The study of immunological markers is essential for the Correct diagnosis and classification of acute lymphoblastic leukaemia (ALL)1. Till the last decade, diagnosis of acute leukaemia was based primarily on the morphology and cytochemistry of leukaemic cells whereas now the technique of immunophenotyping can be applied to confirm the diagnosis as well as to further subcategorize the disease2. The discovery of monoclonal antibodies (McAbs) by Kohler and Milstein in 19753 has made it possible to define the precise stages of differentiation of haemopoietic Cells and resulted in tremendous advancement in the Classification of leukaemia4. Using cytological and cytochemical methods leukaemias designated as acute undifferentiated leukaemia, can now be appropriately classified by McAbs specific for lymphoid, myeloid, erythroid and megakaryocytic cells1,2,5,6. In addition to specificity, the sensitivity of the diagnostic tests has alsy been augmented by the use of McAbs. It is now possible to establish an earlier diagnosis of recurrence of leukaemia in the bone marrow or the detection of lymphoblasts in other sites such as CSF or testicular stroma2,7. ALL blast cells can be immunophenotyped into either B-lineage or T-lineage. B-lineage-ALL includes (I Early-B-ALL: Common ALL antigen (CALM) negative, previously called Null-ALL; (ii) Common-ALL: CALM positive; (iii) Pre-B-ALL cytoplasmic ‘mu’ chains present and (iv) Classical B-ALL: mature, surface immunoglobulin positive blasts. These immunotypes represent progressively maturing stages of B-lineage cells4. The significance of ALL immunotype as an inde pendent predictor of response to treatment has been reported. Common-ALL has the most favourable prognosis both in children and adults. T-ALL has a significantly poorer prognosis than non T-ALL excluding the classifical B-ALL which has the worst prognosis4,8-10. Therefore the precise characterization of leukaemic blasts is critical to establish an accurate prognosis and optimal therapeutical approach. It permits the use of more intensive treatment protocol in those patients who have been identified as having an unfavourable outcome at the time of diagnosis11. The present study was designed to identify the immunological markers on the lymphoblasts of ALL in order to see the frequency of various immunophenotypes of ALL in our country.
PATIENTS AND METHODS
Selection of patients:
This study includes 55 patients, 36 children (S 15 years) and 19 adults (> 15 years). All were newly diagnosed, untreated cases selected from Mayo Hospital and other government hospitals of Lahore. The diagnosis of ALL was made according to the French- American British (FAB) criteria by examining giemsa stained peripheral blood and bone marrow smears supplemented, by special stains which included myeloperoxidase, PAS and estrases12.
Direct bone marrow smears were obtained at diagnosis and airdried for 6 to 24 hours prior to immunolabelling. When required, the smears were stored at -20°C wrapped in aluminium foil. Following.
McAbs were used:
OKDr, OKBCALLA (CD1O), OKB7, OKT11 (CD2), OKT8 (CD8) and OKT4 (CD4) available from Ortho Diagnostics.
A three layer immunolabelling technique was employed. Airdried smears were fixed in acetone for 10 min. They were incubated first with the primary McAb (Mouse Ig), at adequate dilution, for 20 min. at room temperature. Sequentially the smears were incubated for 20 mm. with the linking antibody (goat-anti-mouse Ig) and then with the labelling antibody (peroxidase labelled mouse Ig). The chromogenic substrate used to reveal thc immunological reaction contained 3-amino 9-ethyl carbazol (AEC) and H2O2. The linking antibody, labelling antibody and chromogen system were available together in the Universal Immunoperoxidase Staining Kit (Ortho diagnostics). The smears were counterstained with Mayer’s haematoxylin and coverslipped with glycerol gel. Cases were considered positive when 20% or more of the blast cells were labelled with the specific McAb. Clinical and haematological parameters of all patients were registered at diagnosis including age, sex, lymphadenopathy, hepatomegaly, splenomegaly, Hb, WBC and platelet count.
A large number of studies have been Carried out on the immunophenotypes of ML in western COufl- tries8,10,13-18. In these reports Common-ALL represents the majority of cases in children, i.e., 70-80% and about 50% in adults; T-ALL constitutes 10-20% of ALL cases in children and adults; Early-B (Null) ML is more common in adults - upto 40% than Children - upto 15% and B-ALL is rare comprising less than 2% of childhood and less than 5% of adult cases. In the present study the frequency of ALL immunotypes in adults i.e., Common-ALL 52.6%, T-ALL 15.8% and Early-B-ALL 21.1% is in accordance with the above mentioned reports. The findings in children, on the other hand, are different. Frequency of T-ALL is much higher i.e., 36.1% vs 15% while that of Common-ALL is much lower i.e., 38.9% vs 75%. A high frequency of T-ALL (31%) has been reported in India and in Asian residing in U.K. 19. There maybe a common factor accountingfor this finding in India and Pakistan. A high frequency of T-ALL was also reported in the Gaza Strip of Israel20 and according to this study the pattern of lymphoid malignancy in children is very dependent upon the environment and relates particularly to socio-economic conditions. The frequency of T-ALL has been reported from the southern21 and northern22 parts of Pakistan in previous studies in which the diagnosis of T-ALL was based upon a strongly positive localized acid phosphatase reaction of the blast cells. The study carried out in Karachi21 on 50 children shows a high frequency of T-ALL i.e., 32% and our findings, conform to this study. A low frequency of T-ALL has been reported from Rawalpindi22, only 9.2% of the 65ALL cases being diagnosed as T-ALL These findings show a distinct variation between the northern areas and other parts of the country. Identification of cytoplasmic ‘mu’ chains was not carried out in this study, hence the Common-ALL group is inclusive of pre B-ALL which constitutes about 20% of Common-ALL cases23. Thirteen cases of ALL were subclassified for the thymic stage of maturation; of these 46.2% did not express either of the antigens identified by CD8 and CD4 McAbs and were included in early thymocyte stage; 23% expressed both these antigens simultaneously and were grouped in mid. thymocyte stage; remaining 30.8% expressed only one of these two antigens and were included in late thymocyte stage. A wider panel of anti-T-cell McAbs including CD5, CD3 and CD1 could not be employed, therefore, anomalous phenotypes which do not clearly fall into any one of the three categories of intrathymic maturation cannot be excluded. Previous studies show variable frequencies of T-ALL subclasses; being reported as 70% early, 20% mid and 10% late thymocyte stage in one study24 and as 33%, 37% and 30% respectively in another25. The immunophenotypes of ALL have a definite relationship with prognosis. Excluding the small percentage of B-ALL cases; Common-ALL has the best prognosis and T-ALL the worst5,8-11. T- ALL patients experience significantly shorter duration of complete clinical remission, a higher relapse rate and more frequent relapses in CNS8,14. In general the rank, order of favourable to unfavourable prognosis is: Common-ALL> Early-BALL> Pre-B-ALL> T-ALL> B-ALL5. The high frequency of T-ALL may be an important factor in the poor response of childhood ALL to usual therapeutic protocol in our country. It is important to identify the poor prognosis of T-ALL patients who may need modifications in their treatment. The finding of a high proportion of T-ALL immunotype in the present and previous studies in Pakistan may be related to an indigenous aetiological or predisposing factor - genetic and/or environmental. The present study highlights the need to carry out a more elaborate analysis of ALL immunotypes in our country by including a greater number of patients as well as their follow- up to confirm the prognostic significance of various immunophenotypes.
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