JNMA (142) Final - Journal of Nepal Medical Association

ORIGINAL ARTICLE Journal of Nepal Medical Association, 2002:41:306-310EFFECT OF CHEMOTHERAPY ON PHILADELPHIA CHROMOSOMEIN CHRONIC MYELOID LEUKEMIA (CML) PATIENTSJha C B 1 , Kucheria K 2 , Chaudhary V P 2ABSTRACTChronic myeloid leukemia (CML) is a clonal myeloproliferative disorder as a resultof neoplastic transformation of the primitive hemopoetic cells. It is well known thatthe Philadelphia chormosome (ph) is a specific abnormality found in 90% of CMLpatients. It has been reported that interferon has better effect on disease control andprognosis. Cytogenetic analysis of ph chromosome plays very important role in theprognosis and monitoring of therapy. In this present study 35 diagnosed patients ofCML were considered, which included untreated patients of various age groups (2-62yrs). The cases were refered from haematology clinic of All India Institute of MedicalSciences (AIIMS). Out of 35 patients only 13 patients were available after six monthof therapy for follow-up cytogenetic analysis. Out of 13 patient, 2 were ph negative, 8were 100% ph positive and 3 were ph positive mosaic before therapy. Of the 3 mosaicpatients, 2 remained unchanged after therapy and one patient became 100% phnegative. Though in general significant reduction in ph% by interferon therapy wasseen but minority patients showed complete cytogenetic remission.Key Words: Chronic myeloid leukemia, chemotherapy, Philadelphia chromosome.INTRODUCTIONChronic myeloid leukemia is a clonalmyeloproliferative disorder as a result of neoplastictransformation of the primitive haemopoietic stemcell. It accounts for about 7-15% of all leukemias. 1Males are more commonly affected than female (14:1). 2The Philadelphia (ph) chromosome in themalignant cells is found in more than 90% ofpatients with CML. Nowell and Hungerford 3reported a very small chromosome that appearedto be a partially deleted copy of one of the twosmallest autosome pairs. This small elementbecame known as the Philadelphia chromosome. 41. B. P. Koirala Institute of Health Sciences, Dharan, Nepal.2. All India Institute of Medical Sciences.Address for correspondence : Dr. Chandra Bhushan JhaAssistant ProfessorB.P.K.I.H.S., Dharan, Nepal.Email: chandraj_2000@yahoo.comJNMA, April - June, 2002, 41

307 Jha et. al. : Effect of Chemotherapy on Philadelphia Chromosome ...The genetics of CML plays very important role inthe prognosis of patient's condition and for themonitoring of therapy, which are consistentlyassociated with chromosome abnormality.Cytogenetic molecular analysis of the Philadelphiachromosome provide important information to thephysician that are relevant to the leukemic burdenof the patients.It was planned to study chromosome in clinicallydiagnosed CML patients before and onchemotherapy.MATERIAL AND METHODSThirty- five diagnosed cases of chronic myeloidleukemia were considered for this study, whichincluded untreated patients of various age groups(2-62years). The diagnosis of CML was made onthe basis of hematologic investigations, and caseswere refered from hematology clinic of AIIMS.SAMPLE COLLECTIONBone marrow (BM) aspiration was done by thehaematologist from the posterior superior iliacspine using heparinised syringe. 1ml of aspiratewas collected in a sterile tube containing 5 ml ofRPMI 1640 (Gibco, BRL) culture mediasupplemented with 10-units/ ml of heparin.Samples were transported at room temperature.BONE MARROW CULTUREThe bone marrow aspirate was centrifuged (1000r.p.m; l0 minutes) and washed with RPMI 1640 mediain the same tube in which it was transported. Afterdiscarding the supernatant, the pallet wassupplemented in 5ml of RPMI 1640 reconstituted with20% fetal bovine serum (Gibco, BRL). Two vials wereset up for direct harvesting and for short-term culturefor 24 hours in Co 2incubator at 37° centigrade.CHROMOSOME PREPARATIONHarvesting for bone marrow was done usingcolcemid (Gibco, BRL) treatment for 1 hour at aconcentration of 0.2/ ml. The culture was centrifugedand the cell pallet was suspended in 10ml ofhypotonic solution (0.075m pottassium chloride(KCL) for 30 minutes at 37° c. The suspension wasagain centrifuged and the cells were then fixed inmethanol acetic acid fixative 3:1 for over night at-20°c. Precleaned slides were used for chromosomepreparations. The cell suspension was dropped froman approximate distance of 40-50 cm on the slideplaced at 30°. Conventional staining was done in4% Giemsa (Gibco, BRL) solution.GTG- BANDINGOn slide stored for 2-4 days at room temperature,GTG- banding was done using modified method ofseabright5. Slide was then washed in normal salineand stained with 2% Giemsa (Gibco, BRL) stain.METAPHASE SCREENINGThe preparation was screened under X 10 objectiveusing zeiss light microscope. Well spreadmetaphases were further analysed under xl00 oilimmersion objective. Minimum of 30 well spreadmetaphases were analysed. In samples showingabsence or mosaicism for the Philadelphiachromosome 40-50 metaphases were screened. Thiswas done to detect presence of few malignant cellsand to ascertain the percentage of ph positive cellsrespectively. Two to five spread metaphases werephotographed and karyotyped.MICROPHOTOGRAPHY AND KARYOTYPINGMicrophotography was done under xl00 oilimmersion using automatic exposure system of earlzeiss photo-micrographic equipment. The exposedJNMA, April - June, 2002, 41

Jha et. al. : Effect of Chemotherapy on Philadelphia Chromosome ... 308film was developed with kodak's developer at 20°cusing standard method. Prints were developed inFig. 1standard print developer followed by fixation andwashing. Karyotype analysis was done by cuttingindividual chromosomes from photographs ofmetaphase spreads (Plate land 2). The homologouspairs were arranged according to internationalsystem for cytogenetic nomenclature6on a predesignedformat.RESULTSAt the time of diagnosis, the cytogenetic analysiswas done in all the cases and treatment stared withhydroxyurea(HU) or HU and interferon(IFN)combination chemotherapy (from 15 days- 2yrsduration). The following results were obtained.CYTOGENETIC ANALYSISFig.1. G - banded metaphase spread showing t (9;22)(q34, q11). Arrows indicate 9q+and 22q.All 35 patients were available for cytogeneticanalysis at time of diagnosis, out of 35 patients, 17Fig. 2Fig.2. G - banded karyotype showing t (9;22) (q34, q11). Arrows indicate der (9) and der (22).JNMA, April - June, 2002, 41

309 Jha et. al. : Effect of Chemotherapy on Philadelphia Chromosome ...were 100% ph positive (49.9%), 10 were 50-90%ph positive mosaicism (28.5%) and 3 were 100%ph negative (8.5%). In 14.25% of patients thecytogenetic analysis was failure at the time ofdiagnosis. 49.9% patients showed standard phtranslocation9; 22 (q 34 ; q 11 ).Out of 35 patients, only 13 were available for followupcytogenetic analysis after 6 months. At the timeof diagnosis among these 13 patients, 8 were 100%ph positive, 3 were 50-90% ph positive mosaic and2 were 100% ph negative. Two of the mosaic patientsremained unchanged and one became 100% phpositive. Of the eight 100% ph positive patients, 5became ph mosaic with ph positivity (50-70%) and2 patients remained unchanged, while one patientsbecome 100% ph negative in combinationchemotherapy (IFN and HU).Cytogentic results showed better response withcombination chemotherapy (IFN+HU). Withcytogenetics results, total Leucocyte count (TLC)reduced and hemoglobin increased significantlyafter 3 months of therapy. There was significantchanges at 3rd and 6th months in haematologicalparameters (Fig.3). The platelet count was notFig. 3 : Comparision of pre and On therapyanalysis of peripheral blood samples1494-110.41.4 2.111.60.2 2.4 11.1changed significantly after commencement oftherapy, where as the liver and spleen were foundreduced in size (Fig.4).0.2Pretherapy 3rd M onth 6th M onthHemoglobin TLC (in lac.) Platelet (in lac.)2.2Fig. 4 : Comparision of Liver and SpleenSize-pre and on Therapy1210864201.7DISCUSSION10.21.63.41.33.7Pretherapy 3rd Month 6th MonthLiverSpleenIn the present study, out of 35 patients, only 13patients were available for follow up cytogeneticanalysis after 6 months. The cytogenetic hallmarkof CML, the truncated chromosome No. 22 (phchromosome) which is found due to translocationt (9:22) (q 34 ; q 11 ) in 90-95% cases of CML. Therehave been various reports showing completedisappearance of ph chromosome following therapywith Busulfan or Hydroxyurea accompanied withprolonged haematologic remission. In this study,out of 13 patients 2 were ph negative, 8 were 100%ph positive and 3 were ph positive mosaic beforetherapy, of the 3 mosaic patients, 2 remainedunchanged after therapy and one patients became100% ph positive. Out of eight 100% ph positivepatients, 5 became mosaic with ph positivity (50-70%) and 2 patients remained unchanged, whileone patients became 100% ph negative. Mosaicismin these patients may be transient and induced bytreatment, m studying the ph status it has beendemonstrated the uncomplicated ph in patientslymphnodes stimulated with epstein - Ban- virus.However, ph status was not studied in lymph nodes.Further, a ph reduction was considered good riskJNMA, April - June, 2002, 41

Jha et. al. : Effect of Chemotherapy on Philadelphia Chromosome ... 310factor. 7 Thus, a study has shown a 30-50% phreduction. A minority of patients with CMLachieves a complete cytogenetic remission definedas disappearance of ph chromosome. 8 In the presentstudy one patients has shown completedisappearance of ph chromosome.REFERENCES1. Morrison VA. Chronic Leukemias. CA Cancer J din1994; 44:353-377.2. Hughes TP, Goldman JM. Chronic myeloidleukemia In : Hoffman R, Benz EJ, Jr. Shattil SJ, etal ed. Hematology. Basic principles and practice.New York. Churchil livingstone; 1991: 854-869.3. Nowell PC, Hungerford DA. A minute chromosomein human chronic granulocytic leukemia. Science1960; 132:1497.4. Baikie AG, Court Brown WM, Buckton K.E et al. Apossible specific chromosome abnormality inhuman chronic myeloid leukemia. Nature 1960;188:1165.5. Seabright MA. A rapid banding technique forhuman chromosomes. Lancet 1971:2:971.6. Hamden DG, Klinger HP In. ISCN. An internationalsystem for human cytogenetic nomenclature ed.New York: Karger, 1985.7. Kloki 0, Wandi U, Opalka B et al. A prospectiverandomized comparison of single agent interferonalpha with the combination of IFN-alpha and lowdose 1FN- gamma in chronic myelogenousleukemia - Eur J Haematol 1992; 48:93-98.8. Hochhaus A, Lin F, Reiter A et al. Variable numbersof BCR-ABL transcripts persist in CML patientswho achieve complete cytogenetic remission withinterferon alpha. Brit J Haem 1995; 91: 126.!"!"!NMA wishesto express sincere gratitude to"Mathura Ratna Trust"(Founded by Late Dr. Raghubar Baidya and later augmented byLate Dr. Bharat Raj Baidya in memory of their motherMathura Devi and father Dr. Ratna Das Baidya)for its contribution to publishthis JournalJNMA, April - June, 2002, 41