Lecture 6 and 7

1. Lecture 6 and 7 Myelodysplastic syndromes; Chronic lymphocytic leukemia and related lymphoproliferative disorders AbdulkarimAldosari

2. Objectives • Describe the etiology, pathogenesis, clinical features and treatment of MDS • Describe laboratory diagnosis of MDS • Differentiate the subtypes of MDS based on morphology and laboratory features • Distinguish between MDS and AML on PB and BM smears • Know the International Prognostic Scoring System (IPSS) • List general features of chronic lymphocytic leukemia • List diagnostic criteria of CLL • Explain differential diagnostic criteria used to characterize lymphoproliferative disorders • Name laboratory methods used to study lymphocytes in lymphoproliferative disorders

3. Myelodysplastic syndromes • The Myelodysplastic syndromes (MDS) are a collection of myeloid malignancies characterized by one or more peripheral blood cytopenias. • MDS are diagnosed in slightly more than 10,000 people in the United States yearly, for an annual age-adjusted incidence rate of approximately 4.4 to 4.6 cases per 100,000 people. • They are more common in men and whites. • The syndromes may arise de novo or secondarily after treatment with chemotherapy and/or radiation therapy for other cancers or, rarely, after environmental exposures. http://www.ncbi.nlm.nih.gov/books/NBK66015/

4. Myelodysplastic syndromes In Myelodysplastic diseases, the blood stem cells do not mature into healthy cells • Versus myeloproliferative diseases- the total number of blood cells increases, usually mature cells • A heterogeneous group of clonal hematological malignancies • Characterized by PB cytopenias, dysplastic blood cells, propensity to become acute leukemia • Clinical outcome is variable • Outcome is almost always fatal • No cure except for allogeneic stem cell transplantation

5. MDS etiology and pathogenesis • Mostly affect the elderly – 70+ years • Mostly male • Risk factors – exposure to environmental and occupational products • Ammonia, petrochemicals, low dose irradiation, exposure to benzene, smoking, family history • Mechanism that causes MDS not well known • Genetic mutations/defects • Caused by a somatic mutation → a growth advantage of the neoplastic cell

6. MDS etiology and pathogenesis Which hematopoietic progenitor cell is responsible for the neoplastic clone? • Pluripotent stem cell or an omnipotent stem cell? • Controversial subject > various studies done • Blast cell population in leukemic patients = myeloid or myelomonocytic • Only few cases the blasts are lymphoid • Most studies show that blasts arise from repopulating stem cells committed to myeloid differentiation

7. MDS etiology and pathogenesis The main biological feature of MDS is increased apoptosis –programmed cell death Ineffective hematopoiesis seen in MDS – caused by abnormally high rate of intramedullary apoptosis • Factors that are involved in stimulating apoptosis are increased and those which are anti-apoptotic are decreased • TNF-α and TGF-β, Fas-ligand → initiate apoptosis • GM-CSF - anti-apoptotic • TNF-α and TGF-β are both ↑ in MDS patients • GM-CSF ↓ in MDS patients • The balance between growth-stimulatory and growth-inhibitory cytokines may favor apoptosis

8. MDS etiology and pathogenesis Loss of genetic material is the most common genetic anomaly found in MDS MDS patients are missing key genes essential for maintaining normal hematopoiesis • No single gene found – focus of research Characterized mainly by chromosomal deletions • Either sections of a chromosome or the entire chromosome • Versus chromosomal translocations in acute leukemias Deletions follow a tumor suppressor gene model • Tumor-suppressor genes generally encode proteins that inhibit cell proliferation – (in MDS = apoptosis) • Need mutated allele from both parents – recessive model • Versus oncogene activity of acute leukemias Missing genes → more genetic anomalies → disease progression

9. MDS progression Characteristics of MDS changes as it progresses towards leukemia • Blast cells ↑ • Leukocytosis replaces leukopenia • Hepatoslenomegaly occurs • Becomes a proliferative PB disorder • Progression due to ↓ in medullary apoptosis, ↓ in apoptosis factors (Fas ligand) • Multistep pathogenesis • Normal → clonal and dysplastic → full blown malignancy → acute

10. Morphology of PB and BM in MDS Reliable analysis of blast phenotype of MDS difficult • Blasts are not predominant in the BM and PB • Determined using immunophenotyping • Expression of CD34 > myeloid antigens > myeloid blasts Blast in BM determine type of MDS at initial diagnosis Different types of blasts and sideroblast according to FAB classification • Type 1 myeloblast - variable size without granules or Auer rods • Type 11 myeloblast– slightly larger with 1-20 granules • Type 111 myeloblast –with ~20 granules, basophilic cytoplasm • Type 1 sideroblast – 1-4 cytoplasmic iron-containing granules = 15-50% erythroblasts • Type 11 sideroblast – 2-10 granules, scattered throughout cytoplasm • Type 111 - ringed sideroblast

11. Morphology of PB and BM in MDS MDS characterized by multilineage morphological abnormalities Lineage dysplasia includes: Dyserythropoiesis • In PB – macrocytic/normochromic anemia in 90% of cases, ↓ retic count, macrocytosis, anisopoikilocytosis, basophilic stippling, dimorphic RBC population (normochromic, hypochromic), Pappenheimer bodies, teardrop (dacryocytes), schistocytes, elliptocytes, Howell-jolly bodies, acanthocytes • In BM – Erythroblasts with asynchronous cytoplasmic maturation, internuclear bridging, nuclear budding, Howell-Jolly bodies, Basophilia, ghosts cells, abnormal ringed sideroblasts (types 1 and 11)

12. Morphology of PB and BM in MDS Megaloblastoid change Megaloblastoid erythroid precursors with and multinucleation nuclear irregularities and a multinucleated erythroid precursor

13. Morphology of PB and BM in MDS Characterized by multilineage morphological abnormalities Lineage dysplasia includes: Dysgranulocytopoiesis in peripheral blood • neutropenia in 60% of cases • nuclear and cytoplasmic dysplasia in 90% of cases • neutrophilia possible • pseudo-Pelger-Huët and variable degrees of hyposegmentation • monolobulation (pseudo- Stodtmeister anomaly) • variable degrees of hypogranulation • pseudo-Dohle bodies • rare hypergranulation • abnormal chromatin

14. Stodtmeiser Pelger-Huet anomaly Stodtmeiser anomaly

15. Morphology of PB and BM in MDS Dysgranulocytopoiesis in bone marrow • Pseudo-Pelger-Huët or Stodtmeiser anomaly, • hypersegmentation • ring formation • chromatin sticks • asynchronous nuclear-cytoplasmic maturation • hybrid myelomonocytic cells • vacuolated monocytes/myeloid precursors Pelger-Huët anomaly is considered to be the most specific dysplastic marker for the diagnosis of MDS

16. Morphology of PB and BM in MDS Ring formation Hypersegmentation with nuclear sticks

17. Morphology of PB and BM in MDS Characterized by multilineage morphological abnormalities Lineage dysplasia includes: Dysmegakaryocytopoiesis • In PB – Thrombocytopenia in 60% of cases, platelet gigantism, ballooning, hypogranulation, rare thrombocytosis associated with 5q-syndrome • In BM – micromegakaryocytes(dwarf or mononuclear megakaryocytes), megakaryocytes with multiple small detached nuclei, or nuclei separated by thin strand, “pawn ball” shape, cytoplasmic vacuoles

18. Morphology of PB and BM in MDS Discrete nuclear lobes, or multinucleation Micromegakaryocytes Pawn ball megakaryocyte.

19. FAB classification

20. WHO classification RA and RARS subdivided according to presence or absence of multilineage dysplasia RAEB-t is eliminated; patients considered to have acute leukemia CMML reclassified under a subgroup of myelodysplastic/myeloproliferative disorders Separate out patients with a 5q deletion syndrome

21. Cytochemical abnormalities of MDS • Cytochemical studies included iron stain, periodic acid-Schiff (PAS), peroxidase, butyrate esterase, chloroacetate esterase, and double esterase stains • ↓activity and abnormal positive results of MPO, chloroesterase, alkaline phosphatase, SSB, dual esterase in myeloid and monocytic cells • Especially more difficult to diagnose RARS, RAEB-t, CMML due to major qualitative and quantitative abnormalities, than RA • The iron stain remained most helpful in identifying abnormal ringed sideroblasts, a feature of dyserythropoiesis- supports the diagnosis of MDS • The PAS stain was helpful, if positive, in identifying patients with MDS; when negative did not help distinguish MDS from non-MDS hematologic disorders • The combination of two stains, PAS and iron stain or PAS and double esterase -helpful in excluding MDS– negative results

22. Bone Marrow Histology of MDS Aspirate and biopsy helpful to estimate: • Cellularity – which cells are dysplastic • Histological changes – displacement of hematopoiesis cells from their usual sites • Abnormal localization of immature precursors (ALIP) – may find clusters of immature myeloid cells away from the BM trabeculae = early indicator of leukemic transformation • Myelofibrosis – Rare occurrence, associated with BM hypoplasia Useful to determine dysmegakaryocytopoiesis, not so much for dyserythro- or dysgranulopoeisis

23. Cytogenetics and molecular abnormalities of MDS Clonal abnormalities present in 30-60% of patients with MDS Most frequent abnormalities: 5q- = deletion of the long arm of chromosome 5 • The common deleted region always spans the chromosome band 5q31 • Several genes related to hematopoiesis are found on 5q Genes that encode cytokines and their receptors • More frequent in RA • Clinical features include macrocytic anemia, normal or ↑ platelet, mild leukopenia, monolobular or dwarf megakaryocytes, erythroid hypoplasia • Affects mostly females • Good prognosis – median survival ~8yrs • The prognosis of 5q deletions in MDS is generally favorable if they are not part of complex abnormalities

24. Cytogenetics and molecular abnormalities of MDS Deletion comprise any region located between band 5q13 and 5q33 Band 5q31 is consistently deleted in most patients

25. Cytogenetics and molecular abnormalities of MDS Monosomy 7 (-7) • Abnormalities in chromosome 7 occur in about 20 percent of MDS patients • Common in patients who have prior chemotherapy exposure • Associated with a poor prognosis • Monosomy 7 + complex karyotype = short survival rate and ↑ progression to AML • Rarely seen in RA • In children - > risk of bacterial infections independent of neutrophil count

26. Cytogenetics and molecular abnormalities of MDS Trisomy 8 (+8) • Categorized as intermediate cytogenetic risk group • The most frequent cytogenetic abnormality in de novo MDS in China • Found in 15-20% of MDS • 5-10% of MDS with +8 are treatment-related MDS • Present in each FAB subgroup: up to 25-30% of RARS cases have +8; 15-20% of other subgroups have +8

27. Cytogenetics and molecular abnormalities of MDS Deletion of part of chromosomes 11q, 20q • 20q- alone is associated with a good prognosis regarding survival and potential for AML evolution • pathogenic mechanism by which 20q- alters the hematopoietic stem cells is unknown Anomalies of p17 • Where p53 gene reside • Associated with hypolobulated granulocytes with cytoplasmic vacuoles • Mainly refractory anemia with excess of blasts RAEB/ RAEB-t in MDS Other rare chromosome abnormalities are present in a substantial portion of patients

28. Secondary MDS After significant exposure to chemotherapy, radiotherapy or other toxic agents Laboratory findings, clinical manifestations, evolution similar to new MDS Higher frequency of chromosomal abnormalities Greater tendency to early leukemic transformation

29. Clinical features of MDS • Symptoms associated with progressive bone marrow failure • Depends on the degree of anemia, neutropenia, thrombocytopenia • Patients without excess blast in BM may be asymptomatic for a long time • Abnormal physical findings are not prominent, non specific • Fatigue and malaise result from anemia. • Signs and symptoms of chronic heart failure may develop in patients with underlying cardiac problems– in the elderly • Petechiae, ecchymosis, and nose and gum bleeding- low platelet count. If underlying dysplastic changes were missed initially- may be mistaken for immune thrombocytopenia. • Fever, cough, dysuria, or shock - serious bacterial or fungal infections associated with neutropenia.

30. MDS in children • Rare- less than 5% of all blood cancers in children • The median age at diagnosis in children is 7 years old • RA is the most common subtype of childhood MDS. • About half of all children with MDS are diagnosed with RA • RAEB and RAEB-t subtypes • Symptomatic – fever, pallor, physical weakness/loss of strength • Normocytic, normochromic anemia rather than macrocytic anemia • ↑ risk of development of AML associated with Down’s syndrome, Fanconi’s anemia • Children with treatment-related and secondary MDS tend to have less favorable treatment outcomes than children with primary MDS

31. Prognosis Factors that affect prognosis: • The number of blast cells in the bone marrow. • Whether one or more types of blood cells are affected. • Whether the patient has symptoms of anemia, bleeding, or infection. • Whether the patient has a low or high risk of leukemia. • The nature and number of chromosome abnormality • Whether the myelodysplastic syndrome occurred after chemotherapy or radiation therapy for cancer. • The age and general health of the patient. • The presence of abnormal localization of immature precursors (ALIP)

32. Prognostic scoring systems • The International Prognostic Scoring System (IPSS) - used to help assess the severity of MDS • Based on the IPSS score, the patient's history, and observations, a treatment plan is designed to address the MDS • Based on • The presence of one or more low blood cell counts (cytopenias) • BM blast cell count • Cytogenetic results – chromosomal changes in the marrow cells

33. Prognostic scoring systems

34. Diagnostic problems with MDS Difficult to differentiate from aplastic anemia • Clonal anomaly will confirm MDS • BM smear for dysplastic changes Differentiate from acute myelofibrosis (AML, M7) • 15% of MDS show BM fibrosis – sMDS • Dry tap in BM aspiration, trilineage dysplasia, cytopenias • No organomegaly • Cytogenic abnormalities From essential thrombocytosis JAK2 mutation in ET Cytogenetic analysis – 5q- or other translocation in MDS

35. Diagnostic problems with MDS CMML to be differentiated from CML • CMML, patients have an elevated number of monocytes in the blood • CMML patients have abnormal looking (dysplastic) cells in their bone marrow • CMML has features of both myelodysplastic syndrome and myeloproliferative disorder • Cytogenetic studies to differentiate Differentiate from Acute erythroid leukemia • When the total population of BM erythroblasts > 50% of all nucleated cells • Algorithm to help with diagnosis

37. Treatment of MDS Allogeneic stem cell transplantation – only therapy with potential to treat MDS • Since most are elderly – not as practical • Limited to younger patients • <60yrs with a matched sibling • <55yrs unrelated but matched donor High dose chemotherapy (without transplantation) • Short lived response • Response - < 20% of patients still in remission 2 years after therapy Advances in pathogenesis – development of novel drugs IPSS used to tailor therapy

38. For the elderly - IPSS low or intermediate-1 risk category – treat to improve hematopoiesis • For younger patients – IPSS-2 or high risk – treat to improve survival

39. Review questions The most common dyserythropoietic finding in the bone marrow in MDS • Megaloblastoid development • Impaired hemoglobinization • Pseudo-Pelger- Huet cells • Agranular cytoplasm Ans. a Why should you test B12 and folate levels when entertaining a diagnosis of MDS? Ans. Megaloblastoid changes are often seen in the erythroid and megakaryocytic lines of MDS and are similar to abnormalities of megaloblastic anemia. MDS should not be diagnosed if there is evidence of either a B12 or folate deficiency.

40. Chronic lymphocytic disorders • Clonal proliferations of morphologically and immunophenotypically mature B or T lymphocytes • Diagnosis based on morphology, immunology, cytogenetic, molecular and clinical features • Leukemias = diseases affecting the BM and the PB • Lymphomas = disease affecting extramedullary sites • Chronic lymphoid leukemias have various stages • Early stage – small tumor cells, with low proliferation, prolonged survival • Transformative stage – proliferation in extramedullary sites, increase in large immature cells

41. Chronic lymphocytic disorders • Advances in cytogenetics and molecular biology show that hematopoietic neoplasms are associated with unique genotypic profile • Genotype affects the development and the clinical features of the disease • Advances in the development of monoclonal antibodies → identification of unique immunophenotypic profile for most leukemias and lymphomas • Advances have enhanced accuracy and reproducibility of diagnosis

42. Chronic lymphocytic leukemia • Amonoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. • Most common form of leukemia found in adults in Western countries • In some people with CLL, the disease grows and progresses slowly • May take years for symptoms to appear or for treatment to be needed • Some patients may never need treatment for their CLL

43. Chronic lymphocytic leukemia • In other patients the disease grows more quickly and needs treatment sooner. • Some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years • 90% people diagnosed with CLL are over the age of 50 • Mostly affects men – incidence is twice that of women • When present - symptoms are related to anemia, thrombocytopenia and neutropenia • Signs and symptoms may develop gradually

44. Chronic lymphocytic leukemia • B lymphocyte neoplasm (B-CLL) – T lymphocyte very rare • CLL is a stage of small lymphocytic lymphoma (SLL) -a type of B-cell lymphoma primarily in the lymph nodes • CLL and SLL are considered the same underlying disease but with different clinical presentation • Included in the lymphoproliferative disorders

45. Chronic lymphocytic leukemia Characterized by lymphocytosis in PB and BM • Accumulating lymphocytes in CLL → crowding out of other cells • → neutropenia, anemia, thrombocytopenia • → Organ infiltration → adenopathy, splenomegaly, hypersplenism Neoplastic cells of CLL are more fragile than normal lymphocytes > burst open during smear preparation > smudge cells (bare nuclei) • Smudge cells can also be found in reactive lymphocytosis and other neoplasms • Therefore not diagnostic • Reduce smudge cells by mixing a drop of albumin with a drop of blood prior to making smear

46. CLL morphology CLL in BM, with numerous mature- appearing lymphocytes CLL in PB with smudge cells

47. Chronic lymphocytic leukemia Characterized by altered humoral immunity (antibody mediated immunity) • Due to suppression of all classes of immunoglobulin (Ig) → hypogammaglobulinemia → increased susceptibility to infections • Production of autoantibodies → autoimmune disease - idiopathic thrombocytopenia or autoimmune hemolytic anemia • 15-35% of patients develop autoimmune hemolytic anemia at some time during course of the disease • Immune-mediated destruction of red blood cells and platelets = unique feature that is not present in other kinds of leukemia

48. Chronic lymphocytic leukemia 51% of B-CLL patients positive for Bence-Jones paraproteinemia • Bence Jones proteins are considered the first tumor marker; made by plasma cells; • The presence of these proteins in a person's urine is associated with a malignancy of plasma cells

49. Cause and physiological process of CLL No specific cause known Possibly due to viral infections • B-CLL cells from some patients appear to be a malignant transformation of an antigen-committed B cell responding to the human T-lymphotropic virus type 1 (HTLV-1) infection • HTLV-1 infection seems to precede development of CLL in some patients May be due to failed apoptosis • B-CLL cells have been shown to have high levels of anti-apoptotic agents (protein BCL2) • The proto-oncogene BCL2 is a known suppressor of apoptosis, resulting in a long life for the involved cells • BCL-2 seen in 80% of B-CLL cells • Presence of p53- tumor suppressor gene in CLL –del of 17p