8. Acute leukaemia (Diagnostic approach)

Hematology

1. Rosline Hassan, MD, MMed(Hemato) Professor of Hematology  Acute Leukemia

2. Outline Overview of leukemia Classification of leukemia Understand the pathogenesis Describe the clinical features Able to list down the laboratory investigations required  Understand the basic management of leukemia patients

3. Types of Leukaemia Acute : No maturation beyond blast Chronic : Maturation beyond blast Lymphoid (B or T lineage) ALL CLL Myeloid – granulocytes Erythroids Monocytes Platelets AML CML

4. Acute Leukaemia Define : heterogenous group of malignant disorders which is characterised by uncontrolled clonal and accumulation of blasts cells in the bone marrow and body tissues Sudden onset If left untreated is fatal within a few weeks or months In Malaysia leukemia is the seventh most common cancer Its incidence is 2.9/100,000 population for all leukemia 

5. Classification : Acute leukaemia French-British (FAB) group : Morphology and cytochemistry supplemented by immunophenotyping WHO Clinical Features, Morphology, immunophenotyping and cytogenetic Year 2001, 2008 & 2016 WHO2016 incorporate clinical features, morphology, immunophenotyping, cytogenetics, and molecular genetics

6. Revision of Classification WHO 2016 for Acute Leukemia This revision has been influenced by several factors including the following: 1. Discovery of recently identified molecular features for diagnostic and prognostic markers 2. An integrated approach that includes history, hematologic, morphologic, cytogenetic, and molecular genetic finding WHO2016 : 25 entities and 3 new genetic entities

7. The 2016 revision to the World Health Organization classification of acute leukemia BLOOD, 19 MAY 2016 x VOLUME 127, NUMBER 20

8. FAB Acute Leukemia

9. FAB Acute Myeloid Leukaemia M0 minimal differentiation. <3% MPO positive blasts. Blasts undifferentiated by morphology and cytochemistry. MPO positive by immunocytochemistry, flow cytometry M1 without maturation. >90% blasts in non-erythroid cells (NEC). >3% MPO or SBB positive. M2 with maturation. Blasts 20-89% of NEC. <20% monocytic cells.

10. FAB Acute Myeloid Leukaemia M3 classical - hypergranular promyelocytes. Bundles (faggots) of Auer rods. Bilobed nuclei Cytogenetics: RT-PCR for t(15;17) and PML/RARa transcripts. M3V promyelocytes are agranular with deeply basophilic cytoplasm. Bilobed nuclei   Cytochemistry, cytogenetics and molecular similar to classical M3.

11. FAB Acute Myeloid Leukaemia M4 acute myelomonocytic >20% cells in the granulocytic lineage. >30% cells in the monocytic lineage. CAE and Butyrate esterase stain for both granulocyte and monocyte M4Eo cells Abnormal eosinophil granules are also CAE positive. Cytogenetics & RT-PCR to confirm inv/del/(16)

12. FAB Acute Myeloid Leukaemia M5 : Acute monoblastic leukaemia M5A predominantly monoblastic. M5B shows maturation with clear progression to promonocytes and monocytes. Granulocytes <20% of cells

13. FAB/WHO 2016 Acute Myeloid Leukaemia M6 : Pure Erythroid Leukemia The current definition of PEL requires 80% erythroid precursor marrow involvement with at least 30% of cells being proerythroblasts. Excludes cases arising after prior cytotoxic therapy, are classified as ‘therapy related myeloid neoplasms’. The 2016 WHO classification, elimination of erythroid/myeloid category and the consideration of whether myeloblasts should  The requirement 30% proerythroblasts is needed for AML, 

14. Pure Erythroid Leukemia IHC or flow cytometry distinguish cells based on early stage precursors CD-71 is highly expressed in erythroid precursors, in all stages of maturation Ferritin H is a soluble iron storage protein expressed in early erythroid precursors, These markers may be crucial in diagnosing acute erythroleukemia versus other morphologic forms of myeloid leukemia E-cadherin highly sensitive and specific marker for immature erythroblasts helps in differentiating PEL from erythroid neoplasms American Journal of Hematology, Volume: 92, Issue: 3, Pages: 292-296, First published: 22 December 2016, DOI: (10.1002/ajh.24626)

15. FAB Acute Myeloid Leukaemia M7 acute megakaryoblastic leukaemia >20% megakaryoblasts. Often BM difficult to aspirate. Micromegakaryocytes or abnormal mature forms The only form of AML where the trephine biopsy may be diagnostic. Flow cytometry or immunohistochemistry to identify platelet specific antigens on the blasts.

16. FAB Acute Lymphoblastic Leukemia Acute lymphoblastic leukemia (ALL)* L-1 85% # L-2 14% L-3 (Burkitt's)1% # childhood

17. Specimen requirements 1.PB and BM specimens collected prior to any definitive therapy. Bone marrow specimens for : BM aspirate smears and/or touch preparations; BM biopsy; complete cytogenetic analysis; flow cytometry, with specimen cryopreserved for molecular genetic studies Assessment of blasts 1.Blast percentage in PB and BM is determined by visual inspection. Assessment of blast lineage 1.Multiparameter flow cytometry (at least 3 colors) is recommended; 2.Cytochemistry, such as MPO or nonspecific esterase, may be helpful, particularly in AML, NOS, but it is not essential in all cases. Guidelines for using the revised WHO classification of myeloid neoplasms

18. Assessment of genetic features 1.Complete cytogenetic analysis from BM at initial diagnosis when possible. 2.Additional studies, FISH, RT-PCR, mutational status, should be guided by clinical, laboratory, and morphologic information. 3.Mutational studies for mutated  NPM1 ,  CEBPA , and  FLT3  are recommended in all cytogenetically normal AML Correlation/reporting of data All data should be assimilated into one report that states the WHO diagnosis. Guidelines for using the revised WHO classification of myeloid neoplasms

19. Special consideration AML with recurrent genetic abnormalities Only AML with t(8;21)(q22;q22), inv(16)(p13.1q22) or t(16;16)(p13.1;q22), and APL with t(15;17)(q22;q12) are considered as acute leukemia regardless of blast count in the PB or BM. AML with myelodysplasia-related changes (1) history of MDS or MDS/MPN and have evolved to AML, (2) myelodysplasia-related cytogenetic abnormality, or (3) at least 50% of cells in 2 or more myeloid lineages are dysplastic.

20. Leukemogenesis AML1-ETO, PML-RARA -Block differentiation N or K-RAS, FLT3, BCR-ABL -Increased proliferation Leukemogenesis : multistep process Arises from hematopoietic pluripotent stem These mutations involve multiple genetic pathways: ‘Class I’ oncogenes : proliferative advantage to the leukemic cells ‘Class II’ oncogenes : contribute to myeloid maturation arrest Cooperation between mutations and/or translocations of both Class I and Class II oncogenes are critical to the development of AML

21. Acute Lymphoblastic Leukaemia Commonest in the age 2-10 years Peak at 3-4 years. Incidence decreases with age, and a secondary rise after 40 years. In children - most common malignant disease 85% of childhood leukaemia

22. NCR – LEUKAEMIA 2002 Lymphatic Leukaemia Age specific Cancer Incidence per 100,000 population by sex, Peninsular Malaysia 2002 Specific manifestation : * bone pain, arthritis * lymphadenopathy *hepatosplenomegaly * mediastinal mass *testicular swelling * meningeal syndrome

23. Acute Myeloid Leukemia Arise from myeloid precursor Rare in childhood (10%-15%) The incidence increases with age Specific History : prior therapy prior MDS Myeloid Leukaemia Age specific Cancer Incidence per 100,000 population by sex, Peninsular Malaysia 2002

24. Acute Myeloid Leukemia Specific manifestation : - Gum hypertrophy Hepatosplenomegaly Skins deposit Lymphadenopathy Renal damage DIVC

25. Lab Ix- 1. Confirm Diagnosis Full Blood Picture Bone marrow aspiration

26. 1.   Peripheral blood count Anaemia – normochromic normocytic Normal, Leukopenia or leukocytosis <1.0x10 9 /l to >200x10 9 /l neutropenia Thrombocytopenia <10x10 9 /l

27. How to distinguish AML vs CML from looking at peripheral blood Myeloid cell CML AML normal blasts q q Promyelocytes q myelocytes q Metamyelocytes q bands q neutrophils q q

28. Morphology Lymphoblast Blast size :small Cytoplasm : Scant Chromatin : Dense Nucleoli :Indistinct Auer-rods : Never present Myeloblast Large Moderate Fine, Lacy Prominent Present in 50%

29. Morphology Acute lymphoblastic leukaemia Acute myeloid leukaemia

30. 2. Bone Marrow Aspiration

31. 2.Bone marrow aspiration confirm acute leukemia (blast > 20%) (WHO classification) usually hypercellular

32. Lab Ix- 2. Classify leukemia Classification of leukemia ALL vs AML Subtypes of leukemia Subtypes WHO classification ALL: T or B-ALL AML: MO, M4/M5, M6,M7 AML vs APL

33. Lab Ix: 2.Classify Leukemia Cytochemical staining (LIMITED VALUE) Immunophenotyping Cytogenetic Molecular Genetic

34. 1. Cytochemical staining : AML vs ALL α) Peroxidase :- * negative ALL * positive AML Positive for myeloblast

35. Cytochemical staining : AML vs ALL b) Periodic acid schiff *Positive ALL (block) * Negative AML Block positive in ALL

36. Cytochemical staining: B-ALL vs T-ALL c) Acid phosphatase : focal positive (T-ALL)

37. Cytochemical staining AML M4 vs AML M5 Dual Esterases (CAE & Butyrate esterase ) AML-M4 AML-M5 CAE & Butyrate esterase Butyrate esterase

38. 2. Immunophenotyping ∙   Identify antigens on the blast cells 1.determine lymphoid or myeloid - cytochemical markers are negative or equivocal. E.g : AML-MO, M6, M7) 2.differentiate T-ALL and B-ALL 3. prognostic significance CD10 cells in ALL Rare cases of biphenotypic

39. Immunophenotyping Monoclonal antibodies(McAb) are recognised under a cluster of differentiation(CD).   MONOCLONAL ANTIBODIES USED FOR CHARACTERISATION OF ALL AND AML.   Monoclonal antibodies AML : CD13, CD33 ALL : B-ALL CD10, CD 19, CD22 T-ALL CD3, CD7

40. Lab Ix : 3.Prognostic value Cytogenetic Molecular test

41. Cytogenetic Study of chromosomes detect abnormalities within chromosomes diagnostic or prognostic value 1. Philadelphia chromosome : t(9; 22) : poor prognosis in ALL 2. t(15;17) diagnostic APML

42. (PCR based method) Specific detection of the gene Genetic Testing

43. Sequence in the diagnosis of APL (Acute Promyelocytic Leukemia)

44. Management Supportive care 1. Central venous catheter inserted to : facilitate blood product adm. of chemotherapy and antibiotics frequent blood sampling

45. Management 2. Blood support :- platelet con. for bleeding episodes or if the platelet count is <10x10 9 /l with fever fresh frozen plasma if the coagulation screen results are abnormal packed red cell for severe anaemia (caution : if white cell count is extremely high)

46. Management 3. Prevention and control infection barrier nursed Intravenous antimicrobial agents if there is a fever or sign of infection

47. Management 4.Physiological and social support

48. Specific treatment Used of cytotoxic chemotherapy. Aim : ∙  To induce remission (absence of any clinical or conventional laboratory evidence of the disease) To eliminate the hidden leukemic cells

49. Cytotoxic chemotherapy Anti-metabolites Methotrexate Cytosine arabinoside Act: inhibit purine & pyrimidine synt or incorp into DNA S/E : mouth ulcer, cerebellar toxicity DNA binding Dounorubicin Act : bind DNA and interfere with mitosis S/E : Cardiac toxicity, hair loss

50. Cytotoxic chemotherapy Mitotic inhibitors Vincristine Vinblastine Act : Spindle damage, interfere with mitosis S/E : Neuropathy, Hair loss Others Corticosteroid Act : inhibition or enhance gene expression Trans-retinoic acid Act : induces differentiation

51. What is the fate of acute leukemia Complete remission (CR) :achievement of a morphologic leukemia-free state (bone marrow blasts, <5%; absence of extramedullary disease) recovery of peripheral blood counts (absolute neutrophil count, >1000/µL; platelet count, >100 000/µL) Overall survival (OS) was defined as the time from initiation of therapy to death Relapse-free survival (RFS) was defined as the time from achievement of remission until relapse or death from any cause

52. Lab Ix : 4 Minimal Residual Disease Bone marrow aspirate: blast <5% Immunophenotyping : Leukemia Associated Immunophenotype Profile Cytogenetic / Molecular cytogenetic (FISH)/Molecular for specific mutation or translocation

53. Poor Prognostic Factors ALL AML Age <1 > 60 year TWBC > 50 x 10 9 /l High CNS present present (rare) Sex male male/female Cytogenetic t(9;22) monosomy 5, 7

54. THANK-YOU Thank-you for your attention