January 1990, Volume 40, Issue 1

Original Article

CHROMOSOMAL PATTERN IN ACUTE LYMPHOBLASTIC LEUKAEMIA

Syed Iftikhar Ahmed Abdi  ( Armed Forces Institute of Pathology, Rawalpindi. )
Muhammad Saleem  ( Armed Forces Institute of Pathology, Rawalpindi. )
Manzoor Ahmad  ( Armed Forces Institute of Pathology, Rawalpindi. )

Abstract

Analysis of the chromosomal changes in various neoplasia is being increasingly carried out not only to evaluate its relationship with the prognosis and biological behaviour of the tumour but also for diagnostic purposes in some cases. Leukaemias are one such group of haematological malignancies which have been most extensively studied in this regard. Karyotypic analysis with Giemsa banding techniquewas carried out in 35 consecutive cases of Acute Lymphoblastic Leukaemia. Eighteen cases were in children (less than 15 years age) and 17 cases were in adults. M.F. ratio was 1.8:1. FAB classification of these cases showed 31 cases of Li type and 4 cases of 12 type including one case of T-ALL. Fifteen cases (43%) had no karyotypic abnormality, 6 cases (17%) showed pseudodiploidy, one case each having (—20 + 21), t (9:22), (+ 2-6), (-6+8), while two cases had 6 q-.13 cases (37%) showed hyperdiploidy with 6 cases showing trisomy 8 alone, one case (+ 8+21), one case trisomy 18, one case +15(r), one case trisomy 21 plus t (9:22) and one case with trisomy 21 only. Two cases showed more complex abnormalities i.e. +2+8, t (13:22) and —11 + MR + Min + Min. There was one (3%) case of hypodiploidy showing monosomy 6. The above findings are in agreement with studies carried out in other countries except t (13:22) which is rather a scarcely reported abnormality (JPMA 40: 9, 1990).

INTRODUCTION

Cytogenetic analysis using banding techni­que has been increasingly applied to the study of malignant diseases in recent years. Among the malignant haematological disease Ch. myeloid leukaemia (CML) and acute non lymphohiaslic leukaemia have been most extensively investiga­ted. However within the last few years Ac lym­phoblastic leukaemia (ALL) cases have also been investigated for their chromosomal structure. Ka­neko1 made the following observations.
Clonal chromosomal abnormalitics are pre­sent in more than 60% cases of ALL. Hyper­diploidy is the predominant type of aneuploidy in these eases. Patients with certain abnormalities like Philadelphia chromosome, t (4: 11) and t (1: 19) have bad prognosis. Hyperdiploid cases have better prognosis thin those with other aneuploidy. We studied the clinical and karyotypic feature of 35 ALL cases, mostly belonging to northern parts of Pakistan including northern districts of Punjab and NWFP.

PATIENTS AND METHODS

The patients belonged to northern parts of Pakistan including some districts of North Punjab and NWFP. The patients were interviewed and examined clinically. Out of 35 cases 18 were <15 years and 17> 15 years of age. There were 12 females and 23 males niaking a female to male ratio of 1:1.8. The diagnosis of ALL was made on the basis of peripheral blood and bone marrow ex­amination after Romanawsky staining. The diag­nosis was further confirmed by cytochemical stains and by imniunophenotyping using a panel of monoclonal antibodies. The cases were classified according to the FAB criteria. A blood/bone marrow sample for cytogenetic analysis was ob­tained from all the 35 patients at the time of first presentation. The bone marrow cells were pro­cessed by direct/24 hours cell culture technique without phytohaemagglutinin. The peripheral blood cells were processed by 48 hours cell culture technique without PHA and 72 hours cell culture with PHA to compare between normal and abnor­ ma! cells. RPM! 1640 was used as culture medium to which 20% foetal calf serum, 2 mmol/1 L­glutamine, 25 mmol/l Hepes buffer, 100 units/l Penicillin G and 100 ug/I of Streptomycin was added. For 72 hours blood culture, PHA was added. Hepranazied bone marrow aspirate was taken from each patient under sterile c6nditions and added to two separate culture media bottles, one for direct cell culture and the other for 24 hours culture. 10 ml peripheral blood was drawn into a sterilized tube containing 90 usp heparin. 10-12 drops of blood from this sample were added to two separate bottles containing 10ml of medium, one having 0.1ml of PHA and the other without PHA. All the bottles were incubated at 37°C. The direct bone marrow culture bottles were taken out at the end of 4 hours and 2-3 drops of calcemoid (1 ug/ml) was added. After further period of incubation for 1 hour, whole material was transferred to a conical centrifuge tube and spun at 800 RPM in a swing out centrifuge. Supernatant was discarded without disturbing the bottom. 10 ml prewarmed Hanks balanced salt solution was added to the sediment and mixed gently, centrifuged for another 12 minutes at 800 RPM discarding the supernatant without disturbing the button, 10 ml KCI (0.75 M) was added to the pellet, incubated for further 10 minutes at 37°C. The material was mixed properly and centrifuged again at 800 RPM — supernatant being discarded. After 2—3 washes with fixative (3 parts methanol + 1 part Glacial acetic acid), the materialwas stored overnight at 4°C. Next morning the material was mixed with freshly prepared fixative and slides were made on ice chilled slides and air dried. After artificial ageing at 90°C the slides were trypsinised and Giemsa banding was carried out. The other bone marrow specimen was processed at the end of 24 hours while blood samples were processed at the end of 24 hours and 72 hours incubation. Harvesting and banding was carried out as described above. Slides were anal­ysed on TAS plus image analyser and by conven­tional methods. Photographs and kaiyogranis were made from each specimen. 20 metaphases were analysed from each sample.

RESULTS

Out of 35 cases, 31 cases were FAB type Li where as 4 cases were type L2. There was no L3 case.
Thirty two out of 35 cases had pallor whereas 17 cases had fever, 9 cases had bone pain and 8 cases had haemorrhagicmanifestations in the form of epistaxis and echymosis. Lymphadenopathy was found in 15 cases, splenomegaly in 14 cases where as hepatosplenomegaly was present in 12 cases.
Haemoglobin less than 10 gm/dl was found in 21 cases. Total leucocytes count varied from 5x109/l to 90 x 109/L. A count of more than lOx 10 9/I was found in 16 cases whereas 19 cases had a count of less than 10 x i09/i. Platelet count ranged between 5 x 10 9/I to 110 x 10 9/I. A count of less than 20x 10 9/l was found in 23 cases. Percentage of blast cells in peripheral blood varied between 15 to 90 percent. Less than 30% blast cells were found in 18 cases and more than 30% blast cells were found in 17 cases.
Percentage of blast cells in the bone marrow varied between 65 to 95%.
Cytogenetic Findings Fifteen (43%) cases showed no cytogenetic changes whereas 20 (57%) cases had clonal chro­mosomal changes. Six cases (17%) showed pseu­dodiploidy, one each having (-20 + 21) t (9:22), (+ 2-6), (-6 + 8) where as two cases had 6q-, 10 cases showed a number of abnormalities, most frequent being trisorny-8. Six cases showed 47 chromosomes whereas 3 Cases showed 48 chro­mosomes. These hyperdiploid cases showed a number of abnormalities, most frequently being trisomy 8. Six cases showed trisomy-8 as the only cytogenetic abnormality, and trisomy 8 in com­bination with other abnormalities was seen in 3 more cases. Remaining four cases with 47 chromo­somes showed trisomy 21 in two cases, trisomy 15 in one case and trisomy 18 in one case. Out of 3 cases with 48 chromosome as their modal number, one case had trisomy 2 and trisomy 8, with t (13:22). Trisomy8 and trisomy21 was found in another case whereas one case had complex cytogcnetic find­ings with one marker chromosome and two minute chromosomes alongwith monosomy-li.
Only one case showed hypodiploidy in the shape of 45 XY-6. Two cases showed classical Philadelphia chromosome having t (9:22) and one case had an uncommon Philadelphia chromosome with t (13:22) in that a portion of a q arm of chromosome 22 was translocated to q arm of chromosome 13 (see table for cytogenetic find­ings).

DISCUSSION

The importance of cytogenetic studies in neoplasia in general and in leukaemias in par­ticular has been universally accepted. Cytogenetic study in leukacmia is an important aid in confirm­ing the diagnosis, evaluating the prognosis and to monitor the progress of the case after therapy. Cytogenetic analysis may also be helpful in decid­ing the extent of therapy to be given to a particular patient2. Cytogenetic data in different malignan­cies including acute lymphoblastic leukaemia are available from different parts of the world; how­ever no such data is available from Pakistan. For the last two years cytogenetic studies in various types of leukaemia have been carried out at AFIP Rawalpindi to find out the chromosomal pattern in Acute Lymphoblastic Leukaemia in this region of the country.
In ALL there is remarkable heterogeneity in cytogenetic findings but certain findings are more frequently encountered than the others. In this study many cIassicaI findings like trisomy 8 + 21, monosomy 6 and t (9:22) have been observed but t (4:11), t (1:19) andEt (8:14) as reportedin literature have not been found. t (4:11) is found only in 4-5% cases of ALL mostly in infantile and congenital leukaemia3. Similarly t (1:19) is found in small percentage of infantile ALL4.
Translocation (8:14) is not very common in ALL. Rather it is found in B-cell malignancy5. Recently reported translocation t (10:14) is mostly found in T-ALL6. In this study we did not find these translocations because of the absence of congeni­tal/infantile cases. Similarly there were no cases of t (10:14) and t (8:14) because of the limited number of T-ALL cases and there being no case of L3. In this study 57% cases showed clonal chro­mosomal abnormalities. Twenty metaphases were analysed from each sample and if 5 or more cells had similar abnormality, it was termed as “Clonal” abnormality. Trisomy 8 was the most frequently noted abnormality. Although we have not eva­luated the prognostic aspect in relation to the cytogenetic changes in our cases but it has been well established byother studies that chromosomal changes have definite influence on the course of the disease3. Our study is closely comparable to other international studies except that the t (13:22) is a unique finding which has not been reported in available English language literature.
Although this study has to some extent defined the out-line of chromosomal pattern in ALL in this region a more extensive study is required in various types of Ac leukaemias to ascertain its significance in relation to various clinical patterns and response to various modes of therapy.

REFERENCES

1. Kaneko, Y., Rowley, J.D., Varia Kojis, D., Chilcote, R.R., Check, I. and Sakurai, M. Correlation of Kaiyotype with clinical features in acute lymphoblastic leukaemia. Cancer Res., 1982; 42:2918.
2. Bloomfled, C.D. and Arthur, C.D. Evaluation of leu­kaemic cell chromosomes as a guide of therapy. Blood Cells, 1982; 8:501.
3. Stark,B.,Tehila,U.,Zipora,M., Naomi, G.M., Cohen,J.I., Vogel, M.R. and Rina. Leukaernia of early infancy, early B-cell lineage associated with t (4:11). Cancer, 1985 ; 58: 1265.
4. Jack,I.S.,Seshadri,R.,Garson,M.,Michael,P.,CalIen,D., Zola, H. and Morley, A. RCH-ACV. A lymphoblastic leukaemia cell line with chromosome translocation 1:19 andTñsomy8. CancerGenet. Cytogenet., 1986; 19: 261.
5. Taub, R.I.,Morton, C, Lenoir, G.,Sawn,D.,Tronick,S.A. and Lcdcr, P. Translocation of the C-mycogene into the immunoglobulinheavychainlocusin human Burkitt Lym­phoma and Munne Plasmacytoma ccHs. Genetics, 1982; 79:7837.
6. Dubc, I.D., Baimondi, CS. and Kalousek, K. D. A new Iranslocation 1 (10-14) (q 24: 14), in T cell ncoplasia. Blood, 1986;67: 1181.

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