Acute Myeloid Leukemia

Introduction, Classification, Clinical features, Lab Diagnosis

1. Acute Myeloid Leukemia Dr Abdul Hafeez Kandhro Senior Lecturer B.Sc, M.Sc; Medical Technology, M.Phil Biochemistry Ph.D. Medical Technology (Mahidol University, Bangkok , Thailand)

2. Acute Myeloid Leukemia Malignant neoplastic proliferation and accumulation of immature and nonfunctional myeloid line of blood cells in the bone marrow . Also known as acute myelogenous leukemia or acute nonlymphocytic leukemia (ANLL) Most common Acute Leukemia affecting adults. As an Acute Leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated .

3. In normal hematopoiesis, the myeloblast is an immature precursor of myeloid white blood cells; a normal myeloblast will gradually mature into a mature white blood cell . However, In AML, a single myeloblast genetic changes which " freeze“ the cell in its immature state and prevent differentiation. When such a "differentiation arrest" is combined with other mutations which disrupt genes controlling proliferation, the result is an uncontrolled growth of an immature clone of cells, leading to the clinical entity of AML . Acute Myeloid Leukemia- Pathophysiology

4. The clinical signs and symptoms of AML result from the growth of leukemic clone cells, which tends to displace or interfere with the development of normal blood cells in the bone marrow . This leads to neutropenia, anemia, and thrombocytopenia. The symptoms of AML are often due to the low numbers of these normal blood elements. Acute Myeloid Leukemia- Pathophysiology

5. Acute Leukemia TRKC (15q25) TEL/TRKC t(12;15)( p13;q25) GFR3 (4p16) TEL/FGFR3 t(4;12)(p16;p13) FRK(6q21) TEL/FRK t(6;12)( q21;p13) PCM1/JAK2 t(8;9)(p21-22;p23-24 FLT3 (13q12) ITD (80%), activation loop kinase domain (15%) KIT (4q12) JM region, activation loop kinase domain FLT3 (13q12) Overexpression

6. AML t(8;21) mutation

7. Lab Diagnosis for fusion genes in AML

8. TrkC / Neurotrophic Receptor Tyrosine Kinase 3 NTRTK mutation

9. • Symptoms reflects B.M. failure to produce normally functioning blood cells : Features of anemia: Weakness, shortness of breath and pallor Features of leukopenia: infections (septicemia, pneumonitis) Features of thrombocytopenia: Bleeding and increased hemorrhagic tendency . • Symptoms reflects B.M. hyperplasia and excessive accumulation, infiltration and proliferation of malignant cells : Bone and Joint pain Hepatosplenomegaly Lymphadenopathy Gingival hypertrophy (hyperplasia) and oral lesions Acute Myeloid Leukemia- Signs & Symptoms

10. Chloromas

11. 1- Peripheral Blood: The leukocyte count ranges from < 1X109 to >100X109 . Presence of blast on the blood smear. Neoplastic blast have few granules in the RNA rich cytoplasm . According to the FAB: 1- Type I: typical blast features without granules. 2- Type II: has < 20 granules. 3- Type III: has numerous granules. AML- Lab Diagnosis

12. CBC and blood film examination : RBCs decreased, Hb less than 10g/dl Normocytic normochromic anemia (mild to severe), OR Slightly Macrocytic because of the inability to compete the neoplastic cell for folate and vit-B12 or early release of Retic cells Platelets count: reduced to normal WBC’s count: decreased mature leukocytes The appearance of malignant cells 2- Bone marrow: B.M . hyperactivation and hyperplasia Blasts : represents greater than 20 - 30% depends on the type of Classification and type of Leukemia . AML- Lab Diagnosis

13. AML- Lab Diagnosis

14. Immunological markers (Surface, Cytoplasmic & Nuclear ) TdT : Terminal Deoxynucleotidyl Transferase , differentiate Myeloblasts from lymphoblasts (B & T precursors ) CD markers : e.g. AML: CD11/CD13/CD33/CD117 ALL (B-Cells): CD10/CD19/CD20/CD22 ALL (T-Cells): CD2/CD3/CD5/CD7 Cytogenetics : specific Chromosomal abnormalities Philadelphia chromosome (t(9;22)): common in CML t(8;21 ): AML-M2 t(1;19 ), t(8;22), t(11,14) ALL AML- Lab Diagnosis

15. Classification, Based on: 1- FAB Group Classification: The Morphological criteria of PB and BM . The Immunophenotyping of Leukemic cells . The cytochemistry of Leukemic Cells . For Diagnosis, Blast count in PB or BM is >30% . 2- WHO Classification: Molecular/Genetic Features of Malignancies . FAB Class. Incorporated into WHO Class. For Diagnosis, Blast count in PB or BM is >20% . AML- Lab Diagnosis for Classification

16. AML-M0 (Acute Myeloblastic Leukemia without differentiation): AML with no evident morphological differentiation (no characteristic myeloid features) Bone marrow contains 30 to 90% blast forms with clear cytoplasm (granules free ) The blasts seen in AML and ALL are very similar; with a high nuclear cytoplasmic ratio, immature chromatin, nucleoli, and a variable amount of cytoplasm . < 3% of blasts are MPO and SBB positive Blasts are positive for CD13 and CD33 markers FAB Classification

17. AML-M1 (Acute Myeloblastic Leukemia, without maturation): Poorly differentiated myeloblasts (more than 90% of NEB) Blasts are Type I (Agranular) and Type II ( Granular) MPO & SBB are positive (>3 but < 50%) CAE is with at least 10-20% positivity Blasts Express CD13/CD33/CD117 Auer rods (may present but rarely seen) FAB Classification

18. AML-M2 (Acute Myeloblastic Leukemia, with granulocytic maturation) : E vidence of maturation up to the Promyelocyte stage (represents more than 10% of blasts) >50 % of leukemic cells are MPO and SBB positive. 10-20 % positivity for CAE Blasts Express CD13/CD33/CD117 , Also CD19/CD34 Specific Translocation (t(8;21)), (less specific t(6;9 )) Auer rods are occasionally seen FAB Classification

19. AML-M3 (Acute Promyelocytic leukemia ) Promyelocytes with heavy granulation (> 30% of NEC) Auer rods frequently seen (Faggot cells: cells with bundles of AR) Cells are strongly positive with MPO and SBB Negative with CAE t(15;17 ) is a unique and common feature of AML-M3. AML-M3 variant: showing the characteristic bilobed hypogranular Promyelocytes AML-M3 and DIC ( Thromboplastic Substances ) FAB Classification

20. AML-M4 (Acute Myelomonocytic leukemia): Cells are with granulocytic and monocytic differentiation 20-80 % of cells are positive for MPO, SBB and NSE. Differential diagnosis: serum lysozyme level >3 times 6q deletion or inversion are common. AML-M4 with Eosinophilia Eosinophils represents >5 % of nonerythroid cells immature eosinophils positive for SE and PAS FAB Classification

21. AML-M5 (Acute Monoblastic leukemia): >80 % of NEC are monocytic lineage >80 % of cells are positive for NSE Cells are negative for MPO and SBB (weakly positive in monoblast ). del (11q), t(9;11), t(11;19) Two subclasses: Poorly (M5a) & well (M5b) differentiated M5a : Predominance of monoblasts M5b : Predominance of promonocytes and monocytes FAB Classification

22. AML-M6 (Acute Eythroblastic leukemia): Hypercellularity of B.M. with marked erythroid hyperplasia >50 % of ANC are of erythroid lineage . Blasts commonly show megaloblastoid characterestic features including multi-nucleation, cytoplasm vaculation Most erythroid precursors are positive for PAS (Fine staining) Negative to weakly positive for MPO, SBB and SE AML (M6) against MDS : > 50% or <50% Erythroblast of ANC: With >30% Blast of ANC ---> AML (M6) With <30% Blast of ANC ---> MDS FAB Classification

23. AML-M7 (Acute Megakaryoblastic leukemia): Uncommon form Extensive proliferation of Megakaryoblasts and Megakaryocyte > 50% of blasts are megakaryoblasts Blasts identified by platelets peroxidase Negative for MPO and SBB, while positive for PAS Blasts Express CD41/CD42 t(1;22 ) is common FAB Classification

24. 1- AML with recurrent Cytogenetic abnormalities usually translocation, in most cases the chromosomal rearrangement create a fusion gene, encoding a novel fusion protein I. AML with t(8;21)( q22;q22 );(ETO/AML1): Present morphological as AML with maturation Core binding factor (CBF) also called AML1 is a transcription factor critical for hematopoietic development It is believed that HSC is the origin of leukemia WHO Classification

25. The t(8;21) translocation result in a chimeric protein containing the N- terminal portion of CBF (chromosome 21) and most of the ETO protein from chromosome 8 ( nuclear protein involved in the regulation of transcription ) The fusion protein blocks the normal function of CBF, and induce abnormal gene activation and gene repression, this will lead to increase proliferation with blocked differentiation WHO Classification

26. II. AML with abnormal B.M eosinphils inv (16)(p13;q22) or t(16;16)(p13;q22)(CBFB/MYH11): Present morphology as AML with monocytic and granulocytic maturation and presence of abnormal eosinphils in B.M Combination of acute myelomonocytic leukemia (AMML) with abnormal eosinphilis is morphologically AMML Eo . Abnormal immature ( basophilic ) granules in the eosinphils, promyelocyte , and myelocyte stages. The inv(16 )(p13;q22) and t(16;16)(p13;q22) both result in the fusion of the CBFB gene (16q22) to the smooth muscle mysoin heavy chain gene SMMHC (MYH11 ) at (16p13 ) The CBFB/SMMHC fusion protein binds to AML1/ CBFa and represses it is function as transcription factor. WHO Classification

27. III . AML with t(15;17)(q22;q12)(PML/ RARa ) and variants: it is acute promyelocytic leukemia(APL), an AML in which abnormal promyelocyte predominate. Both hypergranular ( typical APL) and hypogranular or microgranular are seen. The presenting signs are DIC and bleeding . The typical t(15;17) gene rearrangement result in the fusion of (PML/ RARa ) gene and reciprocal ( RARa /PML) gene ( PML/ RARa ) mRNA has been identified in all APL patient while ( RARa /PML ) mRNA in two third WHO Classification

28. The RARa protein is a nuclear hormone receptor that bind to specific DNA sequence and controls transcription. PML is growth suppressor nuclear protein normally found in complex macromolecular structure. The PML/ RARa fusion protein leads to formation of co-repressor complex molecules that enhance the oncogenesis of APL It is believed that HSC is the origin of leukemia WHO Classification

29. IV. AML with 11q23(MLL) abnormalities: These leukemia associated with monocytic features ( monoblasts and promonocyte ). Monoblast and promonocyte can have scattered azurophilic granules and vacuoles. The MLL gene (11q13) is involved in a number of leukemia associated translocation with different partner chromosome. The MLL protein is a DNA binding protein that interact with other nuclear protein and permits the association of transcription factor which regulate transcription. WHO Classification

30. 2- AML with multilinage Dysplasia ( with or without prior MDS) It is an Acute leukemia ( >20% blast) with dysplasia in more than 50% of the cells in two or more myeloid cell lines It is occurs with or following MDS / MPD examples of dysplasia include: hypogranular PMNs, psuedopelger-Huet anomaly, megaloblastic erythrocyte, ringed sideroblast The cytogenetics abnormalities are variable and are similar to these in MDS. HSC is the origin of leukemia poor prognosis WHO Classification

31. 3- AML and Myelodysplastic syndrome, therapy related : These disorder arise as a result of cytotoxic chemotherapy and / or radiation therapy Two major subtypes: I. Alkylating agent/ radiation treatment: initially it start with MDS and eventually evolving AML. II. Topoisomerase II inhibitor treatment WHO Classification

32. 4- Acute Myeloid leukemias not otherwise categorized: Include all AML cases that not fulfill criteria for any of other described The subtypes of this AML are classified according to differentiated on morphology and cytochemical features I. AML Minimally Differentiated II. AML without Maturation III. AML with Maturation IV. Acute Myelomonocytic leukemia (AMML) V. Acute monoblastic leukemia and Acute monocytic leukemia VI. Acute Erythroid leukemia (AEL) VII. Acute Megakaryoblastic leukemia WHO Classification

33. VIII. Acute Basophilic leukemia The most characteristic feature by cytochemistry is metachromatic positivity with toluindine blue IX. Acute panmyelosis with mylofibrosis it is occur with or following chemo and radio therapy X. Myeloid Sarcoma a tumor of myeloblast or immature myeloid cells occurs in the extramedullary site or in the bone WHO Classification

34. No matter how badly someone treat you;;; Never drop down to their level. Remain Calm Stay Strong, and Walk Away