Cfmpter-U X PPKM O'J LITE'M'IV'JfE A :

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2 Cfiap ter-n O f 2.1. LEUKEMIA Leukemia is a progressive, neoplastic disease of the hematopoietic system charaterized by unregulated proliferation of uncommitted or partially committed stem cells. It includes a heterogeneous group of neoplasms that differ with respect to aggressiveness, cell of origin, clinical features, and response to therapy History: In 1845, two individuals, John Bennett in Edindurgh and Rudolf Virchow in Berlin, independently published their observations of leukaemia patients. They are generally credited with defining leukaemia and recognizing its significance as a discrete disease entity (Bennett, 1845; Virchow, 1845). Clinical histories of their patients were similar; with symptoms of increasing wealcness, swelling of the abdomen, and serious nose bleeds in their patients. At autopsy, the two most remarkable findings were a greatly enlarged spleen and a peculiar appearance in the consistency and color of blood. Bennett thought the blood looked as though it was mixed with pus. Microscopically, he affirmed that the blood did contain many large corpuscles similar to those found in pus; however, Bennett remarked that there was no apparent sign of inflammation, a condition that was usually associated with the finding of pus. Virchow preferred the tenn white blood to describe the unusual pale whitish color of the blood. Two years later, the term white blood was translated into Greek, becoming leukemia. As additional cases of leukaemia were found, Virchow recognized that not all leukemias were associated with an increase in the same type of white cell. In some cases, white corpuscles were granular with devided or irregular nuclei, and the spleen was particularly enlarged. In other cases, the corpuscles were agranular with round nuclei, and the lymph nodes of the patient were enlarged. These observed distinctions were probably in reference to what we now classify as myeloid and lymphoid leukemias Types of Leukemia: Leukemia is classified into two broad groups, based on the aggressiveness of the illness: 1. Acute Leukemia (AL): an aggressive form, which, if untreated causes rapid death, usually within months. The AL is a heterogenous group of disease characterized by unregulated, progressive proliferation and accumulation of immature, malignant, hematopoietic precursors in the bone marrow. Eventually, the immature neoplastic cells spill over into the peripheral blood, producing

3 ' # 1 Cfmpter-U X PPKM O'J LITE'M'IV'JfE A : leukocytosis. In the laboratory, the diagnosis of acute leukaemia is suggested when examination of the peripheral blood smears reveals the presence of many undifferentiated or minimally differentiated cells. 2. Chronic Leukemia (CL); a less aggressive form, which, if untreated, causes death in months to years. The chronic leukemias most often have an insidious onset. Diagnosis is frequently made during a routine physical examination for nonspecific patient complaints such as weight loss or weakness. The bone marrow typically exhibits an accumulation of differentiated lymphocytic or myelocytic elements. These cells spill over into the peripheral blood, producing a leukocytosis. The differential count of the bone marrow and peripheral blood are similar with all stages of maturation present but with a predominance of the more mature forms. Both of these major groups are further classified into myeloid or lymphoid according to the origin of the leukemic stem cel! clone. If myelocytic cells or other cells derived from the CFU-GEMM stem cell predominate, the disease is called myelogenous leukaemia. If the lymphoid cells predominate, the disease is termed lymphocytic leukaemia. Thus, using these two classification systems for aggressiveness of the disease and cell of origin, four types of leukaemia are recognized; 1. Acute Myeloid Leukemia (AML) 2. Acute Lym phoid Leukemia (ALL) 3. Chronic Myeloid Leukemia (CML) 4. Chronic Lym phoid Leukemia (CLL) 2,1.3. Epidemiology; Worldwide: Leukemias are the 10 *' most common cancer in men and 12^' in women and constitute 3% of the total global cancer burden (Pisani et cil, 1993). The incidence of leukaemia is highest in North America and Australia/New Zealand and lowest in Sub-Saharan Africa (Parkin el cil, 1997). In the United States, leukemias and lymphoma accounts for 8% of all male cancers and 6% of all female cancers. In 1993, there were about new cases of leukaemia in the U.S. with deaths from leukemia ranging from 3.8 per 100,000 for females to 6.3 per 100,000 for males (Boring et al, 1993).

4 0 ". '-y ip: :",sil Cfiapter-ll <J^E%^'1TW 0 J L F m J m U Z E» * S gsgafflg5a5?!!;iamgmaiagi i.to India: Leukemia are the most common cancers affecting children and it represents >30% of all cancer among childhood cancers. In India, leukemias and lymphoma accounts for 9.9% cases of all cancers in male and 5.6% cases of all cancers in female. Based on the National Cancer Registry Programme, the Indian Council of Medical Research has reported the incidence rate of leukaemia and lymphoma to be 7.1 per 100,000 population for males and 4.4 per 100,000 populations for females (National Cancer Registry Programme, 2001). As per the available information from population based surveys, the incidence of leukemias in India varies from 0.8/100,000 in Barshi (rural area) to 5/100,000 in Delhi. Incidence is highest in the metropolitan cities of Delhi and Mumbai. At the Bombay Cancer Registry, leukemias constituted 3.9% of all registered cancer cases with myeloid leukemia (48%) being commoner than lymphoid leukaemias (40%); this may be related to the under reporting of CLL. Other studies have reported the incidence of leukaemia to be 4.4 and 3.1 per 100,000 population, for males and females respectively. Males are affected more often than females; both for lymphoid and myeloid leukemia. The higher incidence in males may be due to increased exposure to occupational and environmental carcinogens (Jussawalla etai, 1988; Chaudhary eta l, 1996; Yeole et al, 1998). Delhi; Childhood cancer up to 14 years of age in Delhi constituted 4.7% of all cancer registered during the period Leukemia accounts for approximately 34% of all childhood cancers in Delhi. Leukemia showed the highest incidence (4.4 per 100,000 persons for male child and 2.8 per 100,000 persons for female child) followed by brain tumours (1.6 per 100,000 persons for male child and 0.9 per 100,000 for female child). Lymphoid leukemia were found to be the most predominant in children (age < 15 years), both in males and females (Fig. 2.1a and b). In general rates were higher among male children compared to females (2:1 ratio).the incidence of lymphoid leukemia was predominant among male child (Range = 3.0 to 4.5 per 100,000 persons) compared to female child (Range = 1.3 to 2.2 per 100,000 persons). The incidence of myeloid leukemia was the predominant cancer followed by bone, brain and NHL in young adult males (Tyagi et ai, 2006; Delhi Cancer Registry, 2007).

5 C fiapter-ii - sa: a CO c OJ rs Q. o 0> E 3 Z Incidence of leu kem ia by Five Y ear Age Group du rin g in M ale Lymphoid Leukemia M yeloid Leukem ia Age Groups (years) b (A 4^ o ( D. o E 3 z Incidence o f leu kem ia by Five Y e ar Age Group during in Fem ale Lymphoid Leukem ia Myeloid Leukem ia \ 0 - O,^ O? Cb^ 5? Age Groups (years) F igu re 2.1: Lym phoid leukem ia is predom inant in children (< 15 years) w hile m yeloid leukem ia is predom inant in adults. Incidence o f leukem ia is higher in m ale children (a) com pared to fem ale children (b) (2:1 ratio). 8

6 ?<? Cfapter-11 'XEWEfW 0 J L lt Z il^m i'jz E 9 f 2.2. ACUTE LEUKEMIA (AL) Acute leukemia (AL) is a rapidly progressing disease that results in the accumulation of immature, functionless cells in the marrow and blood. The m ait ow often can no longer produce enough normal red blood cells, white blood cells and platelets. Approximately 50% of all leukemias are diagnosed as acute. Although there is some difference in incidence of the AL between countries and regions of countries, the differences are not great. The majority of leukemias (25%) are Acute Lymphoblastic Leukemia (ALL). Most childhood AL are of the lymphoid type (ALL), whereas those occurring in adults are typically myeloid (AML) in origin. The incidence of ALL was highest among children under five years of age (Tyagi et al, 2006). The cell type distribution of leukemias observed in India (Table 2.1) is different from that observed in developed world. Myeloid leukemias predominate in India, while, lymphoid leukemias dominate in the west mainly because of higher incidence of CLL. Table 2.1: Frequency (%) of acute and chronic leukemias in India (Hospital based data) Different Hospitals from India Delhi ( ) (Rani et a l, 1982) C handigarh ( ) (Shome et al., 1985) Bombay ( ) (Advani et al., 1979} Pondicherry ( ) (Prakash e ta i, 1981) Lucknow ( ) (Kushwaha et al, 1981) M umbai ( ) (D'Costa et al., 1989) Calcutta ( ) (Chatterjea e ta i, 1962) Acute Chronic ALL AMI, CLL CMI,

7 .it? Acute Lymphocytic Leiikeiiila (ALL): ALL is the most common malignant disease affecting chidren, accounting for approximately 30% of childhood cancers (Young et al, 1975), Of the estimated 5200 new cases occurring annually in the US, approxmiately75% are children younger than 15 years of age. Before the advent of effective chemotherapy in the 1960s, ALL usually was fatal. Within 20 years, however, more than 50% of children with the disease were in complete remission 5 years from diagnosis (Mouer, 1980) and most of these patients are now considered cured. Unfortunately, therapy that proved effective for most children had little impact on the survival of adults with ALL (Giona et al, 1994). Currently; patients are stratified at diagnosis into prognostic group so that therapy can be tailored to decrease the risk of disease recurrence. Present data indicate that approximately 70-80% children with ALL survive for more than five years without recurrence (Gaynon et al, 2000; Schrappe et al, 2000; Silverman et al, 2000; Pui et al, 2000). ALL is a rare disease in the elderly. The prevalence of ALL in patients >60 years of age is reported to be between 16% and 31% of all adult cases. The biology of ALL in older patients seems to be significantly different from that in younger patients and may, at least in part, explain poor treatment outcome. Immunophenotyping and cytogenetic characteristics are among the most important biological differences in comparison with younger adults. The frequency of pre B-cell ALL and common ALL is higher and T-cell ALL subtype is under-represented in elderly populations compared with younger patients (Robak, 2004) Acute Myelocytic Leukemia (AML): AML is a disease of the elderly with a median age at presentation in the seventh decade and a peak incidence in the U.K. of greater than 20 patients per 100,000 population per yr between the ages of 80 and 84 f Copplestone et al, 1988). AML accounts for about 20% of the acute leukemias seen in children. In contrast to childhood ALL, there has only been a modest improvement in the cure rate of children with AML during the past two decades. Approximately 40% of children treated with chemotherapy alone are long-term survivors (Ebb et al, 1997). 10

8 Cfm pter-n 0 7 L V T B M ^m X E t * Mixed Lineage Acute Leukemia (MI^AL): A minority of acute leukemias have features characteristic of both the myeloid and lymphoid lineage. There is considerable confusion concerning the terminology and diagnosis involving acute leukemias with blast cells that express characteristics of more than one hematopoietic lineage. Such leukemias have been designated with numerous tenns; biphenotypic, bilinea), hybrid, biclonaj, chimeric, mixed, simultaneous, synchronous, metachronous, and lineage switch. The most important distinction is between biphenotypic and bilineal leukemia. Biphenotypic acute leukemia (BAL) is confined to those cases where coexpression of different markers (lymphoid and myeloid) occurs in significant amounts on the same cells. Bilineal leukemia represents those cases in which there are two separate blast populations: one myeloid, the other lymphoid. Bilineal leukemias may be either biclonal or clonal. In a study, 7% cases of the 746 cases of acute leukemia were diagnosed as BAL (Hanson etai, 1993). BAL may present as ALL or as one of the AML subtypes, often Ml or M2 or rarely M4 and M5. Most of the cases exhibit coexpression of B-lymphoid and myeloid markers and less frequently T-lymphoid and myeloid markers. Cases with a B and T lymphoid phenotype or with trilineage differentiation are rare {Matutes et al, 1997). The prognosis of BAL in adults is worse than AML or ALL. Four year overall survival has been quoted at 8%. The most important prognostic features are age (<60 years), absence of Philadelphia chromosome and achievement of complete remission (Killick et al., 1999). A scoring system for BAL was devised by the European Group fo r the Immunological Characterization of Leukemias (EGIL) to distinguish BAL from those leukemias with the expression of a marker from another lineage (Bene et al., 1995). The score encompasses the number and the degree of specificity of the markers expressed by the leukemic cells. The markers considered to be most specific are shown in Table

9 Cfmpter-Il o j L rrz'k ym rj^e "ss» Table 2.2: Scoring system for the definition of BAL*' Points* B"Ilneage T -iineage M yeloid lineage 2 CD79a cyt IgM cyt CD22 CDS (cyt/m) anti-tcra/p anti-tcr y/5 anti-m PO (anti-lysozym e) CD 19 CD2 CD13 1 CDIO CD5 CD33 CD20 CDS, CDIO CD w 65 TdT TdT CD CD 24 CD7 CD 15 CDIa CD64, CD117 BAL is defined wlien scores are over 2 points for the myeloid lineage and for one of the two lymphoid lineages. Each marker scores tsie corresponding point DIAGNOSIS AND CLASSIFICATION OF ACUTE LEUKEMIA The standard methods for establishing the diagnosis of acute leukemias are cytomorphology and cytochemistry in combination with multiparameter immunophenotyping. Cytogenetics, fluorescence in situ hybridization, and PCRbased assays add important information regarding biologically defined and prognostically relevant subgroups, and allow a comprehensive diagnosis of welldefined subentities. Integrated classifications such as that proposed by morphology, immunology and cytogenetics (MIC) cooperative group have resulted in reassessment of the leukemic syndromes and provided new diagnostic, prognostic and therapeutic insights. Recently, the World Health Organization (WHO), in conjunction with the Society for Hematopathology and the European Association of Hematopathology, has proposed a new classification for hematopoietic neoplasms. The WHO classification incorporates genetic aberrations and immunology as major defining features in addition to morphology (Jaffe et al, 2001). Recently, the field of hematopoietic malignancies has seen an explosive increase in the applications of microan-ay technology for a variety of purposes. Several papers have recently been published on the diagnostic application of mlcroarrays for gene expression studies in 12

10 leukemia. These include microarray-based discrimination between lymphoblastic and myeloid leukemias and microarray-based identification of acute lymphoblastic leukaemia. Gene expression profiling by microarray technology may possibly be used routinely for diagnostic purposes in the near future (Haferlach et cil, 2003). In most laboratories in our country, diagnosis of acute leukemia (AL) is based on cytomorphology and cytochemistry (French-American-British (FAB) classification) and Iminunopiieiiotyping (Bennett et al, 1994; Foon et cil, 1986). FAB criteria is widely used but this classification has some limitations, as they are difficult to interpret in several cases Since management may vary with cell lineage in AL, accurate characterization of leukemic ceils using immunophenotyping is essential in these difficult cases. However use of immunophenotyping is limited since a large panel of antibodies is recommended for immunophenotypic characterization of AL. FAB criteria broadly divide AL into Acute Myeloid Leukaemia (AML) and Acute Lymphoid Leukaemia (ALL). AML and ALL differ from one another with regard to clinical presentation, course, and response to therapy. Current management in India is based mainly on morphologic characterization of leukemic cells at diagnosis Freiich-Anierican-British (FAB) Classification of Acute Leukemia: In 1976, in an effort to improve and standardize the classification of AL, a group of French-American-British (FAB) physicians proposed a classification and nomenclature system based on the morphologic characteristics of blast cells on Romanowsky-stained smears and on the results of cytochemical stains (Cytochemistry) (Bennett et ai, 1976). This widely accepted system is known as FAB classification and is only of value in untreated patients because cytotoxic therapy tends to distort both normal and malignant cells, making cell identification difficult. Acute leukaemias demonstrate > 30% blasts in the bone marrow and FAB diagnosis of AL is based on the i) Morphology of the blast cells in peripheral blood and bone marrow and 2) Cytochemistry. Cytochemical stains are used to identify certain enzymes or other substances in the cytoplasm of haemopoietic ceils. 13

11 Cfiapter-Il ^ V lt l W O 'J f i (A) FAB Classification in ALL: In the FAB classification system ALL are divided into three groups, LI to L3, distinguished on the basis of cell size, nuclear shape, number and prominence of nucleoli, and relative amount and appearance of the cytoplasm (Table 2.3) (Fig. 2.2). ALL LI; LI accounts for over 80% of ALL cases in childhood and 30% in adults. It appears to have the best prognosis. The key is homogeneity of lymphoblasts. The lymphoblasts are predominantly small upto twice the size of a small lymphocyte. The chromatin is usually finely dispersed but may appear more condensed in small cells. The nuclear shape is regular with occasional clefts or indentations. Nucleoli are not prominent and may be absent. The cytoplasm is scant and only slightly or moderately basophilic. ALL L2: L2 is the most frequent ALL found in adults. Occasionally, the cells have granular inclusions, making it difficult to distinguish 12 from M2, if cytochemical stains are not perfonned. In contrast to granules in myeloid cells, the granules in lymphoblasts are peroxidase-negative and the cells are positive for TdT. The chromatin pattern is heterogenous varying from finely reticular to condensed. The nucleoli are always present but varying in size and number often they appear very large. The nucleus is irregular with clefting and indentations. The cytoplasm is abundant with variable basophilia. ALL L3: This is the rarest form of ALL. It occurs in both adults and children. L3 makes up 3% to 5% of ALL cases in children and young adults. The lymphoblasts are homogeneous. The cells are large with abundant, intensely basophilic cytoplasm. There is prominent cytoplasmic vacuolization. Vacuoles also may be present in LI and L2 lymphoblasts but it is much less intense than in L3. The nucleus is oval to round, with dense but finely stippled chromatin and one or more prominent nucleoli. (B) FAB Classification in AML: In the FAB classification system AML are subdivided into eight FAB groups, M0-M7, depending on the predominant cell lineage (e.g., granulocytic, moncytic, erythrocytic, megakaryocytic) and the degree of differentiation (Table 2.4) (Fig. 2.3a and b). Evaluation of the bone marrow reveals more than 30% blasts. Myeloblasts can be of three types. The type I blast has an open beady chromatin; one or more distinct nucleoli; and slightly basophilic cytoplasm, with no granules. Type II blasts 14

12 Cfiapter-11 0 f L l't E Iim trm ^ are similar to type I, except they contain a few azurophilic granules (up to 20 granules according to some authors). Type III blasts are similar to type II but have 20 or more granules; lack a Golgi zone; and demonstrate an immature nucleus, which may contain one or more nucleoli. If a Golgi zone is present, the cell should be considered a promyelocyte. Also, the promyelocyte nucleus shows more clumped chromatin and the nucleus may be eccentric. The type III blast is usually seen in AML M2 with the t(8;21). Auer rods are also found in leukemic myeloid elements in the bone marrow. They are usually found in blasts, but may be seen in maturing granulocytic cells. The number seen varies but, if present, indicates myeloid lineage. AM L MO: The AML-MO is minimally differentiated AML and comprises 2% to 3% of AML. The blasts tend to be large with an agranular cytoplasm and have nuclei with open chromatin and prominent nucleoli (can be single). The blasts occasionally resemble FAB L2 lymphoblasts or acute undifferentiated leukemia. Cytochemical reactions are uniformly negative; however, combined esterase staining has been used. MPO can also be detected by using anti-mpo antibody by flow cytometry or by immunohistochemistry. MPO can be detected in small granules by electron microscopy. The diagnosis of MO requires immunologic or ultrastractural methods. AML M l: The AML-Ml is myeloblastic leukemia with poorly differentiated blasts with one or more distinct nucleoli, which makes up 20% of AML. Immunohistochemistry can be helpful in diagnosis. The myeloblast count must be 90% or more of the nonerythi'oid cells. Auer rods may be seen. AML M2: The AML-M2 is myeloblastic leukemia with maturation comprising myeloid cells from promyelocytes to neutrophils (more than 10% of nonerythroid cells) and accounts for 25%-30% of AML. Monocytic precursors must be <20% in the bone marrow. Bone maixow blasts are large with a high frequency of thin, needle-like auer rods and heavy azurophilic granulations. The t(8;21)(q22;q22) is commonly seen in M2. AML M3: The AML-M3 is promyelocytic leukemia and comprises 5% to 10% of AMLs. The abnormal promyelocytes are usually heavily granulated and have bilobed or kidney bean-shaped nucleus with an indistinct nucleolus. Auer rods are often prominent and may occur as a bundle of sticks; such cells are referred to as faggot cells. Myeloblasts are a minor component and abnormal promyelocytes are 15

13 'a Cfiapter-11 ITM 'EW 0!F considered comparable with blasts for the purpose of diagnosis. The t(15;17)(q22' 24;qll-21) is seen in virtually all cases of M3. The genes involved in this translocation are promyelocytic leukemia (PML) on chromosome 15q22 and retinoic acid receptor-alpha (RARa) on chromosome 17q21. A few cases of M3 are cytogenetically nom al, but can be found to have the PML/RARa reamngement by Southern blot or PCR. AML M4." The AML-M4 is myelomonocytic leukemia and makes up 20% to 25% of AMLs. Both granulocytic and monocytic differentiations are present with more than 20% monocytic precursors in the marrow. AML M5; The AML-M5 is acute monocytic leukemia and comprises 10% of AML. M5 consists of two distinct forms: (1) the M5a (poorly differentiated); and (2) the M5b (well-differentiated). The marrow of the former is composed predominantly of immature monoblasts with <20% promonocytes or monocytes, whereas the marrow of the latter contains at least 20% abnormal promonocytes or monocytes, with twisted or folded nuclei, gray-blue cytoplasm, and scattered azurophilic granules. In M5a, the blasts are large with an abundant rim of cytoplasm, rare azurophilic fine granules, and sometimes vacuolated cytoplasm. The nucleus is round to oval with delicate, lacy chromatin. AML M6: The AML-M6 is erythroleukemia, which is a heterogeneous disorder that makes up 5% of AMLs. All subtypes demonstrate 50% or more bone marrow erythrocytic precursors with concurrent erythroid dysplasia. AML-M6 is characterized by >30% blasts of nonerythrocytic elements. Rarely, auer rods are seen in the myeloblasts. AML M7: The AML-M7 is acute rnegakaryoblastic leukemia and makes up 5%~1% of AML. The bone marrow biopsy is invaluable in establishing the diagnosis because of the frequency of extensive fibrosis, which makes it difficult to obtain an adequate aspirate. The megakaryoblasts vary in size and have a high nuclear;cytopiasmic ratio and agranular pale blue cytoplasm. Cytoplasmic blebs resembling platelets may be seen. Cytochemical stains are usually negative except for nonspecific esterases and PAS. Definitive identification of megakaryoblasts requires electron microscopy or immunophenotyping. In addition, staining for the presence of factor VIII antigen may be helpful. 16

14 Cfiapter-11 ^ V l T f W O f L F TU M TU ^J^ 'h ^V 10 ^ g >' M0 f Cytochemistry: Accurate characterization of leukemic blast cells is an important prerequisite of the precise diagnosis of acute leukaemia. Cytochemistry is essential in delineating lymphoid from nonlymphoid leulcemias as well as in subclassification of the latter especially when Romanowsky stains make differentiation impossible. Thus, cytochemistry contributes to the final classification and differential diagnosis of acute leukaemia. Cytochemistry in hematology refers to the staining methods used to identify the chemical composition of cells without significantly altering the cell morphology. Most cellular cytochemical markers are organelle-associated enzymes and other proteins. The cells are incubated with substrates that react with specific cellular constituents. If the constituent is present it reacts with the substrate, yielding a colored product in the cell. Cytochemical staining reactions are of two types: 1. Enzymatic: e.g. Myeloperoxidase (MPO), 2. Non enzymatic: e.g. Sudan black (Stains lipids) PAS (stains glycogen). Following cytochemical stains are used to differentiate/characterize acute leukemic cells: 1. Myeloperoxidase (MPO): MPO is an enzyme capable of catalyzing the oxidation of substances by hydrogen peroxide (H202). MPO activity depends on the oxidation of benzidine by H2O2, in the presence of copper sulphate. This enzyme is present in the primary and secondary granules of granulocytes and their precursorsmetamyelocyte, myelocyte promyelocyte and myeloblast. Monoblasts, monocytes and lymphoblasts are negative. Auer rods of myeloblasts give a positive reaction. Method: Air-dried film is treated with 0.5% copper sulphate followed by 1% benzidine solution and counterstain with 1% saffranin. An insoluble brown black reaction product identifies the site of MPO activity. 2. Sudan Black (SB): Sudan Black B stains lipid membranes of the granules, which contain enzyme myeloperoxidase. SBB also stains the cells of myeloid series. Method: Fixed air-dried film in foimalin vapour is immersed in staining solution consisting of buffered 0.3% sudan black B for 2-4 hours at 56 C followed by washing in 70% ethanol and counterstaining with 1 % saffranin. The reaction product in the cytoplasm is black and the nuclei stain red. 3. Periodic acid Schiff (PAS): The PAS reaction depends on the liberation of carbohydrate radical from combination with protein and their oxidation to aldehyde 17

15 a. Cfiapter-II J^En/rBW O T S» by Schiff s reagent. A positive reaction denotes the presence of glycogen. Lymphoblasts show blocks of PAS+ve material in the cytoplasm. Myeloblasts are negative. Block positivity in lymphoblasts is observed in most of the LI-ALL cases. However PAS positivity has no definite correlation with prognosis. Method: Ethanol fixed slide is treated with 1% periodic acid followed by Schiff s reagent and counter stain with equal part of feme chloride and haematoxylin. 4. Esterases: Esterases are various enzymes in cells of monocytic series. The nonspecific esterase stain (NSE) is used primarily to differentiate the granulocytic leukemias from those that have monocytic origin. This stain is useful to characterize the monocytic component in M4 and M5 AML. NSE stains monocytes, promonocytes and monoblasts. In monocytes non-specific esterase shows a strong positive reaction that is inliibited by sodium fluoride. (A) Cytochemistry in ALL; The diagnosis of ALL by cytochemical methods is by a process of exclusion. However PAS block positivity is suggestive of lymphoid lineage. The PAS positivity is defined by the presence of blocks or coarse granules in 5% or more of the blasts. The sensitivity of a cytochemical staining combination by PAS positivity and myeloperoxidase, Sudan Black B and alpha-naphthyl butyrate esterase negativity in defining cases of lymphoblastic leukaemia is approximately 50%; however the specificity of this combination of lymphoblastic leukemia is 100%. A positive PAS stain, in combination with negative myeloproxidase, Sudan Black B and alphanaphthyl butyrate esterase stains, continues to have a diagnostic role in the distinction between lymphoid and myeloid leukaemia (Snower et al, 1991) (B) Cytochemistry in AML: A paramount feature of myeloid leukemia cells is the presence of MPO in the primary granules. It is done side by side with a Romanowsky stain. When 3% or more of the blasts are MPO-positive by light microscopy, leukemia is classified as myeloid. Three was chosen empirically so that the few normal residual myeloblasts in ALL marrow would not result in every type of leukemia being classified as myeloid. Sudan Black gives similar results as MPO but it is not preferred over MPO as 1.6% of pediatric ALL show positivity. When MPO activity is low, a diagnosis of acute monocytic leukemia depends on non-specific esterase (NSE) staining which 18

16 Cfiapter~ll.Am: shows strong diffuse positivity. NSE stains are useful in differentiating M2 from M4. Some cases of Ml, M5a, M7 and L2 are morphologically similar. In such cases, cytochemical stains can provide an inexpensive and available diagnostic tool. M l is positive for SBB and MPO, M5a is usually NSE positive, whereas SBB and MPO are negative, M7 usually is ANA esterase, PAS and AP reactive and do not stain with SBB, MPO and ANB esterases (Elghetany et al, 1990). Twenty five percent (25%) of pediatric ALL show NSE positivity but it is focal and weak. Table 2.3: FAB Classification of ALL Morphologic al Features Subtypes of ALL LI L2 L3 Cell size Small Large Large Nuclear chromatin Fine or clumped Fine Fine Nuclear shape Regular, may have cleft or indentation Irregular, may have cleft or indentation Regular, oval to round Nocleoli Indistinct or not visible 1 or more/cell; large, prominent 1 or more/cell; large, prominent Amottiit of cytoplasm Scanty Moderately abundant Moderately abundant Cytoplasmic basophilia Cytoplasmic vacuoles Slight Slight Prominent Variable Variable Prominent PAS stain + ± + 19

17 Cfiapter-11 ncet/rbw o ;r L i T E ^ r m ^ E t 9 Table 2.4: FAB Classification of AML Subtypes of AML Morphological Features Auer Mods MPO/ SBB MO (AML minimally differentiated) >30% myeloblasts without granules - - Ml (AML without maturation) M2 (AML witli maturation) M3 (Acute promyelocytic leukemia) M4 (Acute myelomonocytic leukemia) M5 i (Acute monoblastic leukemia without differentiation) M5b (Acute monoblastic leukemia with differentiation) M6 (Acute etythroleukemia) M7 (Acute megakarjfocytic leukemia) >30% myeloblasts, with or without granules. <10% show maturation beyond blast stage. >30% myeloblasts with granule >10% promyelocytes or mature cells; < 20% monocytic cells >30% myeloblasts and promyelocytes with prominent granules Myeloblasts, monoblasts & promyelocytes >30% marrow cells: moncytic cells >20% >80% monocytic cells; >80% monoblasts >80% monocytic cells with monblast, promoncytes (predominant), monocytes, Megaloblastic erythroid precursors >50%; myeloblasts >30% Megakaryoblasts, lymphoid morphology (L1,L2,M1), cytoplasmic budding ± ±

18 C hapter-ii * / «>* B lasts - sm all C ytoplasm scan t, sligh tly or m oderately b asop h ilic N / C ratio - h igh N u clea r outline - regular N u c le o li - poorly visib le C hrom atin - h o m o g en o u s B lasts - larger, p leom orphic C ytoplasm - abundant w ith variable basophilia. N / C ratio - low N u clea r ou tlin e - irregular N u c le o li - clearly visib le C hrom atin - h eterogen ou s varying from finely reticular to co n d en sed B lasts - large, m on om orp h ic C ytoplasm - abundant, in ten sely b asop h ilic N / C ratio - low N u c le u s - oval to round w ith cytop lasm ic vacuolation N u c le o li - prom inent (1 or >1) C hrom atin - d en se but finely stip p led m ore nucleoli Figure 2.2: FAB subtypes o f ALL (L I, L2 and L3) 21

19 C H apter-ii B lasts - large, o cca sio n a lly resem b le L2 C ytop lasm - agranular N u c le u s - o p en ch rom atin N u c le o li - p rom in en t (1 or m ore) C ytoch em istry - uniform ly n egative B lasts - m ed iu m to large C ytop lasm - gran u les + / - N u c le u s - ro u n d /o v a l N u c le o li - 1 or m ore N o gra n u lo cy tic d ifferen tiation B lasts - large C ytop lasm - granular A bnorm al m atu rin g gran u locyte precursors (p rom yelocytes to neu trop h ils) A uer rods + M P O + A bnorm al P rom yelocytes C ytop lasm - H ea v y gran u lation B u n d les o f A uer rods F igu re 2.3a; FA B subtypes o f A M L (M 0-M 3) 22

20 C fiapter-ii O T L ir% < S J\n X )<R ^ ^gt^lbw-w-i '*) _, A M L M 4 B M g r a n u lo c y te s (m y e lo b la sts to n eu tro p h ils) > 20 Vo o f N E C B M m o n o c y tic p recu ro so rs (m o n o b la sts to m o n o c y te s) > 20% o f N E C a n d /o r P B m o n o c y tic c o m p o n e n t > 5 X 10 / I (1) M 5a (p oorly d ifferen tiated ): B la st - large C y to p la sm - a b u n d a n t, rare a zu ro p h ilic g ra n u les N u c le u s - rou n d to oval C h rom atin - d e lica te & la cy (2) M 5b (w ell-d ifferen tia ted ): C y to p la sm - g rey -b lu e N u c le u s - tw iste d or fo ld ed A M L -M 5 (b) E ry th ro b la sts >50% B M - N E B la sts >30% D y sery th r o p o iesis T rilin ea g e d y sp la sia E ry th ro b la sts PA S + M P O F o c a l + G ly co p h o rin -A A b + B la sts - P o ly m o rp h ic N / C - M o d era te C h ro m * a tin - d e n se /r e tic u la r C y to p la sm ic b u d d in g A b n o rm a l p la telets IC C - C D 41, C D 4 2, C D 61, F a cto r V lll-r e la te d A g + E M - P P O + AML F ig u re 2.3 b : F A B subtypes o f A M L (M 4-M 7) 23

21 Cfiapter-ll 09^ Immunophenotyping in Acute Leukemia The FAB classification, which utilizes the morphological and cytochemical criteria usefully, identifies lymphoblastic and myeloblastic leukemia but has some limitations. Since the surface marker characteristics improve the diagnostic specificity the present consensus is that diagnosis of leukemias should be based on morphology, cytochemistry and immunophenotyping. The goal of immunophenotyping is the identification and phenotypic characterization of blast cells. It has been reported that the concordance between experienced observers in the classification of acute leukemia increases from about 70% to greater than 90% when morphologic criteria are supplemented with cytochemical and immunophenotypic data {Kinney et al., 1998). Advances in flow cytometry technology and availability of commercially produced monoclonal antibodies directed against hematopoietic and lymphoid cell antigens have opened new horizons for the diagnosis and classification of acute leukemias (Krause et al, 1988). These antibodies recognize cell surface and cytoplasmic antigen expressed on lymphoid and myeloid cells and their leukemic counterparts. Antibodies are assigned cluster designations (CD) by International Workshops on Leukocyte Differentiation Antigens. List of Relevant CD markers used for immunophenotyping of acute leukemias shown in Table 2.5. In order to establish the lineage of blast cells, immunophenotypic panel must include antigens with high sensitivity that are fully present in a certain lineage (e.g. CD7 in T-cells and CD 19 in B-cells) together with more specific markers (e.g. CD3 for T-cells, or CD33, MPO for AML). Additional progenitor cell markers such as CD34 are used to confirm the immaturity of the pathologic cells. List of most commonly used lineage specific CD markers for the immunophenotyping of AL is given in Table 2.6. The European Group for the Inununological Characterization of Leukemias (EGIL) has proposed an immunologic classification of AL that includes ALL, myeloid antigen positive ALL, AML, lymphoid antigen positive AML, Biphenotypic AL, and undifferentiated AL. 24

22 CftaptCT-Il Table 2^: List of relevant CD markers used for immunophenotyping of AL CD Markers CDla CD2 CD3 CDS CD7 CDIO CDllb CDllc CD13 CD14 CD19 CD20 CD22 CD33 CD34 CD38 CD45 Description and Function Cortical thymocytes, subpopulation of B^cells, dendritic cells MHC-like protein, can associate with p2-microglobulin T-cells, most NK cells Erythrocyte-rosette receptor Surface expression on mature T-cells, cytoplasmic Associated with the T cell receptor, mediates signal expression in immature T cells transduction Thymocytes, mature T cells, subpopulation of B cells Linked to T cell proliferation T-cells, NK-cells, subpopulation of immature myeloid cells 40 icda protein c-all, lymphatic precursor cells, neutrophils subset of mature B cells Common acute leukemia antigen (CALLA), neutral endopeptidase Monocytes, macrophages, neutrophils, NK-cells Adhesion molecule, C3bi receptor Monocytes, neutrophils, NK-cells, subpopulation of B-cells Adhesion molecule, gp 150/95 Myeloid cells Aminopeptidase N Monocytes and neutrophils LPS receptor Precursor B-cells, B-cells Bridge for surface immunoglobulin (Ig) signal Subpopulation of precursor B-cells, B-cells Ion channel, protein kinase C substrate Surface expression on B-cells, cytoplasmic expression in pre-b-cells, Related to neutral cell adhesion molecule, bridge for surface Ig signal Monocytes, myeloid precursor cells, weak expression on 67 KDa glycoprotein neutrophils Myeloid and lymphoid precursor cells KDa glycoprotein Activated lymphocytes, subpopulation of B-cells, plasma 45 KDa glycoprotein cells All leukocytes T200 antigen, protein-tyrosine-phosphatase CD68 Monocytes GP 110 CD79a CD117 Glycophorin A HLA-DR MPO TdT B-lymphocytes, including immature B-cells Ig-o/mbl, part of the B-cell antigen receptor Myeloid precursors cells C-kit, stem cell factor receptor Erythrocytes, erythroblasts and erythroid precursor cells Sialinic acidrich polypeptide B-lymphocytes, activated T-lymphocytes, monocytes. Part of the MHC n complex precursor cells Lysosomal expression in neutrophils and monocytes. Myeloperoxidase including immature myeloid cells Nuclear expression in lymphoid precursor cells Tdt 25

23 Cfmpter-11 o!f Table 2.6: Commonly used lineage specific CD markers for immunophenotyping C ell Lineage CD M arkers B-cell T-cell C D 19, C D 20, CD21, CD22, C D 23, C D 24 C D l, C D 2, CD3, CD4, CDS, CD7, CD8 Lym phoid TdT M yeloid C D 13, C D 33, C D llb, CD15 M onocytic C D 14, C D llb E rythroid G lycop h orin A M egakaryocytic C D 41, C D 42b, CD61 Lineage Independent M arkers (A ntigens) HLA class II HLA-DR L eukocyte Common Antigen(LC A) CD45 Stem C ell Antigen CD34 Common A LL Antigen (CALLA) CDIO 26

24 Cfmpter-ll (A) Immunophenotyping in ALL: Immunophenotyping of blasts has become indispensable in the diagnosis of ALL (Khalidi et al, 1999). Specific phenotypic characterization of ALL blast cells is usually performed using lineage associated maricers such as CD 19 (B-cells) and CD7 (T-cells). ALL is initially divided into B and T lineage with the B lineage further subdivided into B cell, pre B cell and early B-precursor types (Pui et al., 1993). The immunologic classification of ALL is presented in Table 2.7. The correlation of prognosis and choice of therapy with FAB classification alone is much less clear especially for LI and L2, as these include many different immunological subtypes of ALL. Thus, therapy and clinical outcome are better correlated with immunophenotype than morphology alone (Copelan et al., 1995). Childhood precursor B-ALL (CALLA+) do very well and require only standard or less intense therapy to minimise the risks associated with late complication. In adults the overall results are less favourable due in part to increased representation of t(9;22) in the B- precursor group (Crist et al., 1992). Mature B-ALL patients may require more aggressive therapy. T-ALL is less common but has a more favourable prognosis with new treatment regimens particularly in adults (Boucheix et a l, 1994). Table 2.7: Immmunologic Classification of ALL Category Early B-precursor ALL TdT HLA DR Cell Markers CD19 CD20 CDIO Cm* sig# CD7 CD Common ALL + ^ Pre-B-ALL f B-ALL ± ± Early T-precursor ALL T-ALL ± Cytoplasmic mu heavy chain # Surface Ig (with kappa or lambda light chain restriction) 27

25 Cfmpter-ll LITE ^W J^^L 1. B-ALL: B-Precursor ALL is an umbrella term that includes leukemias of immature B cells at different maturation stages often termed early pre-b cell ALL, pre-b cell ALL and pro B cell ALL. It accounts for 65-70% of ALL in infants and children, 55-60% in adolescents and 50% in adults. In children >90% are CDIO positive (+) whereas <50% are positive in infants (Pui et ai, 1993). Among the patients with B- ALL, CD 10+ cases were characterized by a high proportion of children between 1 and 9 years of age (Consolini et ai, 1998). These cells are typically LI or L2 by FAB criteria. Some cases may have very low or absent CD45. The phenotype is positive for TdT, HLA-DR and CD19 along with CD22+, CD34+, CD79a+ and sig negative (-). Two subgroups; CD 10+ with favourable prognosis and CDIO- with poor prognosis have been identified (Pui et ai, 1986). Infants around 1 year of age with ALL that is CD 19+, CDIO- and express myeloid antigen (CD15+) are likely to have a translocation involving llq 23 and a poor prognosis {Morgan et al., 1994; Tritz e ta l, 1995). Pre-B cell ALL phenotype occurs in about 85% of children with ALL. Cells are typically CD 19+, CD24+, HLA-DR+, Cy22+ and TdT±, CD20± and CD34- along with presence of cytoplasmic )i chain (cigm). The pre-b phenotype has been associated with a worse outcome than the early B-precursor phenotype (Pui et al, 1993). Mature B-cell ALL represents 2 to 5% of all and is equivalent to Burkitts lymphoma in leukemic phase. Absence of 34 is an independent prognostic marker in general in B-lineage ALL (Pui et al, 1993). B cell ALL cells have more forward and side scatter than B-precursw cells and may merge with lymphocytes and monocyte region on CD45 side scatter plots. They are L3 by the FAB criteria. The phenotype shows B-lineage antigens CD19, CD20, CD22 and CD24 with bright clonal sig most often IgM. Many cases are CD10+ but the mature antigens and sig distinguish it from earlier B-lineage ALL (Jennings et al, 1997). Rare patients have a mature B- cell acute leukemia without FAB-L3 morphology and they tend to have extensive bone marrow involvement at presentation and an aggressive course. Some patients have FAB-Ll morphology and coexistent t (1; 19) and t (14:18) (Hammami et al, 1991; Rowe et a l, 1996). 28

26 Cfmpter-11 0!F 2. T-A L L : T cell phenotype is presents in 25% of adults (Copelan et al, 1995) and 15% of childhood ALL {Pui et al, 1993). Many cases have significant forward and side scatter and may be in the lymphoblast, myeloblast or monocytic region on CD45 vs side scatter. Most cases demonstrate a thymic phenotype. Pre (pro) T-ALL show positivity for cycd3, CD7, without other T cell antigens and have a poor prognosis. A hallmark of all T-cell neoplasms is the tendency to drop (not display) specific normal T-cell antigens or display aberrant combination (rraiveeit et al., 1993). Adults with a T-cell phenotype tend to have a better outcome (Boucheix et al., 1994). In children, the T-cell phenotype is associated with older age, male gender, a mediastinal mass and CNS involvement. These children do less well than those with pre-b or early precursor phenotype (Shuster et a l, 1990; Crist et al., 1992; Pui et al, 1993). CDIO in T-ALL confers a better prognosis. However in another study prognostic significance of CDIO expression in childhood T-ALL not found (Shuster eta l, 1990; Pui e ta l, 1993). Myeloid associated antigens in ALL: Aberrant expression of myeloid associated antigens is generally considered a poor prognostic indicator, particularly in adult ALL (Pui et a l, 1993); Boldt et al 1994). The frequency of myeloid antigen expression in ALL has been reported from <1% to 54% in different studies (Sobol et al 1987; Traweek et al, 1993). A high frequency of myeloid antigen expression was found with 46% precursor B-cell ALL and 32.2% T-ALL cases expressing 1 or 2 myeloid markers. CD33 was the most frequent expressed myeloid antigen (25%) in the adult cases of ALL followed by CD 13 (23%) cases of ALL. The frequency of myeloid antigen expression was more frequent in L2 (57%) than LI (38%) cases (Khalidi et a l, 1999). However, higher firequency of CD13 (49%) and CD33 (40%) expression has also been reported (Lauria et a l, 1994). Almost half of pediatric cases (49%) expressed myeloid antigen, most frequently CD13 or CD33. The immunophenotypic differences between LI and L2 cases were not clinically significant and other studies showed no significant differences in achieving remission or in survival between patients with ALL with LI or L2 blasts (Reid et al, 1982). 29

27 Cfiapter-ll 0 7 (B) Immunophenotyping in AML: The diagnostic role of immunophenotyping in AML is widely accepted for the minimally differentiated acute myeloid leukemia (AML-MO) where cytochemistry is negative (Bernier et a l, 1995; Kotylo et al, 2000; Bene et a l, 2001). Immunophenotyping is also useful to diagnose other AML subtypes, such as the M6 and M7 and especially M3 (Rothe et a l, 1996; Dunphy et ai, 1999). Although immunophenotyping of AL plays a central role in the determination of clinically relevant subsets, cytogenetic markers are currently the best prognostic markers and immunophenotyping will not replace this modality, as the immunophenotypic pattern is not specific for each genetic marker. Inmiunophenotypic profiles of AML-MO were found to be characterized by an immature myeloid profile and frequently express HLA-DR, CD34 and nuclear TdT in combination with myeloid antigens CD33 and/or CD13. AML-MO blasts have low forward and side scatter and typically merge with lymphoblast region on CD45 vesus side scatter plots and there is an association between CD7 and CD34 expression and worse prognosis in AML (Ball et a l, 1991). Flow appearance of M l is similar to MO and blasts are usually CD13+, CD33+ and HLA-DR+ with partial CD15 expression (Venditti et al, 1997). hi AML-M2, there is a spectrum of cells with varying degrees of light scatter. CD45 and SS can show a continuum of cells from myeloblast region to the maturing myeloid cell regions. CD34 is less prominent (Jennings et al, 1997). Expression of CD19, and less often CD56 m cases of M2 is associated with presence of t(8;21) a favorable prognostic marker in adults (Nucifora et a l, 1995). AML-M3 shows abundant side scatter despite reduced CD45 compared with mature cells and absent HLA-DR in most cases. CD13 and CD33 are generally present; CD34 is less prominent (Rovelli et a l, 1992). The presence of RAR a rearrangements in M3 predicts response to All-trans retinoic acid (ATRA). Complication of ATRA therapy is fatal retinoic acid syndrome, which has been correlated with the expression of CD13 in the pretreatment leukemia population (Warrell et al, 1994; Degos et al, 1995). AML-M4 and M5 cells have more forward and side scatter than MO and ML Phenotypic features are presence of CD 13, CD33, HLA-DR, CD14, and CD15 (Venditti et a l, 1997). Combination of CD33 positivity with negative CD13 and CD34 is highly correlated with an M5 phenotype (Reading 30

28 Cfmpter-Il 0 7 L IT H R ^ T U ^ ^ et al, 1993). AML-M6 shows HLA-DR, CD34 and possibly CD 13 or CD33 are usually present. CD45 side scatter shows a prominent erythroid component (Cuneo et al, 1990). AML-M6 demonstrates a stem cell pattern with TdT and CD34 positivity with CD 13 or CD33. A poor outcome was reported in these cases compared with other AML FAB subtypes (Praxedes et a l, 1994; Venditti et al, 1997; Cohen et a l, 1998). AML-M7 accounts for less than 1% of AML and diagnosed when >30% of nonerythroid cells are megakaryoblasts (confirmed by ultrastuctural demonstration of platelet peroxidase or by immunophenotyping). The prognostic importance of myeloid monoclonal antibodies may be limited to CDl lb; implying poor prognosis and increase in CD34+ blasts, decrease likelihood of complete remission and disease free survival (Bradstock et a l, 1994; Paietta et al, 1998). Furthermore, low response rate has been shown to be associated with CD13 and CD14 (Griffin et a l, 1986) while high response rate has been reported in CD34- and in CD 15+ cases (Campos et al, 1989). In first relapse in AML patients, CD34 expression has been correlated with a lower response rate and shorter survival whereas CD15 was associated with longer survival (Thomas et a l, 1992). Lymphoid associated antigens In AML: The incidence of lymphoid-associated antigen expression in AML is highly variable, ranging from 10%-60% (Drexler et al, 1993; Traweek et a l, 1993; Khalidi et al, 1998). CD7 is most frequently observed lymphoid associated antigen in AML (ll%-29%). There have been reports of the prognostic significance of surface markers in AML. However, the significance of lymphoid associated antigen expression is controversial. Some studies have found no clinical significance to this finding in patients with AML (Bradstock et al, 1994). In some studies, CD7 has been shovra to be an adverse prognostic factor whereas others have not been able to confirm this finding (La Russa et a l, 1992; Del poeta et al, 1993; Kita et a l, 1993; Lauria et al, 1994). Similarly, in some studies, CD2 and CD 19 expression in AML has been reported as a favorable prognostic indicator (Ball et al, 1991) whereas others have found that CD2 and CD 19 expression was a poor prognostic indicator (Cross et al, 1988; Salary et al, 1992). In childhood AML, it has been reported that expression of lymphoid associated antigens lacks prognostic significance (Smith et a l, 1992). 31