Ardeshir Ghavamzadeh MD**, Mina Izadyar MD***

Cytogenetic investigation on malignant tumors is a well-established procedure. In 1960, with the discovery of the Philadelphia chromosome in chronic myelogenous leukemia (CML) patients, Hungerford and Nowell showed that certain neoplasias could have specific chromosome rearrangements.

Progress in cytogenetic techniques such as banding², high resolution³ and molecular cytogenetics including fluorescent in situ hybridization (FISH)^4,5 have revolutionized the interpretation of chromosome rearrangements in neoplasia.

*Acute Myelogenous Leukemia, † Acute Lymphocytic Leukemia, ‡ Lymphoproliferative Disorders,

§ Chronic Myelogenous Leukemia, ║Myeloproliferative Disease

According to the Catalog of Chromosome Aberrations in Cancer⁵, chromosome rearrange- ments have been reported in more than 22,000 types of neoplasia.⁶ Some forms of leukemia have specific chromosome aberrations. For example, translocation 15/17 is specific for AML-M3, while inversion 16 is associated with AML-M4E. However, with progression of the disease, new clonal changes can occur and some of the cytogenetic findings are significant for the prognosis and outcome of the treatment. For example, in AML patients, inversion 16 and translocation 15/17 can have a long remission period and good prognosis but monosomy 5 and 7 are usually associated with poor prognosis in AML.^7-9

In this study, chromosomal investigation was carried out in 35 bone marrow and peripheral blood samples in leukemic patients and chromosomal findings were assessed in relation to diagnosis, stage and prognosis of the disease.

A total of 35 specimens (31 bone marrow and 4 peripheral blood) from patients with leukemia were received from the Genetic Department of Tehran University of Medical Sciences. The patients were referred from the Hematology and Oncology Sections of Shariati Hospital and the Children’s Medical Center, Tehran University of Medical Sciences, and were of five different types of leukemia (acute lymphocytic leukemia, AML; ALL, myeloproliferative disease, CML; MPD and Lymphoproliferative disorders; LPD). Bone marrow aspirate (1mL) was collected in 5mL sterile transport medium containing heparin and antibiotics. In the event of a dry tap and fibrosis, peripheral blood was collected.

Tissue culturing was carried out using standard protocols.¹ Four cultures including overnight clocemid, 24 h, 48 h and synchronized 24 h using FUdR/Uridine block were set up. The cell density used was 10⁶/mL. All cases were routinely evaluated by Giemsa banding (GTG). About 10-30 metaphase spreads were examined. An abnormal clone was defined as a minimum of two cells for structural or a gain in chromosome number and minimum of three cells for a chromosome loss. ISCN guidelines¹⁰ were followed for chromosome nomenclature.

The karyotype was obtained in 28 patients(80%). The number of abnormal karyotypes, subtypes of leukemia, and types of chromosomal abnormalities are shown in Table 1. As it is evident half of the patients with AML had karyotype abnormalities. All of the patients with LPD and more than three-fourth of patients with CML and ALL displayed some kind of karyotype abnormality.

Among the AML patients 5 cases were of AML-M3 of whom 3 had 15/17 translocations. One patient with AML-M2 had an abnormal karyotype containing four different cell lines: 47,XY, +8/47, XY, +13/48, XY, +8, +13/ 46, XY. This 68-year-old patient was diagnosed as MDS at presentation but consequently progressed to AML-M2 and died shortly after.

All patients in the LPD group had abnormal karyotypes. One case was referred as large cell lymphoma, in whom 30% of the metaphase spreads had deletion of the long arm of chromosome 7 at the q36 region. In another case, a 25 year old man, 52% of the cells contained double chromosome abnormalities including loss of chromosome Y and an isochromosome 7q. The last case with LPD was a 16-year-old boy referred as lymphoma. All his cells displayed 6q depletion.

In the CML group, 5 out of 6 patients had the Philadelphia translocation. A 15-year-old boy with clinical features of CML in this group had normal karyotype. One of the specimens was a bone marrow sample obtained from a 34-year-old woman with CML at blast stage. All the metaphase spreads in addition to standard Philadelphia translocation (t 9/22) contained a translocation between chromosomes 7 and 8. On the other hand, this patient contained only the Philadelphia translocation at presentation. The chromosomal changes confirmed the progression of the disease from chronic to blast stage. Another case was a 27-year-old man at CML blast stage. All the metaphase spreads contained the variant Philadelphia translocation involving chromosomes 4, 9 and 22. Two other patients had the standard q/22 Philadelphia translocation.

Finally, one patient was suspected for CML. However this patient was Philadelphia negative. As a result was classified, as MPD and bone marrow transplantation was not carried out.

This study, despite being a pilot inquiry, and in fact due to the small number of cases should be regarded as a case series, signifies the status of chromosomal investigation in the determination of diagnosis, stage and prognosis of disease in patients with leukemia. The cytogenetic findings can have valuable contribution in the management of patients with leukemia.

Regarding the AML cases, Overall, 70% of AML-M3 patients are reported to have 15/17 translocation.¹¹ Both trisomies 8 and 13 have been reported in AML and MDS. Patients with more than one chromosome aberration have poor prognosis.¹²

In the ALL cases, the figures are comparable to similar studies.⁵ Patients with hyperdiploid cells with 50-55 chromosomes are usually pre-B cell ALL and have favorable prognosis.¹³ Patients with dicentric translocation between chromosomes 9 and 12 are usually B-cell type ALL and have also a good prognosis.¹⁴

Five to ten percent of patients with chronic myeloproliferative disorders with clinical findings similar to CML, lack the Philadelphia chromosome but contain the hybrid between C-ab1 and C-bcr at the molecular level.¹⁷ Considering the patient with Philadelphia translocation at the presentation that progressed to blast stage, it is indicated that 75% to 80% of CML patients show new chromosomal changes with evolution of the disease. Such karyotypic changes can occur even six months prior to clinical manifestation of the disease and thus can be valuable in the management of the patients.¹⁸ Five to 10% of CML cases contain a variant Philadelphia translocation.¹⁹

Finally, patients like the one being Philadelphia negative and classified as MPD usually have better prognosis.⁹

It is recommended that extended studies with more number of patients be carried out in order to obtain a more clear view of the chromosomal abnormalities in leukemia in Iran.