ACUTE LEUKAEMIA by DR KAMAL E HIGGY CONSULTANT

ACUTE LEUKAEMIA by DR. KAMAL E. HIGGY CONSULTANT HAEMATOLOGIST

ACUTE MYELOID LEUKAEMIA ETIOLOGICAL FACTORS GENETIC DISORDERS Congenital Defects Marrow Failure Syndromes Down syndrome Bloom Syndrome Monosomy 7 syndrome Klinefelter Syndrome Turner Syndrome Neurofibromatosis Congenital Dysmorphic Syndrome Fanconi Anaemia Dyskeratosis Congenita Schwachman – Diamond Syndrome Amegakaryocytic Thrombocytopenia Blackfan – Diamond Syndrome Kostmann Agranulocytosis Familial Aplastic Anaemia

ACUTE MYELOID LEUKAEMIA ETIOLOGICAL FACTORS Environmental Factors n n n Solvents (benzene) Smoking Ionizing radiation Atomic bomb exposure Nuclear power exposure Medical radiation n n Non ionizing radiation Chemotherapy Alkylating agents Topoisomerase II inhibitors n Other drugs Chloramphenicol Phenylbutazone

Acute Myeloid Leukaemia DEFINITION Acute Myeloid Leukaemia (AML) is currently defined by the FAB criteria. The percentage of blasts, the presence of cytochemical myeloperoxidase, the major cell types present defined by morphology and esterase cytochemistry & the immunophenotype define the 8 FAB subtypes

Acute Myeloid Leukaemia CLINICAL FEATURES n n n Increasing incidence with age. Median age at diagnosis is 50 years. Symptoms are due to marrow failure (anaemia, infection, haemorrhage) or hyperleucocytosis. Rarely presenting symptoms are due to chloromas or CNS involvement.

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS MORPHOLOGY n n Myeloblasts are usually medium sized with an eccentric nucleus and open chromatin. Cytoplasm shows variable basophilia. Few granules or Auer rods may be present. Maturing granulocytes may be normal or show the dysplastic features e. g. Psaudo Pelger (hypogranular bilobed neutrophils). Monoblasts are generally very large with abundant greyblue or basophilic cytoplasm. Nuclei are round or lobulated, and are central in the cell. Fine azurophil granules may be present but Auer rods are rarely, if ever, present. Promonocytes and monocytes show abnormal nuclear maturation, granulation and loss of basophilia.

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS MORPHOLOGY (Cont…) n n Erythroid precursors may be normal or show varying degrees of dyserythropoiesis. Eosinophils may be present in varying numbers, with normal or abnormal morphology. Specific abnormal appearances are linked with cytogenetic abnormalities (e. g. inv 16) Basophils are rarely increased in AML, and if present show abnormal or poor granule formation and nuclear maturation. Megakaryocytes may be reduced or increased. Dysplastic hyperlobated, hypolobated, multinuclear, small and blastic forms may be present

Acute Myeloid Leukaemia FAB Subtypes M 0 -M 5: >20% myelo/monoblasts by morphology or immunophenotype. M 6: >50% erythroid precursors, >20% blasts in non-erythroid cells. M 7: >20% megakaryoblasts present. Note: Cytogenetics, molecular genetics, previous MDS or MPD, previous chemo- or radiotherapy and the presence of trilineage myelodysplasia do not have any place in the FAB system. All these features which contribute to the definition of prognostically important sub-groups is included in the new classification system.

Acute Myeloid Leukaemia >20% FAB Subtypes >20% >20%

M 0 M 1 M 2 M 3 M 4 M 5 a M 5 b M 6 M 7

ACUTE MYELOID LEUKAEMIAS WHO CLASSIFICATION n Acute myeloid leukaemia with reccurent genetic abnormalities Acute myeloid leukaemia with t(8; 21)(q 22; q 22); (AML 1(CBFa)/ETO) Acute myeloid leukaemia with abnormal bone marrow eosinophils inv (16)(p 13 q 22) or t(16; 16)(p 13; q 22); (CBFb/MYH 11) Acute promyelocytic leukaemia (AML with t(15; 17)(q 22; q 12)(PML/RARα) & variants Acute myeloid leukaemia with 11 q 23 (MLL) abnormalities n n Acute myeloid leukaemia with multilineage dysplasia Acute myeloid leukaemia and myelodysplastic syndromes, therapy related

ACUTE MYELOID LEUKAEMIAS n WHO CLASSIFICATION Acute myeloid leukaemia not otherwise categorized Acute myeloid leukaemia minimally differentiated Acute myeloid leukaemia without maturation Acute myeloid leukaemia with maturation Acute myelomonocytic leukaemia Acute monoblastic and monocytic leukaemia Acute erythroid leukaemias Acute megakaryoblastic leukaemia Acute basophilic leukaemia Acute panmyelosis with myelofibrosis Myeloid sarcoma n Acute leukaemia of ambiguous lineage Undifferentiated acute leukaemia Bilineal acute leukaemia Biphenotypic leukaemia

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS CYTOCHEMISTRY n Myeloperoxidase (MPO): identify blast cells as myeloid. Auer rods are detected twice as frequently as on Romanowsky stains. Dysplastic neutrophils may be negative. Eosinophil granules are always positive. Monoblasts and promonocytes may be negative. Sudan Black B gives identical results. Myeloperoxidase (MPO)

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS CYTOCHEMISTRY n Chloroacetate Esterase (CAE) specifically identifies cells of the granulocyte lineage, from the early promyelocyte stage to mature neutrophils. Chloroacetate Esterase

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS CYTOCHEMISTRY n α -Naphthyl Acetate (Nonspecific) Esterase (ANAE) stains monocytes and megakaryocytes at all stages of maturation. Nonspecific Esterase

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS CYTOCHEMISTRY n Periodic Acid Schiff (PAS) stains many cell types. It is not lineage specific but the pattern of staining may be helpful, e. g. positive monoblasts, NRBC, megakaryoblasts…

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS TREPHINE BIOPSY HISTOLOGY n n Helpful when there is severe cytopenia and a dry tap, e. g. in megakaryoblastic AML. Useful for identifying megakaryocyte dysplasia and dyserythropoiesis. Immunophenotyping is possible when the diagnosis is in doubt. Not suitable for fine classification which is based on percentages of cell types and cytological detail.

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS FLOW CYTOMETRY n n Myeloid blasts express combinations of CD 34, CD 13, CD 33, CD 117, HLA-DR, CD 14, CD 15. Glycophorin A and CD 42 b identify erythroid and megakaryocyte lineage blasts respectively. Essential to separate undifferentiated AML (M 0) from ALL. May need supplementation with APAAP staining for cytoplasmic immunoreactive MPO.

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS FLOW CYTOMETRY (Cont…) n n n Essential for diagnosing biphenotypic leukaemias and detecting aberrant (promiscuous) antigen expression. Can identify patient specific unique blast cell phenotypes. Can identify blasts of megakaryocyte, erythroid, monocyte and granulocyte lineages. Can identify leukaemic contamination of 'remission' marrow harvests. Can identify early relapse.

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS MOLECULAR GENETICS n n The 3 major good prognosis translocations are all detectable by RT-PCR, which can be used to detect up to 40% more cases than are found by metaphase cytogenetics. All translocations with cloned breakpoint genes are theoretically detectable by RT-PCR. Real time multiplex PCR may become available for routine diagnosis. RT-PCR is used for monitoring speed of response to treatment and may be predictive for relapse

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS CYTOGENETICS n n An abnormal karyotype is found in 50 -60% of AML cases at presentation. t(15; 17), t(8; 21) and inv/del/t(16) are associated with specific morphology, younger patients and a good prognosis. Complex karyotypes and abnormalities of chromosomes 5 and 7 are associated with older patients, trilineage dysplasia and a poor response to treatment. Other karyotypic abnormalities are prognostically neutral.

Acute Myeloid Leukaemia LABORATORY DIAGNOSIS IMMUNOHISTOCHEMISTRY / IMMUNOFLUORESCENCE APAAP staining for c. MPO, c. CD 3 and c. CD 79 should be done in all cases of undifferentiated acute leukaemia. n Immunofluorescent staining for nuclear PML protein should be done in all cases of suspected acute promyelocytic leukaemia. A microparticulate pattern is diagnostic of APML. n

Acute Lymphoblastic Leukaemia (ALL) Clinical Features ALL can occur at any age but has a peak incidence between 2 - 10 years. It is characterized by: n n n bone marrow failure lymphadenopathy thymic enlargement in T-lineage ALL bone pains which may be associated with radiological changes and fractures tendency to relapse in the CNS and testis.

Acute Lymphoblastic Leukaemia Laboratory Diagnosis FAB Classification

Acute Lymphoblastic Leukaemia Laboratory Diagnosis

Acute Lymphoblastic Leukaemia Laboratory Diagnosis Morphology Blast morphology is variable n some are small with high nuclear / cytoplasmic ratios and indistinct nucleoli (so-called L 1 blasts) n while others are larger with more prominent nucleoli and more abundant cytoplasm (L 2). n A third type (L 3) shows large blasts with moderately abundant highly vacuolated basophilic cytoplasm Blast morphology does not correlate with cell lineage and cytochemistry is of little value.

Acute Lymphoblastic Leukaemia Laboratory Diagnosis - FAB Classification ALL (L 1) ALL (L 2) ALL (L 3)

Acute Lymphoblastic Leukaemia Laboratory Diagnosis CYTOCHEMISTRY PAS Ac. Phos (T-ALL)

Acute Lymphoblastic Leukaemia Laboratory Diagnosis Flowcytometry ALL is derived from precursor lymphocytes that are undergoing antigen receptor gene (Ig and TCR) rearrangement. B-lineage ALL The precursor nature of the cells is established by demonstrating lack of surface Ig, the presence of nuclear TDT and sometimes the expression of CD 34. Sub classification is as follows: n Pre-pre B-ALL: CD 19+ CD 10 - cytoplasmic mu heavy chain negative. n Common ALL: CD 19+ CD 10+ cytoplasmic mu present in <20% of cells n Pre B-ALL: CD 19+ CD 10+ cytoplasmic mu present in >20% of cells n Blasts of all subgroups will express cytoplasmic CD 22 and CD 79 b.

Acute Lymphoblastic Leukaemia Laboratory Diagnosis Flowcytometry T-lineage ALL n n n The precursor nature of the cells is established by demonstrating TDT and sometimes CD 34 positivity and the lack of surface TCR/CD 3. T-cell lineage is demonstrated by the expression of CD 7 and/or CD 1 a. Expression of the other pan-T cell markers is variable.

Acute Lymphoblastic Leukaemia Laboratory Diagnosis CYTOGENETICS Hyperdiploidy is common. A number of balanced translocations have been identified in ALL: n n t(12; 21) - this is the commonest translocation in ALL (30% of cases). It results in the TEL-AML fusion gene and is primarily associated with the common phenotype. t(9; 22) - this is commoner in adults and is associated with a very poor prognosis. t(4; 11) - this translocation results in the MLL-AF 4 fusion gene. It is associated with pre-pre B-ALL and is associated with a poor prognosis. t(1; 19) - associated with pre-B ALL and results in the formation of the E 2 APBX fusion gene. These translocations are demonstrable by RT-PCR techniques. TAL-1 deregulation is the commonest genetic abnormality in T-ALL. This may occur as the result of the t(1; 14) or more commonly due to chromosome 1 p 32 deletions.

Acute Lymphoblastic Leukaemia PROGNOSTIC FACTORS The following are poor prognostic factors in ALL: n n n n age <1 year and >10 years male sex CNS disease at presentation high white cell count t(9; 22) t(4; 11) hypodiploidy

Acute Lymphoblastic Leukaemia OUTCOME AND THERAPY The treatment of ALL consists of the following "phases": n Remission induction - vincristine, prednisolone, daunorubicin, n Consolidation - various combinations of chemotherapeutic agents. n CNS directed therapy - high dose systemic and intrathecal n Maintenance therapy - vincristine, prednisolone, mercaptopurine and asparaginase. methotrexate for 2 years. Childhood ALL is associated with 75% long term survival. Minimal residual disease assessment using PCR based strategies appear to be able to predict relapse although they are not yet in routine clinical use. Allogeneic transplantation is the treatment of choice at relapse. The outlook in adult ALL is poor with approximately 20% long-term survivors. Allogeneic transplantation is advisable in first remission.