3 rd Year – Level 5 – AY Dr. Walid ZAMMITI Msc, PhD MLT Faculty of Applied Medical Sciences Department Of Medical Lab. Technology HEMATOLOGY 2. MLT. 307

Chronic Lymphoproliferative Leukemic Disorders CLLD 2

Objectives 1. Categorize types of CLLD 2. Diagnose based on lab results the following types of Leukemias: CLL; PLL;HCL 3. Discuss characteristics which are used to differentiate those types of CLLD. 4. State the clinical features and predominant cell type for the Leukemias listed in objective #2. 5. Differentiate between a chronic and acute leukemia. 6. Recognize cells typical of Hairy Cell Leukemia. 3

 The CLLD is a proliferation and accumulation of small mature B lymphocyte lineage in the bone marrow and lymphoid organs.  About 99% of these disorders involve B-lymphocytes caused by a decreased rate of destruction of the malignant lymphocyte clone (rather than increased rate of production).  T-lymphocytes cases are very few and tend to have poorer prognosis. 4  This lymphocyte population were affirmed for the proliferation of B lymphocyte by the presence of monoclonal Ig in low intensity on surface of lymphocyte mostly IgM type.

 This main group of diseases (CLLD) compromises 3 major types of leukemias: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PROLYMPHOCYTIC LEUKEMIA (PLL) HAIRY CELL LEUKEMIA (HCL) 5

Chronic Lymphocytic Leukemia (CLL) 6

 CLL is the most common of the chronic lymphoproliferative leukemia disorders.  Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of small mature-appearing CD5+ B lymphocytes in the blood, marrow, and lymphoid tissues.  It is characterized by chronic persistent lymphocytosis which later infiltrate different organs.  Adults affected mostly (90% above 50 years and 65% above 60) with males being more than twice the females  Peak incidence between 60-80yrs.  up to 60% asymptomatic.  9 year median survival, but varies greatly. 7 CLL: Introduction

 The causes of this disease are unknown,  It appears likely that genetic factors contribute to its development.  The leukemic cells from nearly 50% of CLL patients can be found to have clonal chromosomal abnormalities, 9  Del 13q is the most common chromosomal abnormality in CLL, followed in order by trisomy 12, del 11q22.3-q23.1, del 6q21-q23, and 14q abnormalities.  Mutations of the P53 tumor suppressor gene at 17q13 are uncommon except in advanced disease. Etiology (causes)

High familial risk with two-fold to seven- fold higher risk. No documented association with environmental factors. No established viral etiology. In contrast to other forms of leukaemia; there is no higher incidence after previous chemotherapy or radiotherapy. 10 Etiology (causes) (Cont),

11 Clinical presentation: The disease occurs in older subject (above 60),rare before 40 years. Many cases discover routinely. Symmetrical enlargement of superficial lymph node is the most frequent clinical sign.(see Picture) Features of anemia. Splenomegaly & hepatomegaly are usual in later stage. Repeated bacterial or fungal infection. Thrombocytopenia (bleeding). Immunosuppression is a significant problem resulting from hypogammaglobulinaemia and cellular immune dysfunction (caused by a lack of B-lymphocytes and a resulting low level of immunglobulins (antibodies) in the blood).

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 Symptoms usually are very mild and insidious ranging from mild to severe depending on the course of the disease.  Common symptoms in early stages include: fatigue, general weakness, and less tolerance to exercise.  Progressive late stages of the disease can present with severer symptoms all of which are caused by the complications of lymph’s replacement of other normal blood cells such as: pallor, jaundice, fever, easy bruising, weight loss, recurrent infections, bone tenderness, edema, and infiltration of other organs (lymph nodes, liver, kidneys, skin, etc.). 13 Clinical presentation (cont),

14 Lab. diagnosis: CBC: *TWBCs: increased. Diff: lymphocytosis,the absolute lymphocyte count is >5x10 9 /l and may be up to 300x10 9 /l. *RBCs: normocytic normochromic anemia is present in later stages, autoimmune haemolysis. * Platelets : thrombocytopenia may occur. Blood film: 70-99% of white cells are ‘’mature lymphocyte’’= small lymphocytes with minimal cytoplasm and dense chromatin. Smudge or smear cells also present.(These are CLL cells ruptured during smear preparation)

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Smear, Smudge or basket cells are leukocytes that have been damaged during preparation of the peripheral blood smear. 16

17 Cont: Cytogenetic :  The most common abnormalities are: Deletion of 13q14; Trisomy 12; Deletion of 11q23. Bone marrow aspiration: Lymphocytic replacement of normal marrow ( lymphocytes comprise 25% -95% of all cells ). Reduced concentrations of serum Igs are found. Immunophenotyping:  Shows that the lymphocytes are B cells (surface CD19 + )  Expressing surface IgM or IgD. This is shown to be monoclonal because of expression of only one form of light chain (  or only).  cells are also CD5 + &CD23 +

18 The diagnostic criteria for CLL 1) A peripheral blood lymphocyte count of greater than 5 x 10 9 /L, with less than 55% of the cells being atypical 2) The cell should have the presence of B cell- specific differentiation antigens :(CD19, CD20, and CD23) and be CD5(+) 3) A bone marrow aspirates showing greater than 25% lymphocytes

 The major staging systems used for the classification of CLL are the Rai and Binet staging systems  These systems have assisted clinicians in categorizing patients by prognosis, and have provided guidance on designing therapeutic strategies  Survival within each disease stage may vary significantly, particularly in patients with Rai stage 0 and Binet stage A  The median survival of patients with Rai stage 0 disease is approximately 12 years. Patients with Rai stages II through IV have median survival rates of 5 to 8 years Staging CLL 19

StageDefinition Median Survival 0 Low risk Lymphocytosis only (>15x10 9 /l) 11years I Intermediate risk Stage 0+enlarged lymph nodes: Lymphocytosis and lymphadenopathy 11 years II Intermediate risk Stage 0+liver or/and spleen  adenopathy: Lymphocytosis in blood and marrow with spleenomegaly and or hepatomegaly 8 years III High risk Stage 0+anemia  organomegally or  adenopathy: Lymphocytosis and anemia (hemoglobin<11g/dL or hematocrit <33%) 5 years IV High risk Stage 0+thrombocytopenia  organomegally or adenopathy: Lymphocytosis and thrombocytopenia (Platlet count < /mm 3 ) 20 1-Rai staging system

StageDefinition Median Survival HbPlatelet A (50-60%) Enlargment of < 3 lymphoid areas (cervical, axillary, spleen, liver), no anemia or thrombocytopenia 11 years  10  100 B (30%)Enlargment of < 3 lymphoid areas 8 years  10  100 C (<20%) Anemia (hemoblobin <10g/dL or thrombocytopenia), Platlet count < /mm 3 or both 7 years <10 and /or < Binet staging system

PROLYMPHOCYTIC LEUKEMIA (PLL) 22

 PLL is thought to be a rare variant form of CLL due to some similarities between the two.  Similar to CLL, PLL affects adults usually over 60 years old with males more than females.  Nevertheless, PLL and CLL have different symptoms and morphology.  PLL has the worst prognosis among the 3 CLLD (i.e., worse than CLL and HCL) with a mean survival of 1 year. PROLYMPHOCYTIC LEUKEMIA (PLL) 23

 Symptoms tend appear suddenly (acute) and faster than CLL.  The most common finding is the accumulation of lymphoid cells (abnormal prolymphocytes) in spleen, BM, and to a lesser extent, the liver.  Usually, there is an enormous enlargement of the spleen (and to a lesser extent the liver) with no involvement of the lymph nodes.  No lymphadenopathy Symptoms 24

 High WBC counts ( ×10 9 /L) with low, normal, or high differential of neutrophils and monocytes with some nucleated RBC’s (nRBC’s) seen in pb smears.  The malignant cell is a large lymphocyte with round or oval nucleus of coarse chromatin, having 1-2 prominent vesicular nucleoli.  Cytoplasm is of bluish color and usually contains no granules.  BM shows replacement of normal cells by the malignant clone.  Phenotypically, there is an increase in SIg’s with low rosette formation and decreased CD5 expression.  -ve CD5,-ve CD23,+ve CD22, +ve CD79b expression.  About 50% of the cases showed some genetic translocation, involving chromosome 14. Lab. Diagnosis: 25

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HAIRY CELL LEUKEMIA (HCL) 28

 HCL is a very rare disease accounting for 2% of leukemia.  Similar to CLL and PLL, HCL is also an adult disease, striking patients over 50 years old with males 5 times more than females.  The mean survival rate is 5 years from diagnosis.  Death occurs due to complications arising from the infiltration of leukemic cells to BM, spleen, liver, pb, etc.  Lymphadenopathy uncommon HAIRY CELL LEUKEMIA (HCL) 29

 90% of cases showed the presence of the hairy cell in peripheral blood.  The characteristic hairy cell rarely constitutes more than 50% of the pb differential. The cell is a large lymphocyte with round or oval nucleus containing lacy or loose chromatin.  Nucleoli could be present (N:C ratio is variable but usually low).  The cytoplasm is grayish blue in color with no granules, but possessing HAIR-like projections (distinctive feature of HCL cells). Lab finding 30

HCL showing cells with condensed chromatin and a conspicuous single nucleolus, but with more abundant pale cytoplasm with cytoplasmic projections 31

32 Hairy cell Leukemia: numerous irregular cytoplasmic projections that give cells a flagellated or hairy appearance in the bone marrow, spleen, liver, and peripheral blood.

Lab. Diagnosis (cont.) There is an obvious pancytopenia in pb which is supported by the replacement of normal marrow cells by the malignant cells Lymphocyte count is rarely over 20×10 9 /L These cells could be differentially diagnosed by staining them with Tartrate-resistant acid phosphatase (TRAP). Only HCL cells are positive while all other leukemias are negative. -ve CD5,-ve CD23,+ve CD22,+CD103 33

Tartrate Resistant ACP: TRAP + Tartrate

Immunophenotype of the chronic B-cell leukaemias (all cases CD19+). HCLPLLCLL ++ weakSIg --+CD5 ++-CD22/FMC7 --+CD23 +--CD The FMC7 antibody detects an antigen expressed on mature human B cells and is used in immunophenotypic analysis and differential diagnosis of lymphomas and leukemias.

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