EFFECT OF CHEMOTHERAPY ON PHILADELPHIA CHROMOSOME IN CHRONIC MYELOID LEUKEMIA ( CML ) PATIENTS

1. B. P. Koirala Institute of Health Sciences, Dharan, Nepal. 2. All India Institute of Medical Sciences. Address for correspondence : Dr. Chandra Bhushan Jha Assistant Professor B.P.K.I.H.S., Dharan, Nepal. Email: chandraj_2000@yahoo.com


INTRODUCTION
Chronic myeloid leukemia is a clonal myeloproliferative disorder as a result of neoplastic transformation of the primitive haemopoietic stem cell.It accounts for about 7-15% of all leukemias. 1ales are more commonly affected than female (14:1). 2e Philadelphia (ph) chromosome in the malignant cells is found in more than 90% of patients with CML.Nowell and Hungerford 3 reported a very small chromosome that appeared to be a partially deleted copy of one of the two smallest autosome pairs.This small element became known as the Philadelphia chromosome. 4he genetics of CML plays very important role in the prognosis of patient's condition and for the monitoring of therapy, which are consistently associated with chromosome abnormality.Cytogenetic molecular analysis of the Philadelphia chromosome provide important information to the physician that are relevant to the leukemic burden of the patients.
It was planned to study chromosome in clinically diagnosed CML patients before and on chemotherapy.

MATERIAL AND METHODS
Thirty-five diagnosed cases of chronic myeloid leukemia were considered for this study, which included untreated patients of various age groups (2-62years).The diagnosis of CML was made on the basis of hematologic investigations, and cases were refered from hematology clinic of AIIMS.

SAMPLE COLLECTION
Bone marrow (BM) aspiration was done by the haematologist from the posterior superior iliac spine using heparinised syringe.1ml of aspirate was collected in a sterile tube containing 5 ml of RPMI 1640 (Gibco, BRL) culture media supplemented with 10-units/ ml of heparin.Samples were transported at room temperature.

BONE MARROW CULTURE
The bone marrow aspirate was centrifuged (1000 r.p.m; l0 minutes) and washed with RPMI 1640 media in the same tube in which it was transported.After discarding the supernatant, the pallet was supplemented in 5ml of RPMI 1640 reconstituted with 20% fetal bovine serum (Gibco, BRL).Two vials were set up for direct harvesting and for short-term culture for 24 hours in Co 2 incubator at 37° centigrade.

CHROMOSOME PREPARATION
Harvesting for bone marrow was done using colcemid (Gibco, BRL) treatment for 1 hour at a concentration of 0.2/ ml.The culture was centrifuged and the cell pallet was suspended in 10ml of hypotonic solution (0.075m pottassium chloride (KCL) for 30 minutes at 37° c.The suspension was again centrifuged and the cells were then fixed in methanol acetic acid fixative 3:1 for over night at -20°c.Precleaned slides were used for chromosome preparations.The cell suspension was dropped from an approximate distance of 40-50 cm on the slide placed at 30°.Conventional staining was done in 4% Giemsa (Gibco, BRL) solution.

GTG-BANDING
On slide stored for 2-4 days at room temperature, GTG-banding was done using modified method of seabright5.Slide was then washed in normal saline and stained with 2% Giemsa (Gibco, BRL) stain.

METAPHASE SCREENING
The preparation was screened under X 10 objective using zeiss light microscope.Well spread metaphases were further analysed under xl00 oil immersion objective.Minimum of 30 well spread metaphases were analysed.In samples showing absence or mosaicism for the Philadelphia chromosome 40-50 metaphases were screened.This was done to detect presence of few malignant cells and to ascertain the percentage of ph positive cells respectively.Two to five spread metaphases were photographed and karyotyped.

MICROPHOTOGRAPHY AND KARYOTYPING
Microphotography was done under xl00 oil immersion using automatic exposure system of earl zeiss photo-micrographic equipment.The exposed film was developed with kodak's developer at 20°c using standard method.Prints were developed in standard print developer followed by fixation and washing.Karyotype analysis was done by cutting individual chromosomes from photographs of metaphase spreads (Plate land 2).The homologous pairs were arranged according to international system for cytogenetic nomenclature6on a predesigned format.

RESULTS
At the time of diagnosis, the cytogenetic analysis was done in all the cases and treatment stared with hydroxyurea(HU) or HU and interferon(IFN) combination chemotherapy (from 15 days-2yrs duration).The following results were obtained.
Out of 35 patients, only 13 were available for followup cytogenetic analysis after 6 months.At the time of diagnosis among these 13 patients, 8 were 100% ph positive, 3 were 50-90% ph positive mosaic and 2 were 100% ph negative.Two of the mosaic patients remained unchanged and one became 100% ph positive.Of the eight 100% ph positive patients, 5 became ph mosaic with ph positivity (50-70%) and 2 patients remained unchanged, while one patients become 100% ph negative in combination chemotherapy (IFN and HU).
Cytogentic results showed better response with combination chemotherapy (IFN+HU).With cytogenetics results, total Leucocyte count (TLC) reduced and hemoglobin increased significantly after 3 months of therapy.There was significant changes at 3rd and 6th months in haematological parameters (Fig. 3).The platelet count was not changed significantly after commencement of therapy, where as the liver and spleen were found reduced in size (Fig. 4).

DISCUSSION
In the present study, out of 35 patients, only 13 patients were available for follow up cytogenetic analysis after 6 months.The cytogenetic hallmark of CML, the truncated chromosome No. 22 (ph chromosome) which is found due to translocation t (9:22) (q 34 ; q 11 ) in 90-95% cases of CML.factor. 7Thus, a study has shown a 30-50% ph reduction.A minority of patients with CML achieves a complete cytogenetic remission defined as disappearance of ph chromosome. 8In the present study one patients has shown complete disappearance of ph chromosome.

Fig. 3 :Fig. 4 :
Fig. 3 : Comparision of pre and On therapy analysis of peripheral blood samples