. patients with chronic myeloid leukaemia who have maintained Effective targeted therapy is now available in the form of TKIs, complete molecular remission for at least 2 years: the prospective, which can induce deep and prolonged molecular responses multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010; in a large number of patients. TFR has shown moderate 11(11):1029-35. success and has become one of the major goals of treatment. 15. Ross DM, Branford S, Seymour JF, et al. Safety and efficacy of Challenges remain in low and middle income countries related imatinib cessation for CML patients with stable undetectable to accessibility and affordability of investigations and treatment. minimal residual disease: results from the TWISTER study. Blood. Various governmental and non-governmental schemes along 2013;122(4):515-22. with availability of generic medications have gone a long way in 16. Mahon F-X. Treatment-free remission in CML: who, how, and why? Hematology Am Soc HematolEduc Program. 2017;2017(1): ensuring our goal of providing treatment for all. 102-9.

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17. Malhotra H, Radich J, Garcia-Gonzalez P. Meeting the needs of 20. Singhal MK, Sengar M, Nair R. Summary of the published Indian data CML patients in resource-poor countries. Hematology Am Soc on chronic myeloid leukemia. South Asian J Cancer. 2016;5(3):162-5. HematolEduc Program. 2019;2019(1):433-42. 21. Garcia-Gonzalez P, Boultbee P, Epstein D. Novel Humanitarian Aid 18. Ganesan P, Kumar L. Chronic myeloid leukemia in India. J Glob Program: The Glivec International Patient Assistance Program— Oncol. 2017;3(1):64-71. Lessons Learned From Providing Access to Breakthrough Targeted 19. Deotare UR, Chudgar U, Bhagat E. Report of patients with Oncology Treatment in Low- and Middle-Income Countries. JGO. chronic myeloid leukemia, from hematology clinic, Ahmedabad, 2015;1(1):3-45. Gujarat 2000-2010 at 1 stmyelostone meeting: Indian evidence 22. Ganesan P, Sagar TG, Dubashi B, et al. Nonadherence to imatinib of chronic myelogenous leukemia. Indian J Med Paediatr Oncol. adversely affects event free survival in chronic phase chronic 2013;34(3):193-5. myeloid leukemia. Am J Hematol. 2011;86(6):471-4.

MU-229.indd 1471 29-01-2021 15:28:51 CHAPTER

Disability-free Hemophilia in 230 India—Myth or Reality

Sunita Aggarwal, Ranvijay Singh, Sandeep Garg

Abstract Hemophilia is a rare X-linked genetic disorder where a patient bleeds because of lack of clotting factors essential for coagulation. Based on the type of clotting factor deficiency, hemophilia is classified into hemophilia A, which is deficiency of factor VIII and deficiency of a clotting factor IX is called hemophilia B. Currently, replacing the deficient clotting factors with plasma derived and recombinant clotting factor is the widely accepted treatment modality for hemophilia. In many countries, advanced treatment prophylaxis is the accepted modality of treatment, which allows persons living with hemophilia (PWH) to lead a disability-free life. According to recent survey, India has 10% of all the PWH in the world.1 Yet most of the patients have not been adequately treated because of various factors. A void in awareness, unavailability of clotting factors, high treatment costs, poor economic status, and lack of home therapy along with low frequency of physiotherapy exercises are causing big dent in the Indian PWH’s aspiration in achieving a disability-free life. In India, access to prophylaxis and optimal dosages of CFCs is still not up to the established standards of care. This review describes the current scenario of hemophilia care and the challenges faced by PWH in India.

Introduction heterogeneous transformations of the coagulating factor qualities because of the mutation F8C gene and F9 gene Hemophilia is a mostly inherited genetic bleeding causes Hemophilia A and B, respectively. Hemophilia A disorder, first found in the literatures of 2nd century’s constitutes to about 80–85% of the hemophilia patients Jewish-Talmud (Jewish religious script). A more detailed (Table 1).1 explanation was given by the 11th century physician Albucasis. The word “hemophilia” is derived from the Greek word “haima” which means “blood” and “philia” Clinical Features which means “love or attraction.” In the late 18th and Hemophilia is X-linked recessive disease where male is early 19th century, the understanding of hemophilia clinically affected and women are almost asymptomatic. evolved and conceptualized. The European royal families Family history is absent in ~30% of the cases, attributing suffered during the same period, and it led the world to to de novo mutation. Severe the hemophilia earlier the recognize the disease and brought it into prominence. presentation of first bleeding episode, which can present The two types of hemophilia, that is, Hemophilia A and as early as at birth and majority of them are spontaneous Hemophilia B (Christmas disease) were recognized in the bleeds. New born can present as cephalohematoma, mid-20th century. Hemophilia is transmitted as X-linked central nervous system (CNS) bleed, or excessive bleeding recessive pattern where males are usually affected and during medical intervention like venipunctures and females remain as asymptomatic carriers. The lack of circumcision. As child starts walking, joint bleed, bruising

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TABLE 1 Hemophilia clinical classification2

Hemophilia type FVIII/FIX activity (% of normal) Pattern of bleeding episodes Clinical manifestation Mild 6–40 Uncommon Major trauma, Surgery Moderate 1–5 4–6 per year Minor trauma Severe <1 24–48 per year Spontaneous

and musculoskeletal bleed becomes more common. In suspected when patient fails to respond to replacement of older children and adult, presentation is mainly joint and CFC. High titer inhibitor (HTI) and low titer inhibitor (LIT) muscle bleed. Hemarthrosis can affect every joint but are inhibitor classes quantified by Bethesda units (BU). commonly involves knee, elbow, ankle, shoulder, and High titer inhibitor (≥5BU) and low titer inhibitor hips. However CNS, oropharyngeal, and retroperitoneal (<5BU) behave differently and consequently are bleed are life threatening and require immediate therapy. managed differently. LTI may be transient and disappear spontaneously without specific treatment but significant Complications proportion of it gets converted into HTI; hence, demands close monitoring. Treatment of bleed in LTI include Hemophilic arthropathy: Most common and develops in factor infusion at higher dose, porcine rFVIII, which ~50% of patients with severe hemophilia. Mechanism of is not quickly inactivated unlike human FVIII and arthropathy is multifactorial including chronic or episodic Desmopressin (DDAVP) in mild hemophilia A, which synovitis, loss of cartilage, subchondral cyst formation, 5 releases endogenous FVIII. bone cysts, erosion and joint space narrowing leading to HTI are persistent and completely resistant to contractures, pain, and limitation of motion. factor concentrates; hence, their management requires Infection: Found relatively more in plasma-derived avoidance of further FVIII exposure until immune products compare to recombinant factor products and tolerance induction (ITI) is commenced where infusions of elimination of Parvovirus B19, hepatitis A and prion variable doses of FVIII and FIX given over a period of time diseases like Creutzfeldt-Jakob disease is still a challenge. to tolerize the immune response. Use of bypassing agents (rFVIIa and aPCC) and monoclonal antibody emicizumab Pseudotumor: Unique to hemophilia, pseudotumor can be opted.1,5 Emicizumab (Hemlibra) is registered in is a potentially disabling condition that results from over 50 countries around the world for prophylactic use for inadequately treated soft tissue bleeds usually in muscle hemorrhagic episodes in adult and pediatric hemophilia adjacent to bone, which can be secondarily involved. A patients with inhibitors.6 In addition, this drug has Inhibitors: The cumulative incidence (i.e., lifetime risk) recently been approved by FDA for prophylaxis in patients of inhibitor development in severe hemophilia A is in the with hemophilia A without FVIII inhibitors.6 range of 20–30%, approximately 5–10% in moderate or mild disease and hemophilia B has 1–5% lifetime risk of inhibitor development.3,4 Treatment Modalities Inhibitors are neutralizing alloantibodies developed Hemophilia is treated by replacing the deficient clotting following the host immune response to the infusion of factors. Based on source, there are plasma derived clotting clotting factor concentrate, seen by host immune system factors concentrates (PDCFCs) and recombinant clotting as foreign protein. Inhibitors development is highest factors (rFVIII/rIX). Recombinant clotting factors are during the first 20 exposure days (ED) to factors. Frequent inherently safer as the risk of transfusion transmitted screening is required during initial exposure days, diseases is much less compared to plasma derived CFCs. screening every 5ED until 20ED are reached and after There are two modalities in treatment based on timing 150ED frequency of screening can be reduced. Inhibitors of the therapy, i.e., episodic/on demand therapy and are detected either during routine screening or it is prophylaxis therapy.7

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TABLE 2 Dose required in hemophilia2

Type 1 unit/kg factor VIII/IX Dose required (in units) Hemophilia A Raise the plasma FVIII level approximately 2% [Body weight × Desired level]/2 Hemophilia B Raise the plasma FIX level approximately 1% [Body weight × Desired level]

twice weekly. In countries with resource constraints lower Desired level of factors in different sites of bleed in TABLE 3 hemophilia A & B4 dose of prophylaxis maybe given as an interim measure.

Site of bleed Desired level (IU/dL) On Demand versus Prophylaxis Therapy Muscle/joint 40 Manco-Johnson et al. conducted a randomized Iliopsoas bleed 60 controlled study and found prophylaxis is more effective Throat 50–80 in preventing hemarthrosis and is also efficacious in Neck decreasing bleeding and joint damage compared to on demand therapy.8 Another randomized controlled study, GI bleed ESPRIT study, concluded by saying prophylaxis was Genitourinary bleed more effective when started at age 36 months or less with Surgery (Postoperative) PWH having fewer joint bleeds and no radiologic signs 9 Surgery (Preoperative) 100 of arthropathy versus patients treated on demand. The Intracranial bleed MUSFIH study concluded, on demand therapy failed to preserve musculoskeletal functions in PWH.10 All these studies are stressing the importance of prophylactic On Demand Therapy replacement of clotting factors over the on-demand Assuming the baseline factor of persons living with therapy. Even in India, several pilot studies conducted hemophilia (PWH) as 0%, the therapy should raise the across India have shown the evidence of the advantage of 11,12 clotting factor to a certain minimum level in order to stop prophylaxis over on-demand. the bleeding. Although on demand, treatment can stop bleeding, reduce the pain to an extent and restore joint Hemophilia Scenario in India movement. It will not protect the PWH from arthropathy. India has currently 20,321 registered PWH. 17,606 with The target dose of CFC is calculated with the help of hemophilia A and 2,715 with hemophilia B.1 Majority of bleeding site specific desired levels and approximate raise Indian PWH fall into 19–44 years of age. However, as per of plasma factor levels (Tables 2 and 3). the incidence and prevalence estimates, India should have had approximately 100,000 PWH. Many of the patients go 7 Prophylaxis Therapy undiagnosed or unregistered because of lack of awareness Here the treatment is given by intravenous injection of and lack of diagnostic facilities. factor concentrate to prevent anticipated bleeding. It Among the registered patients, a significant percentage prevents life threatening bleeding, joint destruction and of them suffer from joint disease due to recurrent joint preserves normal musculoskeletal function. Prophylaxis bleed. In a study of 148 severe hemophilia A patients has been a standard of care in developed countries. from 5 centers across India, Kar et al. found that about Two well-studied and documented clinical protocols for 94% had some form of disability. Patients from the age prophylaxis are being followed across the world: group 25+ were the most affected with 0% of them being 1 13 „„ Malmo protocol: (high dose: 25–40 IU/kg/dose) free from disability. This pervasive disability has turned „„ Utrecht protocol: (intermediate dose: 15–30 IU/kg/ hemophilia from a mere bleeding disorder to a chronic dose)1 musculoskeletal disorder. Many of these patients in India For hemophilia A, the factors are administered thrice do not complete education, are not gainfully employed a week and for hemophilia B, the factors are administered and become a burden to their families and the society. In

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contrast, in most developed countries, PWH enjoy good qualified treating physicians, nurses, lab technicians and joint health, are not burdened with disability and lead a physiotherapists with adequate experience of handling normal and productive life. PWH.14 HTCs are mainly supported by respective State This stark contrast among the quality of life led by PWH Governments and in some cases by Central Government in India and other countries may be ascribed to several as well.14 Some hospitals also serve as tertiary care centers factors: for hemophilia care where advanced management for „„ Lack of optimum management of bleeds: Bleeds are not inhibitors and even surgeries are provided. However, treated with optimum dosages in many cases. In India, the number of such surgeries is abysmally low. Some of mean per capita factor VIII and IX usage is 0.230 and the tertiary centers have started prophylaxis and home 0.063 IU/population while global mean is 2.40 and 0.37 therapy as pilot projects, albeit at smaller scale. IU/population.1 Also the time from onset of bleed to The Hemophilia Federation of India (HFI) is a patient factor administration is rarely less than 2 hours. This support group, working to help PWH in India in various leads to increased blood in the joint space, increased ways. It also performs a vital task of maintaining a national inflammation and increased time to recovery and in hemophilia registry for all PWH. the long-term results in joint arthropathy. „„ Lack of prophylaxis: Only about 13% of Indian PWH Conclusion have access to prophylaxis.1 Some of the reasons could be lack of awareness, difficulty in venous Persons living with hemophilia (PWH) in India still face many access in young children, training the parents of issues in terms of comprehensive care, inhibitor management, optimum dosage in acute bleeds and optimum prophylaxis PWH and in some cases inadequate supply of clotting regimen. Paving our way toward WFH’s vision of “Treatment factors. Moreover, psychosocial barriers like handling for All”. Awareness, access to safe factors, prophylaxis and the myths, misconception, apprehensions about home therapy should be the focus areas for all the concerned prophylaxis and willingness to endure repeated stakeholders to work in a coordinated fashion and make the venipunctures play a vital role. treatment accessible to all the PWH. With such an approach of „„ Lack of home therapy: Self-administration at home comprehensive hemophilia care and tireless efforts of medical setting demands skills and expertise with proper experts, government, health administrators, and even patients, training of the family members. Lack of motivation we can aspire for a disability-free hemophilia in India. among patients and administrative hurdles reduce the efficiency of home therapy. „„ Lack of diagnostic facilities: Only a few laboratories References across the country can perform factor and inhibitor . 1 Stonebraker JS, Bolton-Maggs PHB, Brooker M, et al. The World assays. This may be one of the reasons why only small Federation of Hemophilia Annual Global Survey 1999-2018. Haemophilia; 2020. portion of the PWH are identified. . 2 Srivastava A, Brewer AK, Mauser-Bunschoten EP, et al. Treatment However, there has been a significant improvement in guidelines working group on behalf of the world federation the hemophilia care in India in the recent years. Out of all of hemophilia. Guidelines for the management of hemophilia. the genetic disorders and hemoglobinopathies hemophilia Haemophilia. 2013;19(1):1-47. have attracted a fair share of support and funding from . 3 Astermark J, Altisent C, Batorova A, et al. European Haemophilia the government. Over the last few years treatment of Therapy Standardisation Board. Non-genetic risk factors and the development of inhibitors in haemophilia: a comprehensive review hemophilia in India has evolved from whole blood and consensus report. Haemophilia. 2010;16(5):747-66. transfusion, fresh frozen plasma and cryoprecipitates 4. Wight J, Paisley S. The epidemiology of inhibitors in haemophilia A: to plasma derived and recombinant clotting factor a systematic review. Haemophilia. 2003;9(4):418-35. concentrates. It is also encouraging to note that hemophilia . 5 Paisley S, Wight J, Currie E, et al. The management of inhibitors is now listed in “The Rights of Persons with Disabilities in haemophilia A: introduction and systematic review of current Act, 2016”. The government is allocating funds, purchasing practice. Haemophilia. 2003;9(4):405-17. 6. Franchini M, Marano G, Pati I, et al. Emicizumab for the treatment of good quality clotting factors and bypassing agents. haemophilia A: a narrative review. Blood Transfus. 2019;17(3):223-8. Many hemophilia treatment centers (HTCs) are opening . 7 Ljung R. Hemophilia and prophylaxis. Pediatr Blood Cancer. across the country to help PWHs. Most HTC have well 2013;60(1):23-6.

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. 8 Manco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophylaxis story from a resource limited country. Haemophilia. 2016;22(3): versus episodic treatment to prevent joint disease in boys with 342-8. severe hemophilia. N Engl J Med. 2007;357(6):535-44. 12. Mohite A, Chandrakala S. Low dose escalating factor concentrate 9. Gringeri A, Lundin B, von Mackensen S, et al. A randomized clinical prophylaxis in patients with severe hemophilia in a comprehensive trial of prophylaxis in children with hemophilia A (the ESPRIT Study). hemophilia care centre of western India. Presented at the 57th J Thromb Haemost. 2011;9(4):700-10. Annual conference of IHSBT, Haematocon-2016 in Jaipur. Abstract 10. Poonnoose P, Carneiro JDA, Cruickshank AL, et al. MUSFIH Study No: BHC-8. group. Episodic replacement of clotting factor concentrates does 13. Kar A, Phadnis S, Dharmarajan S, et al. Epidemiology & social costs not prevent bleeding or musculoskeletal damage—the MUSFIH of haemophilia in India. Indian J Med Res. 2014;140(1):19-31. study. Haemophilia. 2017;23(4):538-46. 14. Berntorp E, Lethagen S. The role of home infusion therapy in 11. Basu D, Biswal N, Ramesh A, et al. A randomized study of very haemophilia. Disease Management and Health Outcomes. low-dose factor VIII prophylaxis in severe haemophilia—a success 2000;7(2):77-81.

MU-230.indd 1476 29-01-2021 15:28:43 CHAPTER

Immunotherapy in 231 Clinical Practice

Vineet Talwar, Amrith BP

Abstract Cancer immunotherapy is rapidly evolving and is gaining a major role in current oncological practice. Advances in understanding of immune surveillance and tumor biology have opened up new therapeutic strategies that can be used for the treatment of many cancers. Immune evasion is a hallmark of property of cancer and cancer immunotherapy uses strategies to augment body’s immunity against cancer. A subset of cancer patients dramatically benefit from this approach and identifying a right patients through a biomarker is need of the hour. In addition, knowledge on tumor response to immunotherapy and toxicity profile of these agents are important as they differ from traditional cytotoxic chemotherapies by virtue of its unique mechanism of action.

Introduction tumor growth despite having a normally functioning host immune system. Various mechanisms for immune escape Advances in understanding of the interlink between include: immune surveillance and tumor biology have opened up „„ Up regulation of immune checkpoints such as PD-1 new therapeutic strategies that can be used for treatment and PD-ligand 1 (PD-L1) of many cancers.1 Nobel prize for physiology/medicine in „„ Alteration of antigen presentation mechanism by loss 2018 was awarded to James P. Allison for the discovery of of MHC class 1 expression or cellular mechanisms cytotoxic T-lymphocyte associated protein (CTLA-4) and that help in transportation of tumor antigens for T cell to Tasuku Honjo for programmed cell death protein 1/ recognition programmed cell death protein ligand 1 (PD-1/PD-L1), a „„ 2 Promotion of immune tolerance by alteration of landmark discoveries in the field of immunotherapy. Cancer cytokines like IL-6, IL-10, and TGF-beta immunotherapy is based on the principle of strengthening Proper understandings of these mechanisms have the host immune system to combat against the cancer. been exploited as the basis of immunotherapy in current clinical practice. Evasion of Immune Surveillance Evasion of the immune surveillance is one of the hall Approaches to Cancer Immunotherapy marks of cancer.3 Adaptive immunity directed at the cancer A number of therapeutic approaches are in practice or antigens is one of the defense mechanism which helps to under investigation to strengthen the body’s immune fight against the cancer. There are several mechanisms system fight against cancer. Various immunotherapy by which the cancer cells escape this natural defense approaches in current practice are enumerated in the known as “immune evasion.”4 Immune evasion helps in Box 1.

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BOX 1 Immunotherapy approaches in current practice

Modalities of immunotherapy Check point inhibitors zz PD-1 and PD legend (PD-L1) inhibitors zz CTLA-4 inhibitors Manipulation of T cells zz Chimeric antigen receptors zz Bispecific T cell engagers Cancer vaccines Cytokines Oncolytic viruses

Checkpoint Inhibitors Programmed Cell Death 1 (PD-1) and PD-Ligand 1/2 Programmed cell death-1 (PD-1), a transmembrane Fig. 1: PD-1/PD-L1 pathway and check point inhibitors protein expressed on immune cells like T cells, B cells, and NK cells. Ligand of PD-1 (PD-L1) is usually expressed initially known as immune confirmed progressive disease on many tissues including tumor cells. Interaction of the (IUPD) commonly known as pseudoprogrssion.9 In such PD-1 with its ligand acts as check point and resists the cell situations the clinical status of the patient should be taken death by inhibition of apoptosis.5 Up regulation of PD-L1 into consideration in deciding the further course of action. expression is seen in many tumor cells and drugs targeting To address these issues many response criteria PD-1/PD-L1 pathway known as check point inhibitors have been developed like immune-related response are an important class of immunotherapy in current criteria (irRC), immune response evaluation criteria in oncological practice. The interaction of the PD-1 with its solid tumors (iRECIST),10 immune-modified response ligand is depicted in the schematic Figure 1. evaluation criteria in solid tumors (imRECIST).11 Detailed Cytotoxic T-lymphocyte-associated protein 4 (CTLA- description of these criteria is beyond the scope of this 4) is another immune check point, which helps in down chapter. regulation of the immune responses against tumor cells.6 Monoclonal antibodies targeting CTLA-4 can also clear Toxicities Associated with Checkpoint Inhibitor these “breaks” in immune surveillance. Ipilimumab, an Immunotherapy Immune-related Adverse Events anti-CTLA-4 antibody was the first immune checkpoint (IRAEs) inhibitor to be approved in patients with malignant The toxicities depend on the class of immunotherapeutic 7 melanoma. agents. Check point inhibitors are the most commonly The various check point inhibitors currently in use are used class of immunotherapy in clinical practice. Though listed in Table 1 with the approved indications and doses. these drugs are well tolerated in most of the cases they are known for unique side effects related to their Immunotherapy Response Criteria mechanism of action. These are known as immune- The pattern of response to immunotherapies mainly related adverse events (irAEs).12 Virtually these can affect checks point inhibitors that can differ from that of classical any organs. However, the important organs affected and cytotoxic chemotherapeutic agents by the virtue of the manifestations are depicted in the Figure 2. The usual difference in mechanism of action of these agents.8 Unlike timeline of appearance of these side effects are shown in chemotherapy the responses may take longer time to the Figure 3. become apparent. The immune infiltration of the tumors The management of irAEs depends on the severity can lead to an apparent increase in the size of the lesion of manifestations. It varies from close monitoring,

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TABLE 1 Check point inhibitors with approved indications

Drug Target Dose Approved indications Nivolumab PD-1 240 mg once every weeks zz Head and neck squamous cell carcinoma Or zz Hepatocellular carcinoma 480 mg once every 4 weeks zz Hodgkin lymphoma zz Melanoma zz Lung cancer zz Renal cell cancer zz Urothelial cancer Pembrolizumab PD-1 200 mg once every 3 weeks zz Cervical cancer (recurrent or metastatic) zz Esophageal cancer (recurrent locally advanced or metastatic) zz Gastric cancer (recurrent locally advanced or metastatic) zz Head and neck cancer, squamous cell zz Hepatocellular carcinoma zz Hodgkin lymphoma, classical (relapsed or refractory) zz Melanoma zz Microsatellite instability-high cancer zz Non-small cell lung cancer zz Renal cell carcinoma zz Urothelial carcinoma Cemiplimab PD-1 350 mg once every 3 weeks zz Cutaneous squamous cell carcinoma, metastatic or locally advanced Atezolizumab PD-L1 Breast cancer: 840 mg Day 1 and zz Breast cancer (triple-negative) Day 15 every 4 weeks zz Lung cancer Others: 1200 mg every 3 weeks zz Urothelial carcinoma Durvalumab PD-L1 10 mg/kg every 2 weeks zz Lung cancer zz Urothelial carcinoma Avelumab PD-L1 800 mg once in every 2 weeks zz Merkel cell carcinoma zz Renal cell carcinoma zz Urothelial carcinoma Ipilimumab CTLA-4 1–10 mg/kg (depending on the zz Melanoma indication) zz HCC zz RCC

interruptions in treatment, dose reductions, and other are engineered by combining antigen-binding and T cell pharmacological management. The pharmacological activating functions into a single receptor.14 So CAR-T cell agents like steroids, infliximab, may be used in the therapy utilizes these T cells engineered with CARs as a management of irAEs.13 therapeutic strategy. CAR-T cells are studied in various solid and Other Forms of Immunotherapy hematological malignancies and have shown a great result in B-cell acute lymphoblastic leukemia (B-ALL). Manipulating T Cells Tisagenlecleucel and axicabtagene-ciloleucel were Immune enhancement by manipulating the T cells is the first two CAR-T cell therapy receiving FDA and another emerging mode of immunotherapy. EMA approval for ALL (tisagenlecleucel) and diffuse- large B-cell lymphoma – DLBCL (tisagenlecleucel and Chimeric Antigen Receptors (CAR) T Cells axicabtagene ciloleucel).15 CAR-T cell therapy involves the modification of the patient’s own T cells to recognize the cancer cells more CD3-Directed Therapies—Bispecific T-cell Engagers effectively and to destroy them. Chimeric antigen receptors Bispecific T-cell engager antibodies (BiTEs) act as linker (CARs) are the engineered proteins that give T cells this between T cells and specific target antigens. These consist new acquired ability. These proteins are chimeric as they of a protein, which contains two separate variable regions,

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Fig. 2: Immune-related adverse events (irAEs)

Blinatumomab is a BiTE having specificity for CD19 found on B cells and the Fc region of the CD3 receptor on T lymphocytes and is approved for Philadelphia- chromosome negative B-ALL.17

Oncolytic Viruses Several viruses are genetically engineered to infect the cancer cells preferentially and to present the tumor associated antigens to immune system. Many virus backbone like attenuated herpes simplex virus 1, adenovirus, reovirus have been studied in clinical trials. talimogene laherparepvec (T-VEC) uses an attenuated HSV-1 to over express granulocyte macrophage colony- stimulating factor (GM-CSF) and mediates the antigen presentation through dendritic cells. Intratumoral Fig. 3: Timeline of immune-related adverse events (irAEs) injections of T-VEC have shown durable responses in Melanomas.18 one recognizing CD3 on T cells and the other recognizing target antigen. Thus, activating cytotoxic T cell-mediated Therapeutic Cancer Vaccine tumor damage. BiTEs functioning in an MHC independent Therapeutic cancer vaccines are a form of immunotherapy, manner and don’t require patient specific processing which educates the immune system about how cancer enabling it to administer to all the patients irrespective of cells look like so that it can fight against it. Vaccine antigen human leukocyte antigen (HLA) type.16 is the most important component of a cancer vaccine,

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which can be tumor-associated antigen or tumor-specific 4. Vinay DS, Ryan EP, Pawelec G, et al. Immune evasion in cancer: antigen. There are mainly three types of vaccine platforms mechanistic basis and therapeutic strategies. Semin Cancer Biol. 2015;35:S185-98. that are being investigated for cancer therapy. These 5. Han Y, Liu D, Li L. PD-1/PD-L1 pathway: current researches in cancer. includes cellular vaccines (autologous or allogeneic), virus Am J Cancer Res. 2020;10(3):727-42. vector vaccines, and molecular vaccines comprised of 6. Seidel JA, Otsuka A, Kabashima K. Anti-PD-1 and Anti-CTLA-4 either peptides, DNA, or RNA. therapies in cancer: mechanisms of action, efficacy, and limitations. The first US Food and Drug Administration approved Front Oncol. 2018;8:86. 19 7. Schadendorf D, Hodi FS, Robert C, et al. Pooled analysis of long- therapeutic cancer vaccine is sipuleucel-T (Provenge). term survival data from phase II and phase III trials of ipilimumab in It was used for metastatic castration-resistant prostate unresectable or metastatic melanoma. JCO. 2015;33(17):1889-94. cancer (mCRPC). Sipuleucel-T was developed by enriching 8. Wolchok JD, Hoos A, O’Day S, et al. Guidelines for the evaluation of the patient derived DCs and their activation ex vivo with a immune therapy activity in solid tumors: immune-related response chimeric protein, GM-CSF fused to the antigen PAP. criteria. Clin Cancer Res. 2009;15(23):7412. 9. Borcoman E, Nandikolla A, Long G, et al. Patterns of response and progression to immunotherapy. Am Soc Clin Oncol Educ Book. Cytokines 2018;(38):169-78. The initial approaches of immunotherapy were mainly 10. Seymour L, Bogaerts J, Perrone A, et al. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. based on alteration of cytokines that influence the Lancet Oncol. 2017;18(3):e143-52. immune system. 11. Hodi FS, Ballinger M, Lyons B, et al. Immune-modified response „„ Interleukin-2(IL-2): evaluation criteria in solid tumors (imRECIST): refining guidelines —— IL-2 plays an important role in activation of to assess the clinical benefit of cancer immunotherapy. JCO. 2018;36(9):850-8. immune system and is an approved treatment for 20 12. Naidoo J, Page DB, Li BT, et al. Toxicities of the anti-PD-1 renal cell carcinoma and melanoma. and anti-PD-L1 immune checkpoint antibodies. Ann Oncol. —— Immunomodulators like lenalidomide and 2015;26(12):2375-91. pomalidomide are an established therapy in 13. Trinh S, Le A, Gowani S, et al. Management of immune-related multiple myeloma act by destruction of Ikaros adverse events associated with immune checkpoint inhibitor therapy: a minireview of current clinical guidelines. Asia Pac J Oncol family proteins, which results in inhibition of IL-2 Nurs. 2019;6(2):154-60. 21 secretion. 14. Hughes-Parry HE, Cross RS, Jenkins MR. The evolving protein „„ Interferon (IFN) alfa-2b has also been used in engineering in the design of chimeric antigen receptor T cells. Int J malignant melanoma. Mol Sci. 2019;21(1):204. 15. Zavras PD, Wang Y, Gandhi A, et al. Evaluating tisagenlecleucel and its potential in the treatment of relapsed or refractory diffuse Conclusion large B cell lymphoma: evidence to date. Onco Targets Ther. 2019;12:4543-54. The immune-oncology has transformed the care of cancer 16. Suryadevara CM, Gedeon PC, Sanchez-Perez L, et al. Are patients. These act in a targeted manner minimizing the side BiTEs the “missing link” in cancer therapy? Oncoimmunology. effects. Checkpoint inhibitors are the mainstay of current 2015;4(6):e1008339. immunotherapy practice in oncology. CAR-T cell therapies and 17. Hoffman LM, Gore L. Blinatumomab, a bi-specific anti-CD19/CD3 personalized cancer vaccine have limited utility at present, BiTE(®) antibody for the treatment of acute lymphoblastic leukemia: which will be added to the immune-oncology armamentarium perspectives and current pediatric applications. Front Oncol. for wider use in near future. 2014;4:63. 18. Conry RM, Westbrook B, McKee S, et al. Talimogene laherparepvec: first in class oncolytic virotherapy. Hum Vaccin Immunother. 2018;14(4):839-46. References 19. Cheever MA, Higano CS. PROVENGE (Sipuleucel-T) in prostate 1. Finn OJ. A Believer’s overview of cancer immunosurveillance and cancer: the first FDA-approved therapeutic cancer vaccine. Clin immunotherapy. J Immunol. 2018;200(2):385-91. Cancer Res. 2011;17(11):3520. 2. Altmann DM. A Nobel Prize-worthy pursuit: cancer immunology 20. Jiang T, Zhou C, Ren S. Role of IL-2 in cancer immunotherapy. and harnessing immunity to tumour neoantigens. Immunology. Oncoimmunology. 2016;5(6):e1163462. 2018;155(3):283-4. 21. Lu G, Middleton RE, Sun H, Naniong M, et al. The myeloma drug 3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. lenalidomide promotes the cereblon-dependent destruction of Cell. 2011;144(5):646‐74. Ikaros proteins. Science. 2014;343(6168):305-9.

MU-231.indd 1481 29-01-2021 15:28:39 CHAPTER

PNH—A Great Masquerader: 232 When to Suspect?

Asish Rath, Jasmita

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterized by partial or complete deficiency of glycophosphatidylinositol anchored proteins (GPI-AP). PNH can present with varied clinical manifestations. PNH can masquerade as an intravascular hemolysis or thrombosis. It can be associated with acquired bone marrow failure. Early diagnosis of PNH is crucial for appropriate clinical management. However, the rarity and diverse clinical manifestations complicate an early diagnosis. Though various tests are available, flow cytometric PNH analysis is considered the gold standard because of its rapidity and high sensitivity. A high index of suspicion and choice of appropriate high sensitivity assay helps in timely diagnosis and prevention of fatal complication in patients.

Introduction (DAF) accelerates destruction of membrane bound C3 convertase where as CD59 (MIRL) inhibits membrane Paroxysmal nocturnal hemoglobinuria (PNH) is an attack complex (MAC) induced lysis. In PNH there is acquired disorder of hematopoietic stem cells caused by a low-level continuous activation alternate complement somatic mutation in X-linked PIGA gene resulting in partial pathway, which in association with GPI-AP causes or absolute deficiency of all glycophosphatidylinositol 1,2 chronic intravascular hemolysis. Classically patients have (GPI) linked proteins. The GPI anchored proteins paroxysms of hemolysis at night, a morning dark colored (GPI-AP) have different functions as enzymes, receptors, 3 urine and gradual resolution over the day. It is primarily adhesion molecules, or complement regulators. the CD59 molecule that is responsible for inhibiting Examples of GPI-AP, which inhibit the complement are CD55 (decay accelerating factor-DAF) and CD59 complement mediated lysis. (membrane inhibitor of reactive lysis-MIRL). PNH can Thromboses cause myriad manifestations ranging from the expected „„ NO (nitric oxide scavenging)—Free hemoglobin intravascular hemolysis to presentation with thromboses 4 released into plasma following intravascular hemolysis or bicytopenia/ pancytopenia due to bone marrow failure. In addition, some patients of PNH present with abdominal scavenges NO. This leads to endothelial dysfunction pains and leg cramps with compensated hemolysis. and platelet activation. NO depletion can manifest in abdominal pain, dysphagia, erectile dysfunction, and Clinical Manifestations and Pathogenesis2,5 thrombosis. „„ Platelet activation by procoagulant microparticles Complement-mediated Intravascular Hemolysis released from GPI-AP deficient cells. PNH red cells are vulnerable to complement mediated „„ Deficiency of GPI-AP like u-PAR (urokinase like lysis due to reduction or absence of CD59 and CD55. CD55 plasminogen activator receptor) and TFPI (tissue factor

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pathway inhibitor) that lead to reduced inactivation of PNH has been subclassified by International PNH the thrombotic pathway. interest group1 into three categories: „„ The classical PNH—clinical and laboratory signs of Bone Marrow Failure intravascular hemolysis without evidence of other diseases of bone marrow failure Approximately 30–45% of PNH patients have associated „„ PNH associated with AA/MDS aplastic anemia or myelodysplastic syndrome (MDS). „„ Subclinical PNH with a minor PNH clone usually less A second hit following PIGA mutation causing immune than 1% destruction of normal stem cells and clonal selection of We will discuss three case scenarios (Table 1) to PNH stem cell may explain coexistence of PNH clones in elucidate the manifestations of the disease and identify acquired aplastic anemia. patients who should be tested for PNH.

TABLE 1 Details of the three clinical cases

Case details Case 1 Case 2 Case 3 Age/Sex 21/Male 28/Female 31/Male Primary complaints symptomatic anemia, Jaundice Petechiae intermittent jaundice, leg cramps Abdominal pain Anemia requiring transfusions Duration of illness 2 years 1 month 2 months Hb (g/dL) 8.1 10.2 5.1 TLC (×109/L) 5.08 8.2 1.2 Platelets (×109/L) 150 99 18 MCV (fL) 115.3 96.5 91.5 Reticulocyte (%) 13.4 3.2 0.9 Additional information - - ANC-120/μL Bilirubin total/Indirect (mg/ 5.2/4.8 2.2/1.9 0.8/0.2 dL) LDH (U/L; Normal reference 890 550 230 220–420) Peripheral blood smear Macrocytes, polychromasia, Normocytic normochromic; mild Pancytopenia with hypersegmented neutrophils polychromasia; Normal platelets normocytic normochromic red cells Bone marrow aspirate Megaloblastic erythroid Normoblastic erythroid hyperplasia Paucicellular with hyperplasia; giant myeloid forms with adequate megakaryocytes predominantly lymphocytes and plasma cells Perl’s stain Absent iron stores 1+ 4+ Bone marrow biopsy Hypercellular; megaloblastic Normocellular with normoblastic Cellularity 10% consistent erythroid hyperplasia erythroid hyperplasia with hypoplastic anemia Other lab tests Negative G6PD, incubated AST>ALT, Negative Direct Coombs test; Chromosomal fragility test osmotic fragility test, Direct Thrombophilia workup normal negative Coombs test including Antiphospholipid antibody syndrome and JAK2V617F mutation Radiology - Hepatic vein thrombosis, mild - hepatomegaly and mild ascites Summary Unexplained hemolytic anemia Hepatic vein thrombosis with evidence Aplastic anemia with secondary folate deficiency of hemolysis

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When to Suspect PNH?5-10 New Delhi of 1501 AA patients, detected PNH clone in 39.7% cases at diagnosis.9 The rarity and diversity of clinical manifestations Various studies have mentioned an excellent complicates the early diagnosis of PNH. Patients may response of AA to immunosuppressive therapies present with anemia, fatigue, dyspnea, chronic kidney in presence of minor PNH clones. Emergence of disease, abdominal pain, pulmonary hypertension, PNH clones and expansion of pre-existing PNH erectile dysfunction, dysphagia, thrombosis or clones post ATG is known. However, clinically hemoglobinuria. Many of these signs and symptoms are relevant PNH clones are seen in less than 5% cases so common that every patient with anemia or thrombosis of AA post ATG causing significant hemolysis. cannot be screened for PNH. Thus, PNH testing should be —— Refractory cytopenia with unilineage dysplasia, advised when some clinical or laboratory findings raise a suspicion. hypoplastic MDS: Up to 50% patients with MDS A PNH test should be ordered in any patient who test positive for a PNH clone. Testing for PNH in presents with: MDS is recommended in cases with refractory anemia subtype, MDS with evidence of hemolysis, „„ Intravascular hemolysis (~25% patients present with hemoglobinuria) as evidenced by hemoglobinuria, or evidence of bone marrow failure such as elevated LDH, reticulocytosis, and elevated plasma hypoplastic MDS. hemoglobin. —— Other cytopenias of unknown etiology after Significant hemoglobinuria is usually present in adequate workup. classical PNH, whereas in cases associated with AA or MDS it may be absent due to a small PNH clone. Diagnosis „„ Evidence of unexplained hemolysis with Diagnosis of PNH is done by detection of the PNH clones. accompanying iron-deficiency, or abdominal pain or Different methods can be adapted. esophageal spasm, or thrombosis, or neutropenia and/ or thrombocytopenia. Complement-based Test—Modified Ham’s Test, „„ Other acquired Coombs’ negative, non-schistocytic, Sucrose Lysis Test5,11,12 non-infectious hemolytic anemia. These tests are based on the sensitivity of PNH cells to „„ Thrombosis with unusual features: activated complement proteins. Though inexpensive, —— Unusual sites—Hepatic veins (Budd-Chiari these tests are laborious, technically challenging, and syndrome)/Other intra-abdominal veins (portal, not accurate quantitatively. As these tests are RBC based, splenic, splanchnic)/Cerebral sinuses/Dermal spurious results may be obtained during hemolytic veins episodes and post-blood transfusion. Also, autoimmune —— With signs of accompanying hemolytic anemia hemolytic anemia and congenital dyserythropoietic —— With unexplained cytopenia anemia may give false positive reports. Hence, these tests —— Young patients PNH is the second most common cause of intra- are no longer recommended as tests of choice for the abdominal vein thrombosis after myeloproliferative diagnosis of PNH.

neoplasms. Thrombosis is the leading cause of mortality 5,11,13 in PNH. About 29–44% of PNH cases experience at Gel Card-based Tests least one thromboembolic event during course of the Gel card-based test is based on antigen-antibody reaction. disease. An early diagnosis and management of PNH If GPI linked proteins, like CD55 and CD59, are not associated thrombosis is paramount. present on RBCs there will be no reaction with anti-CD55 „„ Evidence of bone marrow failure: and anti-CD59. Minor clones and subclinical clones —— Suspected or proven aplastic or hypoplastic cannot be identified using this technique. This test is anemia: Up to 70% patients with acquired AA non-quantitative. Just like complement-based tests, this have a PNH clone and 40% PNH evolves from AA. test also has limited ability to detect PNH clone during Mahapatra et al. in their extensive study at AIIMS hemolytic episodes and in post-transfusion samples.

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Flow Cytometric (FCM) Detection of Though sample can be stored for 7 days at 4°C for PNH Clones5,13-15 RBC analysis, it is preferable to process sample within 48 hours as alteration in scatter and antigen expression in This is a rapid, highly sensitive assay. FCM for PNH is neutrophils over time is known. the gold standard test for detection of PNH clones. FCM Any commercial lysing agent with fixative can be used can pick up subclinical clones and can delineate type I, type II, type III RBCs with normal expression, partial for WBC analysis (ammonium chloride with fixative may deficiency, and complete deficiency of GPI linked protein, be used). respectively. Patients with more than 20% type III red cells 5,14,16 are likely to manifest as hemolysis whereas patients with RBC or WBC majority type II clone without significant type III cells Goal of RBC analysis is to diagnose and quantify cells usually do not show hemolysis. Unlike previous tests, PNH lacking GPI-linked protein (type I/II/III cells). However, can detect PNH clones in RBCs and neutrophils. As in testing only RBCs may not be adequate in cases having multiparametric FCM one can acquire more events/cells active hemolysis or in case of transfusion where it and analyze multiple antigen characteristics of a cell, it can underestimates clone size. So, determining WBC clones help detecting clones as low as 0.01%. More importantly is more important for appropriate quantification of PNH monitoring of patients can be done by highly sensitive clones. technique like FCM.

However, a proper knowledge of monoclonal antibodies 5,14,16,17 to be used, their expression, sample requirement, gating Choice of Antibodies (Table 2) strategy, and sensitivity of the assay is required. GPI-linked protein—simultaneous two GPI-linked proteins for two lineages are recommended. Sample Requirement 14,16 CD55 and CD59 analyses in neutrophils are not Most preferred specimen is peripheral blood in EDTA. recommended as they give a higher false positivity rate for Bone marrow sample is not recommended as immature PNH. myeloid cells may have changes in GPI-linked protein FLAER directly binds to GPI anchors and detects wide expression and in MDS patients there may be altered range of GPI-linked structures in different WBCs. expression of some GPI-linked proteins in neutrophils and CD157 is brightly expressed on both neutrophils and monocytes. monocytes and can replace two different antibodies for A minimum of 1 mL sample is required. But in cases neutrophils and monocytes and is cost effective. with pancytopenia sample as much as 3 mL is required Testing for lymphocytes is not recommended because for higher cell acquisition to detect small subclinical PNH of their longer life span, which may give erroneous results clones. in new onset PNH.

TABLE 2 Antibodies and panels used for the diagnosis of PNH by flow cytometry

Lineage of cells Gating marker Lineage marker GPI-AP

RBC CD235a CD55, CD59

WBC Neutrophils CD45 CD15 CD24, CD157, FLAER

Monocytes CD45 CD64 CD14, CD157, FLAER

PANELS RBC 3-color CD235a/CD55/CD59

WBC 6-color CD45/CD15/CD64/CD24/CD14/FLAER 5-color CD45/CD15/CD64/CD157/FLAER

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Fig. 1: Case 1. Neutrophils gated on the CD15-SSC plot after refinement of initial gate on CD45-SSC plot show a 77.3% PNH clone while monocytes gated on CD64 vs. SSC plot show a 65.5% PNH clone at diagnosis using a high-sensitivity assay. Red cells had a 29% PNH clone primarily type III with complete absence of GPI linked proteins. The clone size was smaller in red cells due to brisk hemolysis

14,16 7,18 „„ Quantification —A laboratory specific lower „„ Monitoring/Retesting —Monitoring of PNH clones limit of detection and lower limit of quantification may be done annually or more frequently if there is should be established. In a high-sensitive FCM-PNH worsening of symptoms. Frequent monitoring may assay, minimum 50 PNH cells should be obtained to also be needed in eculizumab therapy. appropriately quantify. A minimum of 1,00,000 RBCs, Patients with aplastic anemia need serial monitoring 50,000 CD15 positive cells, and 10,000 CD64 positive as minor clone can progress to a hemolytic PNH. Patients cells are recommended to be acquired for sensitivity with no detectable clone should be screened every 6 of 0.05%, 0.1%, and 0.5%, respectively in high sensitive months, decreasing to yearly if no clone appears in the first assays. High sensitivity assays are not required for 2 years. If a clone is present or appears, patients should be classical PNH but small clones in AA/MDS. screened every 3 months until the clone size is shown to be 14 „„ Interpretation — stable for 2 years. —— >1% clone—PNH clone —— 0.1–1%—Minor PNH clone Outcome of the three Cases —— <0.1%—Rare cells with PNH phenotype See Table 3.

MU-232.indd 1486 29-01-2021 15:28:33 PNH—A Great Masquerader: When to Suspect? CHAPTER 232 1487 proteins. Type II clones are sometimes clearly apparent on the CD157-FLAER plots in neutrophils and monocytes on the CD157-FLAER plots in neutrophils sometimes clearly apparent II clones are Type proteins. Both neutrophils and monocytes exhibit predominant type II PNH clones with reduced expression of GPI anchor and related of GPI anchor and related expression type and monocytes II PNH clones with reduced 2. Both predominant neutrophils exhibit 2: Case Fig.

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Fig. 3: Case 3 at the time of diagnosis and follow up. Neutrophils gated on the CD15-SSC plot after refinement of initial gate on CD45-SSC plot show a 0.6% PNH clone while monocytes gated on CD64 vs. SSC plot show a 0.4% PNH clone at diagnosis using a high-sensitivity assay. Red cells do not show any PNH clone on a routine sensitivity assay. After 5 years, PNH immunophenotyping was repeated when cytopenias recurred. PNH clone in neutrophils and monocytes had increased to 28% and 25.8%, respectively while a small clone of 2.9% was seen in red cells, mostly type II. The reason for lower red cell clone was attributed to ongoing packed cell transfusions and mild hemolysis

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TABLE 3 PNH clones and clinical course in the three cases

Case 1 Case 2 Case 3 Summary Unexplained hemolytic anemia with Hepatic vein thrombosis with Aplastic anemia secondary folate deficiency evidence of hemolysis PNH flow cytometry (Fig. 1) (Fig. 2) (Fig. 3) Neutrophil clone 77.3% 36.7%, type II: 27.2% 0.6% Monocyte clone 65.5% 47%, Type II: 37.1% 0.4% Red cell clone 29%, Type III: 28% Not done 0.1% Clinical course Due to unavailability of eculizumab in Patient was treated with Patient responded to immunosuppressive India and lack of response to steroids, full anticoagulation but therapy (ATG + cyclosporine) and attained the patient underwent matched succumbed to death due to a hematological recovery. However, the patient sibling donor transplant; remains second thrombotic episode relapsed with cytopenias 5 years later. Repeat asymptomatic 2 years post-transplant bone marrow revealed hypoplasia with Monosomy 7. PNH-FCM showed increasing PNH clones

Conclusion and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010;78(4):211-30. PNH is a rare and life-threatening disorder, has varied . 8 Dalal B. Paroxysmal nocturnal hemoglobinuria: recommendations presentation like hemolysis, thrombosis at abnormal locations for diagnostic testing. BCMJ. 2016;58(6):306-9. or bone marrow failure. Knowledge of different presenting . 9 Mahapatra M, Singh PK, Agarwal M, et al. Epidemiology, clinico- symptoms and high index of suspicion is needed for an early haematological profile and management of aplastic anaemia: AIIMS experience. J Assoc Physicians India. 2015;63(3 Suppl):30-5. diagnosis. Reliable testing and reporting procedures matter. 10. Scheinberg P, Marte M, Nunez O, et al. Paroxysmal nocturnal Laboratory testing for high sensitivity PNH analysis by FCM in hemoglobinuria clones in severe aplastic anemia patients WBCs and RBCs should be preferred. treated with horse anti-thymocyte globulin plus cyclosporine. Haematologica. 2010;95(7):1075-80. 11. Krauss JS. Laboratory diagnosis of paroxysmal nocturnal References hemoglobinuria. Ann Clin Lab Sci. 2003;33(4):401-6. 1. Parker CJ. Update on the diagnosis and management of paroxysmal 12. Lima M. Laboratory studies for paroxysmal nocturnal hemoglobinuria, with emphasis on flow cytometry. Pract Lab Med. nocturnal hemoglobinuria. Hematology 2014, the American 2020;20:e00158. Society of Hematology Education Program Book. 2016;2016(1): 13. Saxena R, Pati HP (Eds). Hematopathology: Advances in 208-16. Understanding. Springer; 2019. . 2 Brodsky RA. Paroxysmal nocturnal hemoglobinuria. Blood. 14. Sutherland DR, Illingworth A, Marinov I, et al. ICCS/ESCCA 2014;124(18):2804-11. consensus guidelines to detect GPI‐deficient cells in paroxysmal . 3 Kinoshita T. Enzymatic mechanism of GPI anchor attachment nocturnal hemoglobinuria (PNH) and related disorders part clarified. Cell Cycle. 2014;13(12):1838-9. 2–reagent selection and assay optimization for high‐sensitivity . 4 Young NS, Meyers G, Schrezenmeier H, et al. The management testing. Cytometry B Clin Cytom. 2018;94(1):23-48. of paroxysmal nocturnal hemoglobinuria: recent advances in 15. Correia RP, Bento LC, Bortolucci AC, et al. Technical advances in diagnosis and treatment and new hope for patients. Semin flow cytometry-based diagnosis and monitoring of paroxysmal Hematol. 2009;46(1 Suppl 1):S1-S16. nocturnal hemoglobinuria. Einstein (Sao Paulo). 2016;14(3):366-73. 5. Manivannan P, Ahuja A, Pati HP. Diagnosis of paroxysmal nocturnal 16. Brando B, Gatti A, Preijers F. Flow cytometric diagnosis of hemoglobinuria: recent advances. Indian J Hematol Blood Transfus. paroxysmal nocturnal hemoglobinuria: pearls and pitfalls—a 2017;33(4):453-62. critical review article. EJIFCC. 2019;30(4):355. . 6 Dezern AE, Borowitz MJ. ICCS/ESCCA consensus guidelines to 17. Zhang Y, Ding J, Gu H, et al. Diagnosis of paroxysmal nocturnal detect GPI‐deficient cells in paroxysmal nocturnal hemoglobinuria hemoglobinuria with flow cytometry panels including CD157: data (PNH) and related disorders part 1–clinical utility. Cytometry B Clin from the real world. Cytometry B Clin Cytom. 2020;98(2):193-202. Cytom. 2018;94(1):16-22. 18. Killick SB, Bown N, Cavenagh J, et al. Guidelines for the diagnosis . 7 Borowitz MJ, Craig FE, DiGiuseppe JA, et al. Guidelines for the and management of adult aplastic anaemia. Br J Haematol. diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria 2016;172(2):187-207.

MU-232.indd 1489 29-01-2021 15:28:37 CHAPTER

JAK2 Mutation—What 233 Physicians Should Know?

Tulika Seth

Abstract Myeloproliferative disorders include polycythemia vera, primary myelofibrosis, and essential thrombocytosis. These disorders have an increased risk of thromboembolic complications and may progress post fibrotic state or transform into acute myeloid leukemia. Understanding of the molecular pathogenesis has resulted in newer targeted therapies and better risk stratification of patients.

Introduction We are entering an era of molecular medicine. All of us were earlier taught about the variability of disease presentation, severity, and varied responses to treatment. We are now aware that variability even in infectious diseases and much more so in non-infectious diseases has a genetic basis. We know that different mutations, polymorphisms of genes, and even the allele burden are implicated in diseases. Knowledge about JAK2 mutation associated diseases is increasing. The classic JAK2 diseases are myeloproliferative conditions—polycythemia vera (PV), essential thrombocytosis (thrombocythemia) (ET) and primary myelofibrosis (PMF).1 But the JAK2 Fig. 1: Different myeloproliferative disorders and mutation has been found in many other conditions and their risk of transformation to make things more complicated the classic conditions of PV, ET, and PMF may also occur without JAK2.2 as PV, PMF, chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS),3 etc. (Table 1). The JAK2 Mutation presence of the mutation is not diagnostic of any disorders; When we speak of JAK2 mutation we commonly refer to it has to be taken in context with other clinical and JAK2 (V617F) mutation (Fig. 1). This mutation is acquired, laboratory abnormalities. To make a diagnosis we need and is present in the myeloid lineage of the hematopoietic to evaluate the clinical presentation and hematological cells. JAK2 mutation has been associated with a wide parameters. Many of the conditions typically associated variety of myeloproliferative/myeloid disorders—such with JAK2 can also arise without JAK2 mutation.

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TABLE 1 Conditions associated with JAK2 V617F mutation TABLE 2 Types of JAK2 abnormalities and pathogenesis

Conditions associated with JAK2 V617F Frequency of JAK2 Rearrangements

zz Polycythemia vera (PV) PV: >90% JAK2 can be rearranged with other genes: JAK2 exon 12 = PV: 2–3% zz TEL/ETV6: t(9;12) (p24;p13) reported in myeloproliferative neoplasm (MPN), also in T-cell ALL zz Essential thrombocytosis/ ET: 60% zz BCR: t (9;22) (p24;q11.2) reported in few MPN thrombocythemia (ET) zz PCM1: t (8;9) (p22;p24) reported in MPN, also in AML and ALL zz NF-E2: der (9) t(9;12) (p24;q13) reported in MDS zz Primary myelofibrosis (PMF) PMF: 60% Point mutations zz Systmemic mastocytosis (SM) 25% V617F G >T at nucleotide 1849 on exon14, the classical MPNs zz Chronic neutrophilic leukemia (CNL) 17–33% T875N reported in AML (M7)

zz Hypereosinophilic syndrome (HES) 0–2% Deletions/Insertions

zz Myelodysplastic syndrome (MDS) 1.5–5% Exon12: In 4% PV patients there are >8 reported mutations including deletions and insertions in codon 538 to 543 zz Chronic myelomonocytic leukemia 3–13% Numerical (CMML) It can present as trisomy (+9) or be overexpressed due to zz Unclassified MPD (UN) 20% amplification. zz HVOTO/ Budd-Chiari Syndrome (BCS) Varied Blood. 2005;106(6):2162-8; Blood. 2005;106(10):3370-3; Blood. 2005;106(4):1207-9. different genetic alterations (Table 2), the interplay of various genetic and cytokine abnormalities results in the Abbreviations: CMML, chronic myelomonocytic leukemia; CNL, chronic 3-5 neutrophilic leukemia; ET, essential thrombocythemia/thrombocytosis clinical disease state and its severity. (ET); HES, hypereosinophilic syndrome; MDS, myelodysplastic syndrome; PMF, primary myelofibrosis; PV, polycythemia vera; SM, systemic mastocytosis; UN, unclassified MPD. What is JAK2? The JAK2 gene is a member of a family of genes that includes four Janus kinases 1, 2, 3, and tyrosine kinase What are the Implications of JAK2? 2. The protein group was named Janus kinases after the JAK2 associated diseases are a wide variety of chronic Roman God Janus with two faces, as the non-receptor diseases in the myeloproliferative neoplasms (MPN) or kinases have two similar “active” and “inactive” domains. MDS group of diseases. They are characterized in MPN The JAK gene has domains, which binds to type by increased production of red blood cells, platelets, 1 cytokine receptor, it plays a role in trafficking of the or one type of white blood cell; or in MDS by increased (erythropoietin) EPO receptor (EPOR). production of dysplastic hematopoietic cells, resulting The JAK2 protein in contact with the cytoplasmic in cytopenias.3,4 They are associated with constitutional domain of the receptor catalyses tyrosine phosphorylation symptoms, high symptom burden, poor quality of life, and and leads to activation of signal transducer and activator decreased life expectancy and risk of transformation to of transcription (STAT) molecules that act as transcription acute myeloid or other leukemia (Fig. 1). factors and modifies other key regulatory pathways and cytokine signaling3-5 (diagram illustrating functional JAK Why Do Various Conditions Evolve from STAT pathway—Fig. 2). The V617F mutated JAK2 spontaneously activates the Same Mutation? downstream STAT mediated transcription, and activation The JAK2 gene is a signaling molecule for many cytokines of ERK/MAP kinase and P13K/AKT pathways. The including: INF-γ, erythropoietin (EPO), prolactin, wildtype JAK2 has an autoinhibitory activity, and does thrombopoietin, G-CSF, GM-CSF, and IL-3 via activating not mediate such events. The hematopoietic stem cells many signaling pathways like: MAPK, PI3, ERK20, and in patients of myeloproliferative/myeloid disorders are importantly STAT. JAK2 abnormalities present with extremely sensitive to growth factors; proliferation is

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occur secondary to other conditions, so exclusion of renal, pulmonary causes of increased hemoglobin, or causes of reactive thrombocytosis etc. are necessary. Myelofibrosis is often missed or under diagnosed as the huge splenomegaly may be confused with other medical conditions. The bone marrow fibrosis results in massive splenomegaly, hepatomegaly, and extramedullary hematopoiesis as a compensatory mechanism. In India, patients with lower hemoglobin levels than standard criteria may be recognized; the other molecular tests and bone marrow evaluation are useful to identify them so that they can receive appropriate therapy. MPN patients Fig. 2: Steps of JAK-STAT pathway particularly PV may show iron deficiency, this may be due to many reasons, but one important factor is disorder of triggered by JAK2 signaling. Patients who are homozygous iron metabolism, with the inflammation caused by PV for JAK2 mutation have advanced disease and increased counteracting Hepcidin suppression.11 Iron therapy is risk of progression to secondary myelofibrosis compared controversial as the HU used in cytoreductive therapy in to heterozygous patients.3-6 high-risk PV patients is more effective in iron-deficient than in iron-replete patients. Types of JAK2 The diagnosis of MPNs is made in a symptomatic JAK2 V617F occurs more frequently in a specific JAK2 patient and requires a combination of parameters and 12,13 haplotype, named JAK2 46/1 or GGCC, which is tagged by tests. rs10974944 (C/G), rs12343867 (T/C), and rs4495487 (T/C). Other JAK2 mutations are more commonly associated Diagnostic Criteria 6-9 with Budd Chiari and other disorders. The diagnosis of MPNs is made in a symptomatic patient and requires a combination of parameters and tests. Allele Burden and Disease The usual criteria followed are the WHO 2016 diagnostic Allele burden has different implications in different criteria. diseases, a high V617F allele burden in PV is associated The diagnosis of MPN should be based on the 2016 with a more aggressive phenotype, but a low allele burden WHO diagnostic criteria.12 Criteria include specific in myelofibrosis is associated with reduced survival.10 findings from the CBC, blood smear, bone marrow analysis, correlated with clinical history, as well as the JAK2 as a Target for Therapy presence of certain molecular markers and the exclusion We now have agents, which are targeted inhibitors JAK2, of other disorders. and also has inhibits JAK1. This is approved for therapy of patients with intermediate- or high-risk MF, including Laboratory Tests primary MF (PMF), post-polycythemia vera MF (PPV-MF), All patients should have a CBC, differential count test; and post-essential thrombocythemia MF (PET-MF), and this should be repeated to ensure no fallacy. Tests to fulfill PV patients with hydroxyurea (HU) failure or intolerance. diagnostic criteria of WHO criteria12 should be done. In Newer more specific JAK2 inhibitors are in phase 3 trials— PV serum erythropoietin levels and evaluation to exclude fedratinib. causes of secondary polycythemia are required. In all patients, a detailed peripheral smear with evaluation Clinical Features of MPN for blasts, base line tests for RFT, LFT, evaluation for The MPN are characterized by increased counts comorbidity, history of smoking, medications, etc., should and constitutional symptoms (Table 3). These may be performed. Cardiovascular risk assessment is essential.

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TABLE 3 Clinical features of JAK2 positive myeloproliferative diseases

MPN Clinical presentation Features

PV zz Median age 60 years, rare before 40 years. Male to female zz High number of RBCs (high hemoglobin, high hematocrit, red ratio 1.2:1. Median survival 14 years cell mass), bone marrow fibrosis, risk of thrombosis zz Facial plethora, headaches zz Risk of post-PV myelofibrosis (MF) ET with poor outcome zz Heaviness of head, fatigue, weight loss, congestion of eyes, blurring vision or diplopia, and burning (erythromelalgia) sensation or itching in hands and feet, often worse after water (aquagenic pruritus). Increased risk of thrombosis— venous and arterial

ET zz Median age 65 years, but can be seen at any age. Female zz High platelet number, and risk of thrombosis. Bone marrow to male ratio 2:1 shows increased numbers of large, atypical megakaryocytes zz Median survival 20 years zz Post ET myelofibrosis is rare, need to rule out if previous zz Headache, dizziness, fainting, chest pain, temporary vision diagnosis of ET was actually early-phase of PMF changes zz Numbness and tingling of hands and feet zz Redness, throbbing, and burning pain in hands and feet (erythromelalgia) zz Increased risk of thrombosis

PMF zz Mean age 60 years. Male to female ration 1.5:1. Median zz De novo/primary MF (PMF) (rather than from the survival 6 years. Increased constitutional symptoms in transformation of PV to MF) comparison to the blood count abnormality. Fatigue, zz Leukoerythroblastic blood smear with dacryocytes if marrow weakness, shortness of breath, pain or fullness of abdomen is fibrotic (due to massive splenomegaly, hepatomegaly), early zz Bone marrow fibrosis might be absent or minimal in early- satiety, unintentional weightloss. stage/prefibrotic PMF zz Fever, excessive sweating, fever, easy bruising, itching, zz Reticulin or collagen fibrosis is considerable in overt PMF bleeding, or bone pain

CNL Fatigue, weight loss, night sweats, bone pain, easy bruising, zz Sustained neutrophilia in peripheral blood (cells are pruritus, or symptoms of gout not immature), hepatosplenomegaly. Bone marrow is hypercellular with predominance of myeloid proliferation zz CSF3R mutations may be present but not specific

CEL, NOS Fatigue, fever, cough, muscle pain, itching, diarrhea, periorbital zz Eosinophilia due to clonal proliferation swelling, angioedema zz Evidence of clonality; and abnormal bone marrow morphology

MPN-U Same as other classic MPN zz Cases with features of an MPN but do not meet diagnostic criteria, either because features are not fully fulfill subtype or if not clear due to advanced stage or another diseases MF, myelofibrosis; MPN-U, myeloproliferative neoplasm unclassifiable; NOS, not otherwise specified.

Diagnostic Molecular Testing with ET or PMF.12,13 If PV is still suspected in those with Testing for BCR-ABL1 mutation is required as by definition negative JAK2 V617F mutation results, testing should these conditions are BCR-ABL negative. Then evaluation be performed for JAK2 exon 12 mutations. In those with for other MPN mutations should be performed. Molecular ET or PMF but without JAK2, MPL, or CALR mutations testing is used to assess clonality and to detect MPN- (triple-negative MPNs), testing for mutations in ASXL1, specific mutations. First testing for JAK2 V617F mutation CBL, CSF3R, DNM3TA, EZH2, IDH1, IDH2, LINK/SH2B3, should be performed in all MPN suspected patients; it is SF3B1, SRSF2, TET2, TP53, and U2AF1 genes should the most common mutation and occurs more than 95% then be considered. A comprehensive next generation patients with PV. sequencing (NGS) myeloid panel can be useful for If the JAK2 V617F mutation is not detected, testing for this, instead of single-gene tests and provides details of CALR and then MPL mutations should follow for patients additional prognostic genes.13

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Bone Marrow should be offered allo-hematopoietic stem cell transplant Bone marrow examination is very important and a bone as a curative option. Patients with low-risk disease marrow aspiration and biopsy is necessary confirm a will have a longer life and supportive treatments like 15,16 diagnosis of MPN, and for prognosis by assessment of hydroxyurea and ruxolitinib are acceptable. All of these blast percentage and evidence of fibrosis.12,13 treatments are palliative. For high-risk patients curative options of allogeneic hematopoietic stem cell transplant Cytogenetics (bone marrow transplant, BMT) should be discussed and Cytogenetic analysis is important in distinguishing MPN planned. subtypes. The chromosome analysis also serves to provide evidence of clonality and clonal evolution, it may be Glossary repeated if concerns of progression exist. „„ A gene is polymorphic if more than one allele occupies the gene’s locus within a population. A polymorphic Prognosis variant of a gene can lead to the abnormal expression The overall survival of patients with MPNs is variable or abnormal form of protein. These may cause or be depending on the subtype. Several scoring systems associated with diseases. and prognostic models have been developed for the „„ An allele is any of the possible forms in which a gene risk stratification of patients with MPN. The dynamic for a specific trait can occur. In humans, two alleles for international prognostic scoring system (DIPSS) is each gene are inherited, one from each parent. The commonly used for its ease and ability to stratify patients variant alleles arise by mutation and are found at the 14 into risk groups. Treatment decisions are made per the same place as the original gene on the chromosome. risk stratification of the patient, higher risk patients are „„ Wildtype gene is in its normal state, mutated gene has more likely to develop progression from their baseline acquired or inherited alterations. MPN this can mean transformation to acute myeloid „„ Allele burden One, called the gene-dosage hypothesis, leukemia or post-fibrotic myelofibrosis. These secondary postulates a correlation between disease phenotype cases are more difficult to treat and have poorer prognosis. and the proportion of JAK2 (V617F) mutant alleles Therapy introducing the concept of allele burden, that is, the ratio between mutant and wildtype JAK2 in These patients must get a hematology consultation hematopoietic cells. periodically due to their risk of thrombosis, progression, „„ Haplotype—A haplotype is a group of genes, which is and symptom load. Shared care with primary care inherited together by an organism from a single parent. physicians is possible and desirable for low-risk patients. Therapy of MPN depends on the diagnosis, age, risk classification, and whether complications of thrombosis Conclusion 15-18 have occurred or not. JAK2 associated conditions of PV, ET, and PMF may be Patients of PV and ET require low-dose aspirin to underrecognized and underdiagnosed. The rare conditions decrease the risk of thrombosis. Younger PV patients can associated with JAK2 are also often missed. High index of be well managed by phlebotomy, and/or interferon alpha, suspicion, appropriate tests, and timely referral can change the but older patients or those with history of thrombosis scenario. Correct diagnosis and treatment can prevent serous require hydroxyurea (HU). HU is titrated to response; thrombotic complications and allow access to recommended those who fail or are intolerant to HU can be given treatment will help the patient. ruxolitinib (a JAK2 inhibitor). In ET along with low-dose aspirin, additional medications to reduce the platelet References counts like hydroxyurea, anagrelide, and interferon alpha 17,18 . 1 Levine RL, Gilliland G. Myeloproliferative disorders. Blood. are used. 2008;112:2190-8. Myelofibrosis low-risk patients may be given HU, . 2 Reilly JT. Pathogenetic insight and prognostic information thalidomide-prednisone or ruxolitinib. High-risk patients from standard and molecular cytogenetic studies in the BCR-

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ABL-negative myeloproliferative neoplasms (MPNs). Leukemia. 10. Tefferi A, Lasho TL, Schwager SM, et al. The clinical phenotype 2008;22(10):1818-27. of wild-type, heterozygous, and homozygous JAK2 (V617F) in . 3 Donal McLornan, Melanie Percy, Mary Frances McMullin. JAK2 polycythemia vera. Cancer. 2005;106(3):631-5. V617F: a single mutation in the myeloproliferative group of 11. Ginzburg YZ, Feola M, Zimran E, et al. Dysregulated iron metabolism disorders V617F mutation. Ulster Med J 2006;75(2)112-9. in polycythemia vera: etiology and consequences. Leukemia. . 4 Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2 2018;32(10):2105-16. V617F mutation in chronic myeloproliferative disorders. Blood. 12. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World 2005;106(6):2162-8. Health Organization classification of myeloid neoplasms and acute . 5 Alabdulaali MK. The role of JAK2 abnormalities in hematologic leukemia. Blood. 2016;127(20):2391-405. neoplasms. Hematol Rev. 2009;1(1):e10. doi: 10.4081/hr.2009.e10. 13. NCCN Clinical Practice Guidelines in Oncology: Myeloproliferative PMCID: PMC3222247. Neoplasms. Version 3.2019. Fort Washington, PA: National . 6 Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function Comprehensive Cancer Network. mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 14. Passamonti F, Cervantes F, Vannucchi AM, et al. A dynamic 2005;352(17):779-90. prognostic model to predict survival in primary myelofibrosis: . 7 Jelinek J, Oki Y, Gharibyan V, et al. JAK2 mutation 1849 G>T is rare in acute leukemias but can be found in CMML, Philadelphia a study by the IWG-MRT (International Working Group for chromosome-negative CML, and megakaryocytic leukemia. Blood. Myeloproliferative Neoplasms Research and Treatment). Blood. 2005;106(10):3370-3. 2010;115(9):1703-8. 8. Steensma DP, Dewald GW, Lasho TL, et al. The JAK2 V617F activating 15. Tefferi A. How I treat myelofibrosis. Blood. 2011;117(13):3494-504. tyrosine kinase mutation is an infrequent event in both ‘atypical’ 16. Tefferi A. Primary myelofibrosis: 2019 update on diagnosis, myeloproliferative disorders and myelodysplastic syndrome. Blood. risk-stratification and management. Am J Hematol. 2018;93: 2005;106(4):1207-9. 1551-60. 9. Patel RK, Lea NC, Heneghan MA, et al. Prevalence of the activating 17. Spivak JL. How I treat polycythemia vera. Blood. 2019;134(4):341-52. JAK2 tyrosine kinase mutation V617F in the Budd-Chiari syndrome. 18. Beer PA, Erber WN, Campbell PJ, et al. How I treat essential Gastroenterology. 2006;130(7):2031-8. thrombocythemia. Blood. 2011;117(5):1472-82.

MU-233.indd 1495 29-01-2021 15:32:14 CHAPTER Approach to a Case of Polycythemia: More 234 Blood May Be Bad….

Tuphan Kanti Dolai, Prakash Singh Shekhawat, Malini Garg

Abstract A 59-year-old male teacher reported with painful erythematous finger swelling and excessive itching without any rash of 3-week duration. Physical examination shows multiple excoriations, splenomegaly, and erythematous swelling of hands. Vitals were normal. Complete blood count shows Hb 17 g/dL, MCV 87 fL, TLC 14 × 109/L, and platelet 780 × 109/L. Biochemistry shows normal LFT and urea, creatinine while serum EPO was 2.4 U/L (range 7–20 U/L). He had JAK2V617F mutation and was started on regular phlebotomy and Tab Aspirin 75 mg PO OD. His hand swelling and pruritus improved.

Introduction Etiology and Pathogenesis Polycythemia means an abnormal increase in hemoglobin Primary polycythemia is caused by dysregulation or hematocrit. It is categorized as absolute and relative. in erythropoietin sensing mechanism and is mainly Absolute polycythemia has an increased red blood cell associated with low erythropoietin levels. Primary familial mass (RCM), the classical example being polycythemia and congenital polycythemia has an EPOR gene mutation, vera (PV). There is a mild rise in hematocrit without which disrupts down regulation of JAK2 pathways. PV raised RCM in cases of relative polycythemia like severe is due to an acquired, somatic mutation of multipotent dengue fever, severe diarrhea, or other conditions with a hematopoietic stem cell triggering clonal proliferation moderate increase of hematocrit secondary to reduced plus suppression of normal polyclonal hematopoiesis. plasma volume. Absolute polycythemia is categorized Most common mutation is JAK2V617F found in 95–98% into primary and secondary.1,2 PV is the standard example of cases.4 It causes persistent activation of EPO receptor for primary absolute polycythemia. Secondary absolute signaling. In vitro development of erythroid colonies in polycythemia has hypoxia, because of chronic lung absence of EPO is characteristic of PV.3 PV can occur at any disease, carboxyhemoglobinemia due to smoking and age because JAK2V617F expression is age-independent.5 renal cell carcinoma producing erythropoietin. PV is a Secondary polycythemia has increased erythropoietin clonal malignant disorder arising from a mutation in production because of hypoxia. Secondary familial and multipotent hematopoietic stem cells with an increase in congenital polycythemia may have mutations that exist blood cell production independent of cytokine. It is the in genes encoding hypoxia inducible factor (HIF), von commonest myeloproliferative neoplasm (MPN) in the Hippel-Lindau (VHL) proteins, or prolyl-hydroxylase United States and has a male preponderance with median domain (PHD) enzymes, which regulate renal oxygen age of diagnosis in the seventh decade.3 Annual incidence sensing and EPO production. Secondary acquired ranges from 1–2.5 per 100,000 persons in different causes have increased EPO levels either secondary to countries. tissue hypoxia (pulmonary illness, CO poisoning, high

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altitude, high-affinity binding hemoglobinopathy) or due evidence of PV the differential diagnosis includes early to overproduction (stenosis of renal artery, kidney cyst, or mild polycythemia vera, primary pure erythrocytosis tumors with ectopic EPO production). Smoking history or secondary erythrocytosis.6 The history and physical or occupational exposure to hydrocarbon may lead to examination may give clues to the presence of a cause for increased carboxyhemoglobin and increased EPO levels.4 secondary erythrocytosis. Additional laboratory testing Separation of primary and secondary polycythemia is is often helpful. An oxygen saturation < 92% indicates important as primary polycythemia has risk of leukemic/ hypoxemia as the cause for polycythemia.6 HPLC and fibrotic transformation. Phlebotomy is rarely needed in oxygen dissociation curve can pick high oxygen affinity secondary polycythemia unlike primary polycythemia.3 hemoglobins which usually has a family history. Renal ultrasound, intravenous pyelography, or computed Diagnosis tomography is essential to rule out suspected renal Polycythemic patients are often diagnosed incidentally lesion. In selected patients hepatic lesion is diagnosed by having elevated hemoglobin or hematocrit found in CBC ultrasound, CT scan, or radionuclide scan. Similarly, for during baseline evaluation of other complaints. History cerebellar hemangioblastoma a CT brain with stress on of smoking, high altitude stay, congenital heart disease, the posterior fossa is helpful and can determine the source alcohol, and drug abuse along with clinical examination of ectopic EPO production. Commercially available serum becomes very important to distinguish secondary from erythropoietin radioimmunoassay is helpful in differential primary causes. diagnosis. Overall, increased erythropoietin suggests Clinical spectrum varies from no symptoms to life secondary erythrocytosis, whereas a normal value is threatening thrombosis. Genesis of these symptoms uncertain as occasionally erythropoietin may be increased could be related to increased RCM or the underlying in secondary erythrocytosis. Erythropoietin assay may disease process. Increased RCM may cause hyperviscosity be most useful in those lacking obvious clinical sign of 6 resulting in symptoms like vertigo, tinnitus, headache, PV and no apparent cause for secondary erythrocytosis. visual disturbance, and hypertension. Both usual and In PV patients endogenous colony formation occurs unusual types of arterial and venous thrombosis can be without added erythropoietin, whereas for other causes seen. Thrombotic symptoms depend upon the site and of polycythemia exogenous erythropoietin should be the extent of thrombus. Polycythemic patients may have added.6 Flowchart 1 shows the approach to polycythemia. characteristic ruddy complexion. WHO defines polycythemia vera as a myeloproliferative Although aquagenic pruritus and peptic ulcer disease neoplasm with hemoglobin level >165 g/L (16.5 g/dL) for are common associates of polycythemia, commonly males and >160 g/L (16 g/dL) for females or hematocrit they may have signs and symptoms of the underlying levels >49% in men or >48% in women or increased red cell disease like chronic obstructive airway disease (COAD) mass (RCM) >25% from baseline (Table 1). or cyanotic heart disease. Apart from the signs of the underlying disease patient may have splenomegaly, which Treatment of Polycythemia Vera is rarely seen in early phase of the disease.4 Age, sex, initial presenting symptoms along with blood Pretreatment correct diagnosis is essential. Overt PV is picture must be considered for individualized therapy. easily revealed by clinical findings.6 Splenomegaly is seen Goal of therapy is to decrease symptoms, reduce likelihood in 75% of the cases and is rare in other varieties.6 Around of thrombus, and prevent/delay transformation. 30% cases have hepatomegaly. Pruritus occurs in 40% of PV cases and is rare in other varieties of polycythemia. Phlebotomy: Initially phlebotomy is needed in all patients Associated thrombocytosis, leucocytosis, or basophilia to keep hematocrit <45% to reduce the risk of thrombotic indicates the diagnosis of polycythemia vera. Panmyelosis and cardiovascular complications.8,9 In young fit, a 450 is seen in bone marrow.6 Diagnostic marrow studies mL phlebotomy is safely done on alternate days till commonly show reticulin fibrosis and absent iron stores. this goal is reached. In older, unfit patients smaller Bone marrow cytogenetics shows clonal markers in phlebotomies approximately 200–300 mL twice weekly 13–31% of untreated patients.6 In patients lacking definite can be considered.6 A standard unit of phlebotomy

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Flowchart 1: The approach to polycythemia

TABLE 1 WHO 2016 PV diagnostic criteria7 TABLE 2 Polycythemia vera risk stratification7

Major criteria zz Hb >16.5 g/dL in men (or Hct > 49%), Hb >16 g/ PV risk stratification Age ≤60 years Age >60 years dL in fematles (or Hct >48%), or >25% increase in red cell mass No history of thrombosis Low risk High risk zz Bone marrow biopsy showing characteristic panmyelosis and pleomorphic megakaryocytes History of thrombosis High risk High risk zz Mutation in JAK2V617F or JAK2 exon Minor criteria Serum EPO level below the normal reference decreases Hct by 3%. Polycythemia Vera Study Group range (PVSG) recommends use of myelosuppressive treatment. Diagnosis zz All three major criteria or Cytoreduction is usually not needed in low risk group zz First two major criteria and the minor criterion whereas it is used in high risk group (Table 2).

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Hydroxyurea (HU): HU, a ribonucleotide reductase due to sleep apnea and chronic obstructive pulmonary inhibitor, is effective and first-line recommendation for disease.18 Reducing weight may be helpful in obesity- cytoreduction in high risk group. Need of cytoreduction or hypoventilation syndrome. Surgical removal of EPO other therapy should be individualized, and this reminds producing lesions usually causes improvement and us of Dameshek. “There is a tendency in medical practice— resolution of the polycythemia. Likewise, polycythemia by no means limited to hematologists—to treat almost any reversal may be seen after correcting underlying benign condition as vigorously as possible. In hematology, this renal lesion. Some secondary polycythemics require consists in attempting to change an abnormal number— phlebotomy. Preoperative elective phlebotomy should whether this number is the hematocrit, white cell count, be considered in these cases.6 The ideal hematocrit level or platelet count to get normal values, whether the patient in these patients is a tough call, to maximize the oxygen needs it or not!”10 Overall hydroxyurea is the commonest carrying capacity, and minimize the deleterious side cytoreductive drug used for ET and PV cases, based on effects of hyperviscosity.6 The ideal hematocrit differs with the results of the PT-1 randomized controlled trial in 809 the main disorder and usually differ amongst patients with high-risk patients with ET, in which HU proved superiority the same illness. Usually hematocrit >60% is expected to anagrelide in the rates of serious hemorrhage, arterial to be harmful and they must undergo phlebotomy, thrombosis, and myelofibrosis progression11,12 especially when sign/symptoms of poor oxygenation are Interferons: Interferon-α is an important drug if HU is seen.6 There is decreased arteriovenous oxygen difference, no longer suitable for a patient. Newer longer acting improvement in pulmonary artery resistance, better right pegylated-α-2a is available and can allow less frequent ventricular function along with improved hemodynamics administration. IFN-α has anti-clonal activity and also and exercise tolerance.6 Maintaining hematocrit between 13,14 helps in histological improvement. 50–55% is likely to help hypoxic lung patients. Acute Although it does not have leukemogenic effect and is hemodynamic effects of phlebotomy can be managed by safe for long-term use but flu-like symptoms and mood isovolumic phlebotomy. Myelosuppressive treatment is changes often limit its use. best avoided in cases of secondary erythrocytosis. Ruxolitinib: The JAK1/2 inhibitor ruxolitinib is approved therapy for PV cases where HU is intolerant or refractory, Treatment of Relative Polycythemia based on RESPONSE and RESPONSE-2 studies in patients with or without splenomegaly, respectively.15-17 It is given Numerous factors are responsible for the relatively in 15–20 mg BD doses for symptom control. increased hematocrit. Abstaining from alcohol intake and smoking may be helpful. Strict hypertension management Treatment of Secondary Polycythemia is essential. Hydration status should be well supported. Phlebotomy is not recommended in cases of relative Cause and underlying mechanism will help in deciding polycythemia. treatment for polycythemia, especially secondary causes. TABLE 2 Polycythemia vera risk stratification7 Aggravating factors like smoking and dehydration must be rectified. Smokers must be encouraged to refrain Conclusion PV risk stratification Age ≤60 years Age >60 years from tobacco usage. Diuretics can reduce plasma Polycythemia included variety of causes from smoking, high No history of thrombosis Low risk High risk volume, increase hyperviscosity, and thus are avoided altitude, dehydration, congenital heart disease to the clonal History of thrombosis High risk High risk where possible. Drugs like Androgens must be carefully polycythemia vera, which has a risk of leukemic progression. discontinued or lower doses used if feasible. Smoking They have an increased likelihood of vascular manifestation cessation helps in reversal of polycythemia related to and risk of thrombosis. Goal is to keep Hct <45% and a carboxyhemoglobinemia and it can improve chronic cardiovascular risk factor modification. Phlebotomy and aspirin obstructive pulmonary disease associated polycythemia.6 forms important part of treatment along with hydroxyurea or interferons in selected cases. Ruxolitinib is approved for HU Continuous low flow oxygen therapy may reduce resistant or intolerant cases. hematocrit and it improves status of hypoxemic patients

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