1. LEUKEMIAS By Dr Aamer Aleem Consultant Hematologist Prof. of Medicine College of Medicine & KKUH Riyadh, Saudi Arabia

2. Leukemias Leukemias are a group of cancers of the blood/ bone marrow and are characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). Leukemia is a broad term covering a spectrum of diseases. Any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs and which is usually accompanied by anemia and thrombocytopenia

3. Classification of leukemias Two major types (4 subtypes) of leukemias Acute leukemias Acute lymphoblastic leukemia (ALL) Acute myelogenous leukemia (AML) (also "myeloid" or "nonlymphocytic") Chronic leukemias Chronic lymphocytic leukemia (CLL) Chronic myeloid leukemia (CML) (Within these main categories, there are typically several subcategories)

4. Myeloid maturation myeloblastpromyelocytemyelocytemetamyelocytebandneutrophilMATURATION Adapted and modified from U Va website

5. Hematopoietic stem cell Neutrophils Eosinophils Basophils Monocytes Platelets Red cells Myeloid progenitor Lymphoid progenitor B-lymphocytes T-lymphocytes Plasma cells naïve ALL AML

6. Acute leukemia-definition Disorders with clonal expansion of precursor cells (myeloid or lymphoid) with reduced capacity to differentiate. 1. Maturation arrest at the blast stage 2. Bone marrow infiltration by blast cells causing suppression of normal precursors (bone marrow failure)

7. Myeloid vs Lymphoid Any disease that arises from the myeloid elements (white cell, red cell, platelets) is a myeloid disease ….. AML, CML Any disease that arises from the lymphoid elements is a lymphoid disease ….. ALL, CLL

8. Acute vs. chronic leukemia Acute leukemias: Young, immature, blast cells in the bone marrow (and often blood) More fulminant presentation More aggressive course Chronic leukemias: Accumulation of mature, differentiated cells Often subclinical or incidental presentation In general, more indolent (slow) course Frequently splenomegaly Mature appearing cells in the B. marrow and blood

9. Acute vs. chronic leukemia Leukemias are classified according to cell of origin: Lymphoid cells ALL - lymphoblasts CLL – mature appearing lymphocytes Myeloid cells AML – myeloblasts CML – mature appearing neutrophils On a CBC, if you see: Predominance of blasts in blood consider an acute leukemia Leukocytosis with mature lymphocytosis consider CLL Leukocytosis with mature neutrophilia consider CML

10. Acute leukemias Definition:Malignancies of immature hematopeotic cells. (> 20% blast cells in the bone marrow) Types: Acute Myeloid Leukaemia (AML) Acute Lymphoblastic leukemia (ALL) Groups:Childhood ( 80% ALL Adult (> 15)> 80% AML Elderly (> 60 years)

11. Epidemiology of AML Incidence – 2.7 per 100,000 –12.6 per 100,000 in those over 65 yrs –median age of presentation : 67 yrs More prevalent in: –Males –European descent –Hispanic/Latino background (promyelocytic leukemia, AML-M3)

12. Etiology of acute leukemias Mostly unidentifiable cause i.e., de nove (≥80%) Drugs & chemicals Alkylating agents (Chlorambucil, N mustard, Melphalan) Topoisomerase inhibitors (Etoposide) Benzene Ionizing radiation Myelodysplastic syndrome Myeloproliferative disorders Genetic disorders Down’s syndrome Bloom syndrome Faconi anemia Wiskott Aldrich syndrome

13. Clinical presentation Symptoms Usual 1-3 Month History : MDS – 1yr (Features of BM failure) Anemia Low platelets Low neutrophils

14. Clinical presentation Symptoms (Features of BM failure) Fatigue, malaise, dyspnea (anemia) Bleeding eg after dental procedure Easy bruisability Severe epistaxis Fever (bacterial & fungal infections) Bone Pain

15. Clinical Presentation Signs Signs  Pallor  Hemorrhage from the gums, epistaxis, skin, fundus, GI tract, urinary tract  Hepato-splenomegaly  Enlarged lymph nodes  Gum (hypertrophy) or skin infiltration (M5)  Fever (sepsis, pneumonia, peri-rectal abscess)

16. Differential Diagnosis 1.Aplastic anemia 2.Myelodysplastic syndromes 3.Multiple myeloma 4.Lymphomas 5.Severe megaloblastic anemia 6.Leukemoid reaction

17. Laboratory Tests 1.CBC a. Anemia b. Trombocytopenia c. WBC High Normal Low 2.Coagulation Studies (AML-M3 DIC) 3.Biochemical Studies (U/E, LFT) Cont..

18. Peripheral Blood Smear – blasts in almost all cases Bone Marrow Examination (>20% blasts) Diagnosis of Acute Leukemia

19. 4.Peripheral Blood smear – blasts in almost all cases 5.Bone Marrow Examination (>20% blasts) 6.Flow cyometry (Surface immunophenotype of blast cells) 4.Cytogenetics (chromosomal analysis) 5.CSF analysis (all ALL patients, some AML)

20. Diagnostic methods of importance Bone marrow aspirate & Romanowsky stain (morphology) Enumeration of blasts, maturing cells, recognition of dysplasia Cytochemistry Myeloperoxidase, Sudan Black B, esterases to determine involved lineages Immunophenotyping Defines blast cell lineage commitment as myeloid, lymphoid or biphenotypic Cytogenetics & molecular studies (FISH, PCR) Detects clonal chromosomal abnormalities, including those of prognostic importance

21. M3* M4 M5 M6 M0, M1, M2 58% 7% M0, M1, M2 M7 M6 M5 M4 M3* 52% 9% Adults Children Frequency of Acute Myeloid Leukemia Subtypes

22. WHO Classification of AML AML with recurrent genetic abnormalities - t(8;21), t(16), inv(16), chromosome 11 changes - t(15;17) as usually seen with AML M3 AML with multilineage dysplasia (more than one abnormal myeloid cell type is involved) AML related to previous chemotherapy or radiation AML not otherwise specified - undifferentiated AML (MO) - AML with minimal maturation (M1) - AML with maturation (M2) - Acute myelomonocytic leukemia (M4) - Acute monocytic leukemia (M5) - Acute erythroid leukemia (M6) - Acute megakaryoblastic leukemia (M7) - Acute basophilic leukemia (M8) - Acute panmyelosis with fibrosis - Myeloid sarcoma (also known as granulocytic sarcoma or chloroma) Undifferentiated or biphenotypic acute leukemias (leukemias that have both lymphocytic and myeloid features. Sometimes called ALL with myeloid markers, AML with lymphoid markers, or mixed lymphoid lineage leukemias).

23. WHO Classification of AML AML not otherwise specified - undifferentiated AML (MO) - AML with minimal maturation (M1) - AML with maturation (M2) - Acute myelomonocytic leukemia (M4) - Acute monocytic leukemia (M5) - Acute erythroid leukemia (M6) - Acute megakaryoblastic leukemia (M7) - Acute basophilic leukemia (M8) - Acute panmyelosis with fibrosis - Myeloid sarcoma (also known as granulocytic sarcoma or chloroma)

24. Blood Film-Normal

26. Normal BM cells

27. BM aspirate-Normal

30. AML

31. Bone marrow biopsy-Normal

32. Trephine biopsy in AML

33. AML

34. AML Auer rods

35. AML-M3 (Acute promyelocytic leukemia)

37. ALL

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

39. Chloracetate (Specific) Esterase Myeloid Cell Line Naphthol-ASD-chloracetate CAE > Free naphthol compounds + Stable diazonium salt (eg, Fast Corinth) > Red deposit

40. Non-Specific Esterase Monocytic Line  Naphthyl acetate ANAE > Free naphthyl compounds +Stable diazonium salt (eg, Fast blue RR) > Brown deposits

41. Discover the latest technology from Bayer Discover the latest technology from Bayer <> Bloodline Home About Educational Features Image Atlas Case Studies Private Lectures Conference Reviews Journal Articles Book Reviews Glossary Resources Conference Calendar Grants & Fellowships Hematology Links Full Text Journals Classifieds Specialties BMT/Stem Cell Cord Blood Thrombosis Hemostasis Laboratory Malignancies Pediatrics Red Cell Disorders Transfusion Medicine Veterinary News Hematology News BloodLink Newsletter Blood News Update Discussion Today's Discussion Create New Topic List by Topic Plasma Cells, Acute myelomonocytic (M-4) leukemia Clump of Plasma Cells most of which are small with a deep basophilic blue cytoplasm. Two cells in the center are partially smudged and show a paler cytoplasm and less dense and redder staining nuclear chromatin. Acute myelomonocytic (M-4) leukemia. Marrow - 100X Image ID: 0147-094 Copyrigh t 2001 - Carden Jennings Publishing Co., Ltd. All rights reserved. The material available at this site is for educational purposes only and is NOT intended for any diagnostic, clinically related, or other purpose. Carden Jennings Publishing Co., Ltd., assumes no responsibili ty for any use or misuse of this material and makes no warranty or representat ion of any kind with respect to the material available at this site. Carden Jennings Publishing Co., Ltd.

42. Immunophenotyping in acute leukemias Immunophenotyping detects the presence or absence of white blood cell (WBC) antigens. Most of the antigens that immunophenotyping detects are identified by a CD (clusters of differentiation or cluster designation) number, such as: CD1a, CD2, CD3, CD4, CD8, CD13, CD19, CD20, CD33, CD61, etc.

43. Immunophenotyping in acute leukemias Flow cytometry is performed by processing blood or marrow samples and adding specific antibodies tagged with fluorescent markers. These antibodies attach to corresponding antigens on the WBCs when the antigens are present, and are analyzed. Results are then graphed and compared to "normal" results and to patterns that are known to be associated with different leukemias

44. Flow Cytometry First labeled with fluorescent dyes. Forced through a nozzle in a single-cell stream passing through a laser beam The laser is focused to a known wavelength Excitation of a specific fluorochrome Photo-multiplier tubes detect the scattering of light and emission from the fluorescent dye

45. Flow cytometry Forward and Side Scatter Side Scatter (granularity) Forward Scatter (Size) Neutrophils Monocytes Lymphocytes

46. Immunophenotyping in leukemias

47. Application of Cytogenetic, FISH and Microarray Analysis in the Diagnosis of Leukemia

48. ISCN: 46,XX,t(8;21)(q22;q22)[19]/46,XX[1]

49. Fluorescence in situ hybridization (FISH)

50. FISH with AML1/ETO –DF probe: 76% of cells show one fusion, two red and two green signals Three way translocation of t(8;21)(q22;q22) 45,X,-Y,t(8;20;21)(q22;p13;q22),del(11)(q21q25)

52. FISH: Dual fusion probe for t (8;14): IgH (14), MYC (8), Cen8 Aqu nl 8 der(8)t(3;8;14) nl 8 der(14)t(3;8;14) der(3)t(3;8;14) der(18)t(14;18) der(14)t(14;18)

53. Age Above the age of 50 years the complete remission rate falls progressively Cytogenetics Three risk groups defined –Good risk: patients with t(8;21), t(15;17) and inv/t(16) –Intermediate risk: Normal, +8, +21, +22, 7q-, 9q-, abnormal 11q23, all other –Poor risk: patients with -7, -5, 5q-, abnormal 3q and complex karyotypes Acute Myeloid Leukaemia (AML) Prognostic factors in AML 1.Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999; 107: 69-79 2.Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

54. Treatment response –Patients with >20% blasts in the marrow after first course of treatment have short remissions (if achieved) and poor overall survival Secondary AML –Patients with AML following chemotherapy or myelodysplasia respond poorly Trilineage myelodysplasia –Patients with trilineage myelodysplasia have a lower remission rate Acute Myeloid Leukaemia (AML) Prognostic factors in AML 1.Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999; 107: 69-79 2.Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

55. Intensive chemotherapy –Patients < 55 years old: 80% remissions –Patients > 55 years old: progressive reduction in remission rate Bone marrow (stem cell) transplantation –Autologous and allogeneic transplants reduce the relapse rate Importance of cytogenetics for prognosis in children and adults < 55 years old Good risk cytogenetic group –91% remissions, 65% five year survival Treatment and prognosis of AML 1.Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999;107: 69-79 2.Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

56. Treatment of acute leukemias 1.Specific therapy (chemotherapy) 2.Supportive treatment Stages of Therapy a.Induction b.Consolidation c.Maintenance

57. (Treatment of acute leukemias) Supportive Care 1.Vascular access (Central line) 2.Prevention of vomiting 3.Blood products (Anemia, ↓Plat) 4.Prevention & treatment of infections (antibiotics) 5Management of metabolic complications

58. Intensive chemotherapy –Patients < 55 years old: 80% remissions –Patients > 55 years old: progressive reduction in remission rate Bone marrow (stem cell) transplantation –Autologous and allogeneic transplants reduce the relapse rate Importance of cytogenetics for prognosis in children and adults < 55 years old Good risk cytogenetic group –91% remissions, 65% five year survival Acute Myeloid Leukaemia (AML) Treatment and prognosis of AML 1.Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999;107: 69-79 2.Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

59. Acute Lymphoblastic Leukemia

60. Acute Lymphoblastic Leukemia (ALL) Poor Prognostic Factors Age 10 yrs Male sex High WBC count ( > 50 х10 9 /L) Presence of CNS disease Cytogenetics Good risk Poor risk Hyperdiploid (>50 ch) Hypodiploid, t(9:22), t(4:11) Bone Marrow: Blasts present on day 14 Day 28:No complete response Prognostic factors in ALL

61. Bone Marrow-ALL

62. ALL

63. Treatment of acute leukemias 1.Specific therapy (chemotherapy) 2.Supportive treatment Stages of Therapy a.Induction b.Consolidation c.Maintenance

64. (Treatment of acute leukemias) Induction Obtained by using high doses of chemotherapy 1.Severe bone marrow hypoplasia 2.Allowing regrowth of normal residual stem cells to regrow faster than leukemic cells. Remission  Normal neutrophil count  Normal platelet count  Normal hemoglobin level Remission defined as < 5% blast in the bone marrow

65. (Treatment of acute leukemias) Consolidation Repeated cycles of different or same drugs to those used during induction Higher doses of chemotherapy Advantage: Delays relapse and improved survival

66. (Treatment of acute leukemias) Maintenance Smaller doses for longer period Produce low neutrophil counts & platelet counts Objective is to eradicate progressively any remaining leukemic cells.

67. Treatment-ALL vs AML ALL  Induction  Consolidation  Maintenance  CNS prophylaxis all patients AML  Induction  Consolidation  No maintenance  CNS – Selected group only

68. Definition: Neoplastic proliferations of mature haemopoeitic cells. Types: Chronic lymphocytic leukemia (CLL) Chronic myeloid leukemia (CML) CHRONIC LEUKEMIAS

69.  Neoplastic proliferations of mature lymphocytes.  Distinguished from ALL by a. Morphology of cells. b. Degree of maturation of cells. c. Immunologically immature blasts in ALL. d. CLL affects mainly elderly. CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL)

70. SYMPTOMS OF CLL  May be entirely absent in 40%  Weakness, easy fatigue, vague sense of being ill  Night sweats  Feeling of lumps  Infections esp pneumonia

71. PHYSICAL EXAMINAITON-CLL  Pallor  Lymphoadenopathy a. Cervical, supraclavicular nodes more commonly involved than axillary or inguino-femoral b. Non-tender, not painful, discrete, firm, easily movable on palpation  Splenomegely, mild to moderate  Hepatomegaly

72. Stage  (0-1) - lymphocytosis  LNS.  (II) - above + hepatosplenomagely.  (III-IV) - Anaemia. Hb< 10 g/l Thrombocytopenia. Platelet count : <100x10 9 /L. CLINICAL STAGING-CLL

73. LABORATORY TESTS-CLL CBC Lymphocyte count > 5 x 10 9 /L (5 -500 x 10 9 /L ). Platelets may be decreased Hb may be low Blood film PB immunophenotyping Bone marrow biopsy (needed before starting treatment) Imaging

74.  Observation  ChemotherapyOral chlorambucil Fludarabine, Cyclo  ImmunotherapyAnti-CD 20 (Rituximab),  Anti-CD 52 (Alemtuzumab)  FC-R is the current standard in younger fit patients Indications for starting treatment a. Progressive Symptoms b. Progressive Anemia or Thrombocytopenia c. Bulky LN, large spleen d. Recurrent Infections TREATMENT OF CLL

75. Four new drugs approved for CLL recently (usually 2nd or subsequent line of therapy)  Ibrutinib (effective in P53 mutation)  Obinutuzumab  Idelalisinib  Ofatumumab TREATMENT OF CLL

76.  CML is a clonal stem cell disorder characterised by increased proliferation of myeloid elements at all stages of differentiation.  Incidence increases with age, M > F. CHRONIC MYELOID LEUKEMIA

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

78. Symptoms  Asymptomatic (50% of patients)  Fatigue  Weight loss  Abdominal fullness and anorexia  Abdominal pain, esp splenic area  Increased sweating  Easy bruising or bleeding CLINICAL PRESENTAITON OF CML

79.  Splenomegaly (95%) ( 50% of patients have a palpable spleen ≥ 10 cm BCM, Usually firm and non-tender )  Hepatomegaly (50%) SIGNS OF CML

80. Chronic phase  Peripheral blood – neutrophil leukocytes 11,000 - >500, 000/  L basphilia  LAP score blasts < 5% Nucleated RBCs Thrombocytosis Anaemia DIAGNOSIS OF CML

81. CYTOGENETICS OF CML  Philadelphia (Ph) chromosome is an acquired cytogenetic abnormality in all leukaemia cells in CML  Reciprocal translocation of chromosomal material between chromosome 22 and chromosome 9. t(9;22)

83. Karyotype 46,XX,t(9;22)(q34;q11.2) -- Ph chromosome

87. CML-Treatment Response Criteria Hematological response Normalisation of blood count Cytogenetic response Major cytogenetic response 1-35% Ph +ve cells in metaphase Minor cytogenetic response 36-65% Ph +ve cells in metaphase Molecular response Absence of BCR/ABL gene

88. CML-Principles of Treatment Control & prolong chronic phase (non- curative) - Tyrosine kinase inhibitors-Imatinib (Glivec) - Alpha-Interferon - Oral chemotherapy (Hydroxyurea, ARA-C) Eradicate malignant Clone (curative) - Allogeneic BM/stem cell transplantation - Alpha Interferon? - Imatinib? 2 nd line TKIs

89. Tyrosine kinase inhibitor (TKI) Imatinib (Glivec) is the first line treatment In resistent cases 2nd line TKIs ( Nilotinib, Dasatinib, Bosutinib ) very useful (approved as 1st line) Allogenic bone marrow trasnsplantation can be curative in patients resistant to TKIs but has significant complications & mortality Accelerated and blast phase Imatinib, 2nd line TKIs Treat like AML or ALL followed by BMT TREATMENT OF CML

90. 1. LA P Score 2. Philadelphia Chromosome 3. Basophilia 4. Splenomegaly CML VS LEUKEMOID REACTION

91. Bone marrow or PBSC transplantation in leukemias Types of transplant 1.Autologous transplant 2.Allogeneic Transplant Purpose of transplant Autologous -To deliver a high dose of chemo to kill any residual cancer (lymphoma, multiple myeloma) Allogeneic -To eradicate residual leukemia cells -Graft vs leukemia effect

92. Bone marrow or PBSC transplantation in leukemias Technique of transplantation  MHC + HLA matching  Chemotherapy  Total body irradiation  GVHD prophylaxis Complications of transplantation Prolonged BM suppression (graft failure) Serious infections Mucositis Graft versus host disease (GVHD)

93. Complications of BMT