Leukemias-basic pathology

Leukemias-basic pathology[AML M0-M7]

1. BY- JAIMIN MANEK LEUKEMIAS Guided by: Dr.Ami shah Moderated by: Dr.Priyanka gohel Presented by : Jaimin Manek

2. What is “Leukemia ???

3. Definition Leukemia : is characterized by abnormal proliferation of blood cells,usually WBCs(Leukocytes). Acute leukemia : rapid increase of immature blood cells . Chronic leukemia : excessive build up of relatively mature, but still abnormal blood cells.

4. Causes Leukemia results - From somatic mutations in the DNA. - By activating oncogenes or deactivating tumor suppressor genes. FACTORS: Ionizing radiation Viruses : Human T- lymphotropic virus (HTLV- 1 ) Chemicals : Benzene,chemotherapy . Smoking : slight increase in leukemia incidence. Genetic predisposition : Down syn.,Fanconi anemia

5. Haematopoiesis

6. Pathogenesis: Normal hemopoiesis is finely tuned by hemostatic feedback mechanisms involving cytokines and growth factors that modulate the marrow output of red cells, granulocytes and platelets. These mechanisms are deranged in marrows involved by myeloid neoplasms . Loss of control on growth & survival and suppressor fuctions . CSBRP-SDUMC-Oct-2014

7. Pathogenesis :

8. Leukemias - classification Main groups: 1-Myeloid (acute / chronic) 2-Lymphoid (acute / chronic) 3-Mixed lineage leukemias CSBRP-SDUMC-Oct-2014

9. The most common malignant neoplasms of childhood are Leukemias Which one is the commonest type of childhood Leukemias ? AML 10% ALL 80% CML 2-3% JCML 1-2%

10. Acute Myeloid Leukemia Myeloblast P romyelocyte M yelocyte M etamyelocyte B and N eutrophil MATURATION Adapted and modified from U Va website Myeloid maturation:

11. Classification of AML Two types of classifications : 1- FAB classification - degree of maturation & lineage of blasts - usage of cytochemistry & IHC 2- WHO classification - degree of maturation & lineage of blasts - Immunophenotyping - cytogenetic & molecular features - clinical outcome CSBRP-SDUMC-Oct-2014

12. Acute myeloid leukemias (AML) Classification - FAB M0: minimally differentiated M1: myeloblastic leukemia without maturation M2: myeloblastic leukemia with maturation M3: hypergranular promyelocytic leukemia M4: myelomonocytic leukemia M4Eo: variant, increase in marrow eosinophils M5: monocytic leukemia M6: erythroleukemia ( DiGuglielmo's disease) M7: megakaryoblastic leukemia

13. WHO classification AML with recurrent genetic abnormalities AML and MDS AML - Tx related AML not otherwise categorised Myeloid Sarcoma Myeloid proliferation related to Down Syndrome

14. Signs and Symptoms Most of the signs and symptoms are due to: 1-Anemia. 2-Leukopenia. 3-Thrombocytopenia. Bicytopenia , Pancytopenia . All symptoms associated with leukemia can be attributed to other diseases, consequently, leukemia is always diagnosed by laboratory investigations.

15. Gum bleeding CSBRP-SDUMC-Oct-2014

16. Gum hypertrophy CSBRP-SDUMC-Oct-2014

17. Hepatosplenomegaly CSBRP-SDUMC-Oct-2014

18. Cervical lymphadenopathy CSBRP-SDUMC-Oct-2014

19. Cerebral hemorrhage CSBRP-SDUMC-Oct-2014

20. Specific Presentations AML M0, M1, M2 : Chloromas AML M3 : DIC AML M4, M5 : Gum hypertrophy AML M7 : Mediastinal mass (germ cell tumors ) CSBRP-SDUMC-Oct-2014

21. Diagnosis

22. Diagnostic Studies CBC a. Anemia b. Trombocytopenia c. WBC High Normal Low Coagulation Studies (M3-DIC) Biochemical Studies

23. Bone marrow (>20% blasts) - Morphology - Cytochemistry - Immunophenotyping - Cytogenetics Peripheral blood examination blasts in almost all cases Molecular Genetic Analysis

24. Requisits for diagnosis of AML The diagnostic requisite of 20 percent type I and II myeloblasts in the peripheral blood or bone marrow. BLAST Equivalents: In AML (M3), the predominant leukemic cell is promyelocyte In AML (M5A), the predominant proliferating cell is the monoblast In AML (M5B), the predominant cell is the promonocyte . The megakaryoblasts of acute megakaryoblastic leukemia vary in morphology but uniformly lack the cytochemical properties of myeloblasts . CSBRP-SDUMC-Oct-2014 20% BLASTS

25. Myeloblast < 1% of the normal bone marrow, not observed in normal blood Vary in size, but are usually large Nucleus is delicate, large, round or oval, with prominent nucleoli. Stain purplish red with Wright stain. Chromatin stains evenly Small to moderate amount of bluish nongranular cytoplasm Three major types: Type I, II, and III

26. Type I Fine nuclear chromatin 2 to 4 distinct nucleoli Moderate rim of pale to basophilic cytoplasm Without azurophilic granules Type II Delicate azurophilic granules in the cytoplasm (up to 20) Type III Numerous azurophilic granules in the cytoplasm

27. Typical Features of Blast Cells Monoblast Myeloblast Erythroblast Megakaryoblast Lymphoblast

28. CYTOLOGIC FEATURES OF BLASTS IN AML & ALL A M L A L L Blast size Medium to large, uniform Variable Small to medium Cytoplasm Fine granules may be present Usually scant, a few coarse granules may be seen Auer rods Present in 60-70% of cases absent Nuclear chromatin Finely dispersed Fine to coarse Nucleoli 2-4, prominent 1-3, indistinct

29. Auer rods Is an azurophilic linear structure (Single or multiple) present in numerous blasts or in rare cells Present approximately 60 to 70 % of cases of AML. MPO, SBB, and CAE positive The presence of an Auer rod in one or more blasts is definitive evidence of AML. (The finding is not specific for any one type of AML.)

30. CYTOCHEMICAL PROFILES OF ACUTE LEUKEMIAS MPO / SBB CAE NSE PAS AP ALL _ _ + / - Focally + 75% + / - Focal in T-ALL AML + + + Monocytic -diffuse - / + + MPO - myeloperoxidase , SBB -Sudan balck B, CAE - chloracetate esterase, NSE -non specific esterase, PAS -periodic acid schiff , AP -acid phosphatase CSBRP-SDUMC-Oct-2014 cv cv

31. Myeloperoxidase (MPO) p- Phenylene diamine + Catecol + H 2 O 2 MPO > Brown black deposits

32. Sudan Black B It is a direct stain phospholipid in granular membrane. Auer rods are MPO and SBB positive.

33. Chloracetate (Specific) Esterase Myeloid Cell Line Naphthol -ASD- chloracetate Free naphthol compounds + Stable diazonium salt Red deposit

34. Immunophenotyping FAB Immunological marker AML with minimally differentiated CD13, CD34 , HLA-DR , CD33,CD117,CD2,CD7,TdT AML without maturation CD13,CD14,CD33, CD34 AML with maturation and with t(8;21) CD34 , CD56 Acute promyelocytic leukemia CD13,CD33, HLA-DR absent, CD34 negative Acute myelomonocytic leukemia with abnormal eosinophils and inversion 16 CD13, CD34 , CD11b,CD11c,CD14 ,CD33 Acute monocytic leukemia and 11q23 abnormalties CD14 ,CD4, CD36,CD64 Erythroleukemia Glycophorin 7, Transferrin receptor CD71 Acute Megakaryocytic leukemia c CD41, c CD42b, c CD61

35. Chromosomal Abnormality Leukemia t(8;21) M2 t(15;17) M3 inv, del, t(16q) M4 t(9;11) M5 (M5a); M4 Cytogenetics

36. AML M0 5% of AML cases No definite evidence of myeloid differentiation can be given by morphology & cytochemistry . Blasts resembles M1 blast and L2 Lymphoblast PS: Large cells with pale grey blue CYTOPLASM NUCLEUS has opened fine chromatin with >= 1 nucleoli Cytochemistry : < 3% blasts reactive for MPO, SBB or NSE Immunophenotyping : 20% blasts express one or more myeloid antigens: CD13, CD14, CD33 may be TdT positive ;

37. AML-M0 Bone marrow smear, May-Giemsa stain, x1000

38. AML M0 Bone marrow smear, cyMPO stain, x1000

39. AML- M1 15-20% of all AML cases M1 is differentiated from M2 by the fact that >90% blasts of non erythroid cells <10% of marrow nucleated cells are promyelocytes or more mature neutrophils PS : Blasts have variable N/C ratio with pale basophilic CYTOPLASM NUCLEUS has 1-4 nucleoli Cytochemistry :>3% blasts reactive for MPO or SBB. Immunophenotyping : Blasts express myeloid antigens: CD13, CD14, CD33 .

40. BLOOD SMEAR BONE MARROW SMEAR BLAST WITH PALE TO BASOPHILIC AGRANULAR CYTOPLASM , NUCLEI WITH FINE CHROMATIN & PROMINENT NUCLEOLI

41. AML M1 CASE-4 BM 1000X

42. AML- M2 Commonest (30%) of all AML Evidence of maturation to promyelocytes and more mature neutrophils in 10 percent or more of the cells. PS: Blasts are large with pale basophilic CYTOPLASM NUCLEUS: oval to indented with 2-3 nucleoli Cytochemistry : MPO, SBB, CEA + ve Immunophenotyping : CD15, CD13, CD33 + ve In t(8,21) associated cells: Pathognomonic 40-80% are positive for CD19 20% are TdT positive

43. AML M2 CASE 1 Bone marrow smear, MGG x200

44. AML M2 CASE 1 Bone marrow smear, MGG X1000

45. AML M3( APL) A form of AML characterized primarily by a proliferation of abnormal promyelocytes . It is usually accompanied by DIC t(15;17) The disease presents in two morphologic types : 1) hypergranular APL predominant cell is an abnormal promyelocyte with markedly increased and coarse azurophilic granules 2) microgranular or hypogranular APL predominant cell is an abnormal promyelocyte with diminished or small azurophilic granules.

46. AML M3 The most common presenting symptoms, occurring in 90 % of patients, relate to hemorrhagic manifestations and include easy bruisability , bleeding gums, hemoptysis , epistaxis , petechiae , symptoms of gastrointestinal & intracranial hemorrhage . Basic pathology is DIC .

47. BONE MARROW SMEAR HYPERGRANULAR Nucleus : Folded, lobulated , granular obscure border. Cytoplasm : Prominent Azurophilic granules. Auer rods : Frequent, FAGGOT cells ( cells with bundles of auer rods)

48. MICROGRANULAR Nucleus : Irregular, Folded. Mostly binucleated . Cytoplasm : Fine small granules, “Dusky “ appearance. Auer rods : Rare.

49. AML M3 , MGG x1000

50. DIC CSBRP-SDUMC-Oct-2014

51. AML M4 (AMML) ACUTE MYELOMONOCYTIC LEUKEMIA Nearly 20% of all AML cases Both MYELOBLAST and MONOBLAST co exist M.c . Acute Leukemia in INFANTS P.S : Both Myeloblast and monocytic components ( monoblasts , promonocytes , monocytes ) seen Cytochemistry : monocytic cells – NSE stain myeloid cells – Mpo /CAE stain IFT : Monocytic component- CD64,CD14 Myeloid component- CD13,CD33,CD15

52. Peripheral smear MYELOBLAST MYELOCYTE PROMONOCYTE Large monocytoid cells with Cytoplasm: Pale blue agranular Nuclei: Ovoid to reniform

53. AML M5 (Acute monoblastic leukemia ) > 80% of leukemic cells are monocytic lineage Neutrophil component may constitute <20% Acute monoblastic M5a Vs monocytic leukemias M5b : PS : Monoblasts >80% in monoblastic leukemia Promonocytes are predominant in monocytic leukemias Cytochemistry : SBB – Fine scattered granules in monoblast NSE – Diagnostic IFT: CD14, CD11b, CD11c, CD64, CD68

54. M5a( Acute Monoblastic Leukemia ) BLOOD SMEAR BONE MARROW SMEAR MONOBLAST 80% or more are MONOBLAST Abundant cytoplasm Round nuclei with nucleoli MONOBLAST WITH ABUNDANT CYTOPLASM WITH FINE GRANULES

55. M5b( Monocytic Leukemia ) BLOOD SMEAR BONE MARROW SMEAR PROMONOCYTES <80% Monoblast Mature monocytes or promonocytes predominate

56. AML M6( ACUTE ERYTHROID LEUKEMIA ) Def : Erythroleukemia by definition involves both the granulocytes and erythroid cells TYPES : M6a Erythroleukemia M6b PURE erythroleukemia Erythroblast: Relatively high nuclear/ cytoplasmic ratio Nucleus round with slightly condensed chromatin; Nucleoli variably prominent Deeply basophilic cytoplasm that may be vacuolated

57. M6a ( ERYTHROLEUKEMIA) 5% of AML cases More COMMON THAN pure erythroid leukemia. Bimodal distribution- <20 yrs and >60yrs. CRITERIA FOR DIAGNOSIS > 50% of nucleated marrow cells are erythroid lineage > 20% of nonerythroid cells are myeloblast Dyserythropoiesis is prominent Cytochemistry : MPO + ve , PAS + ve IFT: Glycophorin A +, CD13, CD33, CD117

58. BLOOD SMEAR BONE MARROW SMEAR ERYTHROID PRECURSOR

59. M6b ( PURE ERYTHROID LEUKEMIA ) Very rare Also called ERYTHEMIC MYELOSIS , ACUTE Di GUGLIELMO SYNDROME > 80% of marrow cells are erythroblast No significant myeloblastic component

60. Bone marrow smear Abnormal erythroid precursors

61. AML M6 Case-3 Bone marrow smear, May-Giemsa stain, x1000

62. History: 47year old man with a history of renal transplant as well as refractory anemia with ringed sideroblasts diagnosed 1 year back. Now he has fatigue and loss of weight. He is on cyclosporin and prednisolone. Source: CAP AML M6b case-5

63. Investigations: Blood count revealed anemia and thrombocytopenia and rare blasts. BM revealed 81 % erythroid precursors, marked dysplastic changes and “block-and-blush” PAS positivity of erythroid lineage. There were 12 % myeloblasts and minimal dysplasia of granulocytic cell line.   Diagnosis: Acute erythroleukemia-M6b (pure erythroleukemia ). AML M6b case-5 Source: CAP

64. AML M7 (ACUTE MEGAKARYOBLASTIC LEUKEMIA) 10% of AML in children & 5% of adult AML Bimodal distribution- Infancy and elderly Most common leukemia seen in Down’s Syndrome. May be associated with mediastinal germ cell tumors CRITERIA FOR DIAGNOSIS Megakaryoblast 20% or more in BM Bone marrow fibrosis MK blast: 12-18µm Round nucleus with reticular chromatin 1-3 nucleoli Cytoplasm is basophilic and agranular Cytoplasmic blebs May resemble Lymphoblast

65. ACUTE MEGAKARYOBLASTIC LEUKEMIA Morphologically confused with - L2 subtype of ALL - AML M1. Diagnosis depends on Elevated serum Lactate Dehydrogenase level. Marked leucocytosis Cytochemistry : MPO, SBB, TdT are negative Some scattered PAS positivity Immunophenotyping : CD41, CD61, Gp IIb / IIIa

66. Blast show distinct cytoplasmic blebs or psedopods formation Peripheral blood – fragments of megakaryoblast micromegakaryocytes Or dysplastic large platelets seen

67. Promegakarocytes , irregular nuclei , coarse chromatin BONE MARROW SMEAR

68. Malignant germ cell tumor of anterior mediastinum CSBRP-SDUMC-Oct-2014

69. Prognostic Factors in AML Favorable: younger age (<50) WBC <30,000 t(8;21) – seen in >50% with AML M2 inv(16) – seen in AML M4 eos t(15;17) – seen in >80% AML M3 Unfavorable: older age (>60) Poor performance status WBC >100,000 Elevated LDH prior MDS or hematogic malignancy CD34 positive phenotype, MRD1 postive phenotype del (5), del (7) trisomy 8 t(6;9), t(9;22) t(9;11) – seen in AML M5 FLT3 gene mutation (seen in 30% of patients)

70. CHRONIC MYELOID LEUKEMIA

71. CHRONIC MYELOID LEUKEMIA CML is a clonal stem cell disorder characterised by increased proliferation of myeloid elements at all stages of differentiation. 15% of leukaemias . It occurs most often between 40–60yrs Incidence increases with age, M > F.

72. CYTOGENETICS OF CML Philadelphia chromosome Present in >80% of those with CML. It is a hybrid chromosome comprising reciprocal translocation between the long arm of chromosome 9 and the long arm of chromosome 22—t(9;22) forming a fusion gene BCR/ABL on chromosome 22, which has tyrosine kinase activity

73. CML is characterised by 3 distinct phases Chronic Phase: Proliferation of myeloid cells, which show a full range of maturation. Accelerated Phase: decrease in myeloid differentiation occurs. Blast crisis: (acute leukemia)

74. FAB Classification 1) Chronic granulocytic leukemia Classical CML with Ph chromosome + ve / - ve But bcr / abl 1 + ve 2) Atypical CML or Ph – ve CML Monocytosis is intermediate b/w Classical CML & CMML ( 3-10%) bcr / abl 1 – ve 3) Chronic myelomonocytic leukemia (CMML) Ph – ve CML with predominance of monocytes in blood ( >10%)

75. CLINICAL PRESENTAITON OF CML Symptoms Asymptomatic (50% of patients) Fatigue Weight loss Abdominal fullness and anorexia Abdominal pain, esp splenic area Increased sweating Easy bruising or bleeding Signs Splenomegaly (95%) ( 50% of patients have a palpable spleen ≥ 10 cm BCM, Usually firm and non tender ) Hepatomegaly (50%)

76. DIAGNOSIS OF CML Approximately 85% of patients are diagnosed in the chronic phase and then progress to the accelerated and blast phases after 3-5 years. The diagnosis of CML is based on Histopathologic findings in the peripheral blood & Philadelphia chromosome in bone marrow cells Investigation CBC with differential peripheral blood smear bone marrow analysis US using for liver/spleen

77. Peripheral blood – neutrophils 20,000 - >500, 000/  L - basphilia -  LAP score - blasts < 5% - Nucleated RBCs - Thrombocytosis - Anaemia Neutrophils and Myelocytes are PREDOMINANT cells with blasts being < 3%.

78. PS shows increased Myelocytes and Mature Polymorphs

79. Two basophils in blood smear

80. Leukemoid reaction CML WBC High High Anemia (-) (+) PBS Shift to the Left Toxic granulation Dohle bodies Shift to the left (blast) Eosinophilia , basophilia LAP score High Low Philadelphia chromosome (-) (+)

81. Summary – Acute leukemias Leukemias are clonal disorders Mutations in oncogenes is the most common underlying pathology Present with : Anemia, Petichiae , infections, hepatosplenomegaly , Lymphadenopathy There may be normal, low or elevated total white count.

82. Summary AML is a heterogeneous disease Blast count should be 20% in BM There are blast equivalents The presence of an Auer rod is definitive evidence of AML WHO classification is well accepted Detection of genetic abnormalities dictates Tx and Prognosis

83. Identify…. AML or Normal bone marrow A B

84. Identify…. AML or CML A B

85. Thank You!! To Be Continued..