Ahmad Sh. Silmi Msc Haematology, FIBMS The Acute Leukaemias Ahmad Sh. Silmi Msc Haematology, FIBMS

What Is Acute Leukemia?(1) The acute leukaemias are a heterogeneous group of malignant disorders, which are characterized by the uncontrolled clonal proliferation and accumulation of poorly differentiated blast cells in the bone marrow and other tissues. Thus replacement of normal bone marrow elements with abnormal (neoplastic) blood cells.

What Is Acute Leukemia?(2) These leukaemic cells are frequently (but not always) present in the peripheral blood stream. Subsequently there is a raised total blood count and evidence of bone marrow failure (i.e. anaemia, neutoropenia, thrombocytopenia) are ensues. In the acute leukaemias the blasts commonly invade reticuloendothelial tissues including the spleen, liver and lymph nodes. They may also invade other tissues, infiltrating any organ of the body. If left untreated, leukaemia eventually causes death.

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

Myeloid maturation MATURATION myeloblast promyelocyte myelocyte metamyelocyte band neutrophil MATURATION Adapted and modified from U Va website 5

Acute Leukemia accumulation of blasts in the marrow 6

Marrow failure Neutropenia: infections, sepsis Anemia: fatigue, pallor Thrombocytopenia: bleeding 7

Demographics of Leukemia Patients (2001 Data) CLL=Chronic Lymphocytic ALL=Acute Lymphocytic CML=Chronic Mylogenous AML=Acute Mylogenous Total Reported Cases = 31,500 Sources from Leukemia, Lyphoma, Myeloma Facts 2001

Acute Leukaemogenesis (1) The acute leukaemias are thought to develop as a result of a genetic alteration within a single stem cell in the bone marrow. Which means, a blockage of maturation at certain level; this reveals an abnormal regulatory mechanism, and the cell is incapable for further proliferation and differentiation, but it still has the ability to divide.

Acute Leukaemogenesis (2) Successive mitotic division in the progeny of the cell produces a clonal population, which once it is sufficient a clonal mass has been achieved, and gives rise to the clinical manifestation of the disease.

Two-hit model of leukemogenesis Loss of function of transcription factors needed for differentiation eg. AML1-ETO CBFb-SMMHC PML-RARa Gain of function mutations of tyrosine kinases eg. FLT3, c-KIT mutations N- and K-RAS mutations BCR-ABL TEL-PDGFbR differentiation block enhanced proliferation Acute Leukemia +

Development of Leukemia in the Blood stream Stage 1- Normal Stage 2- Symptoms Stage 3- Diagnosis Stage 5a- Anemia Stage 5b- Infection Stage 4- Worsening Legend White Cell Red Cell Platelet Blast Germ Sources from Leukemia, by D. Newton and D. Siegel

Acute leukemias - laboratory findings 1. Blood examination - anaemia. - thrombocytopenia. - variable leukocyte count, usually increased. - blood morphology: presence of blast cells. 2. Bone marrow morphology - presence of blast cells. - suppression of normal hematopoiesis.

Acute leukemias - Laboratory findings (2) 3. Cytochemical stains 4. Immunophenotyping 5. Cytogenetics 6. Molecular studies

Classification of the immature cells involved may be done by: 1- Morphology – an experienced morphologist can look at: the size of the blast. the amount of cytoplasm. the nuclear chromatin pattern. the presence of nucleoli. the presence of auer rods. N.B: Auer rods are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes.

Myeloblasts with auer rods AML – The myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli. 10-40% of myeloblasts contain auer rods.

Morphology, cont. ALL – in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, and no auer rods Lymphoblast

FAB In an attempt to improve the reproducibility and comparability of the classification process, a grouped of expert haematologists from French, America, and Britian (FAB) collaborated to define a more objective criteria for the classification of acute leukaemias. The initial FAB study was based on the examination of more than 200 different cases of acute leukaemia by expert morphologists, in addition, a myeloperoxidase or suddan black stains should be used to facilitate the recognition of myeloblasts.

Classification of leukemia Main classification Chronic leukemia Acute leukemia FAB Lymphoid Myeloid Lymphoid Myeloid L1 L2 L3 AML M0 M1 M2 M3 M4 M5 M6 M7

FAB Characterization for ALL L1: Homogenous population of small lymphoblasts with scanty cytoplasm and scanty nucleoli. Nucleus occasionally cleft. L2: Heterogeneous population of large lymphoblasts with moderately abundant cytoplasm & or more nucleoli. Nucleus commonly intended or cleft. L3-Burkitt’s type: Homogenous population of large lymphoblasts with prominent nucleoli & deeply basophilic, vacuolated cytoplasm.

Morphologic subtypes of acute lymphoblastic leukemias (FAB classification) Subtype Morphology Occurrence (%) L1 Small round blasts 75 clumped chromatin L2 Pleomorphic larger blasts 20 clefted nuclei, fine chromatin L3 Large blasts, nucleoli, 5 vacuolated cytoplasm

Acute leukemias Acute lymphoblastic leukemia – They may be classified on the basis of the cytological features of the lymphoblasts into: L1 - This is the most common form found in children and it has the best prognosis. The cell size is small with fine or clumped homogenous nuclear chromatin and absent or indistinct nucleoli. The nuclear shape is regular, occasionally clefting or indented. The cytoplasm is scant, with slight to moderate basophilia and variable vacuoles.

ALL-L1 (peripheral Blood)

ALL-L1 (Bone Marrow)

ALL – L2 L2 – This is the most frequent ALL found in adults. The cell size is large and heterogeneous with variable nuclear chromatin and prominent nucleoli. The nucleus is irregular, clefting and indented. The cytoplasm is variable and often moderate to abundant, the basophilia is variable and may be deep, and vacuoles are variable.

ALL-L2 (peripheral Blood)

ALL-L2 (Bone Marrow)

Acute leukemias L3 – This is the rarest form of ALL. The cell size is large, with fine, homogenous nuclear chromatin containing prominent nucleoli. The The nucleus is regular oval to round. The cytoplasm is moderately abundant and is deeply basophilic and vacuolated.

ALL-L3 (peripheral Blood)

ALL-L3 (Bone Marrow)

Acute leukemias Incidence – ALL is primarily a disease of young children (2-5 years), but it can also occur in adults. Clinical findings – pancytopenia with resulting fatigue, pallor, fever, weight loss, irritability, anorexia, infection, bleeding, and bone pain. L1 occurs in children, L2 in adults, and L3 is called Burkitts leukemia.

Acute leukemias Prognosis – age, WBC count, and cell type are the most important prognostic indicators Patients younger then 1 and greater than 13 have a poor prognosis If the WBC count is < 10 x 109/L at presentation, the prognosis is good; If the WBC count is > 20 x 109/L at presentation the prognosis is poor T cell ALL (more common in males) has a poorer prognosis than any of the B cell ALLs which have a cure rate of 70%.

AML

Acute leukemias Acute myeloid leukemia (also called acute granulocytic leukemia) – classification depends upon Bone marrow blast morphology Degree of cell maturation Cytochemical stains Immunophenotyping AML is divided into 7 different classifications:

Acute Non-Lymphoblastic Leukaemia Diagnosis Alternative Bone Marrow Appearance M0 Identified by ultrastructural myeloperoxidase activity or immunophenotyping. M1 AML without maturation Monomorphic with one or more distinct nucleoli, occasional auer rod and at least 3% myeloperoxidase positivity. M2 AML with maturation 50% OR > myeloblasts & promyelocytes and common single auer rod. Dysplastic myeloid differentiation may also be present. M3 APL Dominant cell type is promyelocyte with heavy azurophilic granulation. Bundles of Auer rods confirm diagnosis. Microgranular variant exist (M3v) M4 AMMoL As M2 but > 20% promonocytes & monocytes. M5 AMoL M5a:> 80% monoblasts is poorly differentiated. M5b:> 80% monoblasts, promonocytes or monocytes is well differentiated. M6 AEL >50% bizzar, dysplastic nucleated red cells with multinucleate forms and cytoplasmic bridging. Myeloblasts usually > 30%. M7 AMegL Fibrosis, heterogeneous blasts population with cytoplasmic blebs. Platelet peroxidase positive.

AML - M1 M1 – myeloblastic without maturation The bone marrow shows  90% blasts and < 10% promyelocytes The disease occurs in older adults

AML – M1 Note the myeloblasts and the auer rod:

Acute leukemias M2 – myeloblastic with maturation The bone marrow shows 30-89% blasts and > 10% promyelocytes; This is characterized by an 8,21 chromosomal translocation This occurs in older adults

AML – M2 Note myeloblasts and hypogranulated PMNs:

AML - M3 M3 – hypergranular promyelocytic This form of AML has a bone marrow with >30% blasts Is more virulent than other forms Occurs with a medium age of 39 The WBC count is decreased Treatment causes a release of the granules and may send the patient into disseminated intravascular coagulation and subsequent bleeding It is characterized by a 15,17 chromosomal translocation

AML – M3 Note hypergranular promyelocytes:

Acute leukemias M3m – hypogranular promyelocytic – The bone marrow has > 30% blasts The WBC count is increased. Like the M3 type, treatment causes a release of the granules and may send the patient into disseminated intravascular coagulation and subsequent bleeding. It is characterized by a 15,17 translocation

AML – M3m Note hypogranular promyelocytes:

AML-M4 M4 – acute myelomonoblastic leukemia Both myeloblasts and monoblasts are seen in the bone marrow and peripheral blood Infiltration of extramedullary sites is more common than with the pure granulocytic variants

AML – M4 Note monoblasts and promonocytes:

Acute leukemias M5 – acute monoblastic leukemia >80% of the nonerythroid cells in the bone marrow are monocytic There is extensive infiltration of the gums, CNS, lymph nodes and extramedullary sites This form is further divided into M5A - Poorly differentiated (>80% monoblasts) M5B - Well differentiated (<80% monoblasts)

AML – M5A Note monoblasts:

AML-M5B Note monoblasts, promonocytes, and monocytes:

AML-M6 M6 – erythroleukemia This is rare and is characterized by a bone marrow having a predominance of erythroblasts It has 3 sequentially morphologically defined phases; Preponderance of abnormal erythroblasts Erythroleukemia – there is an increase in both erythroblasts and myeloblasts Myeloblastic leukemia – M1, M2, or M4 Anemia is common

AML – M6 Note M1 type monoblasts

Acute leukemias M7 - Acute megkaryoblastic leukemia This is a rare disorder characterized by extensive proliferation of megakaryoblasts, atypical megakaryocytes and thrombocytopenia.

2- The Cytochemistry Cytochemistry AML ALL Myeloperoxidase + - Sudan black + - Non-specific esterase + (M4,5) - PAS + (M6) + c-ALL Acid phosphatase + (M6) +T-ALL

Acute lymphoblastic leukemias - reactivity with special stains Subtype Peroxidase or Non-specific Periodic Sudan black esterase acid-Schiff L1 - - +++ L2 - - +++ L3 - - +++

3- The immunophenotyping Significance: used for determining B cell or T cell lineage. Principle: antibodies made against specific surface markers. Procedure: They constitute what we would call the primary antibody and in an indirect assay they are allowed to react with the cells and unbound antibody is then washed away. Fluorescently labeled antibody (secondary antibody) against the primary antibody is added and allowed to react and then unbound secondary antibody is washed away. The cells are then sent through a flow cytometer that will determine the number of cells that have a fluorescent tag and which are thus positive for the presence of the surface marker to which the primary antibody was made. In a direct assay, the primary antibody is fluorescently labeled.

Direct versus indirect labeling of antigens

Flow cytometer

Terminal deoyxtidyl transferase This is a unique DNA polymerase present in stem cells and in precursor B and T lymphoid cells. High levels are found in 90% of lymphoblastic leukemias. It can also be detected using appropriate antibodies and flow-cytometry.

3- Classification upon The Immunological Approach First: The AML FAB classification : CD13+, CD33+ and TdT-. M6 has glycophorin A + M7 has CD41,42,61+.  

Second: The ALL-FAB classification is:   Precursor B-ALL which is CD19+, cytoplasmic CD22+ and TdT+, includes three subtypes, common-ALL which is CD10+, null type which is CD10- and pre-B-ALL which shows intracytoplasmic m chains ( and may be CD10+ 0r CD10- ). B-ALL which shows surface immunoglobulin (Ig) and TdT-. T-ALL which shows T-cell antigens CD7+, cytoplasmic CD3+ and TdT+.  B-ALL usually corresponds to the morphological L3 type whereas the CD10+, null, pre-B or T types may all be L1 or L2 and are morphologically indistinguishable.

Immunologic classification of acute lymphoblastic leukemias B- lineage (80%) Markers Pro-B CD19(+),Tdt(+),CD10(-),CyIg(-). Common CD19(+),Tdt(+),CD10(+),CyIg(-). Pre-B CD19(+),Tdt(+),CD10(+),CyIg(+),SmIg(-). Mature-B CD19(+),Tdt(+),CD10(±),CyIg(±),SmIg(+). T-lineage (20%) Pre-T CD7(+), CD2(-), Tdt(+). Mature-T CD7(+), CD2(+), Tdt(+).

B cell maturation

T cell maturation

4- Cytogenetics Cytogenetics – cytogenetics studies can now be used for diagnosis and for prognosis of hematologic malignancies. Many leukemias (and lymphomas) are characterized by specific chromosomal abnormalities, including specific translocations and aneuploidy. The specific type of malignancy can be identified based on the specific abnormality or translocation. These may be identified by Looking at the karyotypes of the chromsomes from the abnormal cells DNA based tests – these tests are very useful for following the course of the disease A normal karyotype is usually associated with a better prognosis.

Chromosomal translocation

Chromosome karyotyping

4- Cytogenetics FAB Type Chromosomal Abnormality Type of Leukaemia L1,L2 t (9;22 ) ALL L1 t (1;19 ) Pre-B ALL L3 t (8;14 ) t (8;22) t (2;8 ) B ALL t (11;14) T ALL M2 t (8;21) AML M3 t (15;17) APL M5 del/t (11) AmoL M4 inv16,and t (16;16) AMMoL M6 t (8;16) AEL

t(9;11) and other translocations involving 11q23 (MLL) 4- Cytogenetics M2 t(8;21) (q22;q22) AML1(CBFa )/ETO M3 t(15;17)(q22;q12) PML/RARa M4Eo Inv(16)(p13q22) CBFb /MYH1 M5 t(9;11) and other translocations involving 11q23 (MLL)

Chromosomal/molecular abnormalities with prognostic significance in ALL Better prognosis - normal koryotype - hyperdiploidy Poor prognosis - t (8; 14) - t (4; 11) Very poor prognosis - t (9; 22); BCR/ABL (+)

Chromosomal/molecular abnormalities with prognostic significance in AML Favorable t(8;21), t(15;17), inv(16) Intermediate (Most patients) normal, +8, +21, +22, del(7q), del(9q), Adverse -5, -7, del(5q), abnormal 3q, complex karyotype (> 3 -5 abnormalities)

Pathophysiology of the Acute Leukaemias Acute leukaemia causes morbidity and mortality through three general mechanisms: Deficiency in normal blood cell number or function. Invasion of vital organs with impairment of organ function. Systemic disturbances shown by metabolic imbalance.

First: Deficiency in normal blood cell number or function Infection Haemorrhage Anaemia

Constitutional symptoms fever and sweats common weight loss less common 72

2nd:Infiltration of tissues/organs enlargement of liver, spleen, lymph nodes gum hypertrophy bone pain other organs: CNS, skin, testis, any organ 73

Gum hypertrophy 74

Gingival Infiltration in Monocytic (AML M4 eos) Variant of AML Mani, A, Lee, DA. Leukemic Gingival Infiltration. N Engl J Med 2008; 358(3): 274. Copyright ©2008 Massachusetts Medical Society

Chloromas A B C NEJM 1998 76

Leukostasis Leukostatic tumours accumulation of blasts in microcirculation with impaired perfusion lungs: hypoxemia, pulmonary infiltrates CNS: stroke only seen with WBC >> 50 x 109/L (Hyperleucocytosis ) 77

Sanctuary site relapse Leukaemic infiltration of the testes and meninges provide an effective sanctuary for resident laeukaemic blasts because cytotoxic drugs poorly penetrate them. This provides a source for relapse. Meningeal and testicular relapse are most commonly seen in childhood ALL.

3rd:Metabolic Disturbances hyponatraemia is relatively common in ANLL secondary to the production of a vasopressin-like substance by myeloblasts. Hypokalaemia is also common, especially in AMMoL and AmoL secondary to the renal damage. Spontaneous lysis of blast cells causes the release of abnormally large amounts of purines into the plasma. This results to an increase uric acid or hyperuricaemia. This abnormality may be severe enough to require dialysis, particularly where renal damage is present.   Several of the cytotoxic drugs are nephrotoxic and therefore may increase the severity of minor metabolic disturbances secondary to massive cell lysis.

Notes to Remember

Laboratory features WBC usually elevated, but can be normal or low blasts in peripheral blood normocytic anemia thrombocytopenia neutropenia DIC 81

Bone marrow in acute leukemia necessary for diagnosis useful for determining type useful for prognosis Acute leukemias are defined by the presence of > 20% blasts in bone marrow (% of nucleated marrow cells) 82

Distinguishing AML from ALL light microscopy AML: Auer rods, cytoplasmic granules ALL: no Auer rods or granules. flow cytometry special stains (cytochemistry) 83

AML 84

AML 85

Auer rods in AML 86

ALL 87