CYTOGENETICS FINDINGS OF PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA IN WEST INDIAN REGION

Pankaj Gadhia 1 , Nitisha Parekh 1 , Pratik Chavda 1 , * Garima Bhatia 2 and Salil Vaniawala 1 . 1. Cytogenetic Unit, S. N. Gene Laboratory and Research Centre, President Plaza-A, Ring Road, Surat-395001. 2. M.Sc. student from School of Biotechnology and Bioinformatics, D. Y. Patil deemed to be University, New Mumbai-400614.a ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in India. Detection of chromosomal abnormalities by conventional karyotyping and Fluorescent in situ hybridization (FISH) plays a very important role in classification and treatment of ALL patients. The aim of this study was to define the frequency and types of chromosomal aberrations in B-ALL patients of West India. In this study, we analyzed bone marrow samples of 35 (18 adults and 17 childhood) West Indian patients with B-ALL using the conventional Gbanding and FISH technique. Total 24 patients (68.57%) showed chromosomal alterations, including numerical and/or structural abnormalities. Hyperdiploidy was most common (11.43%) numerical abnormality while the most common fusion oncogenes were BCR-ABL1/t(9;22) in adults (33.33%) and ETV-RUNX1/t (12;21) in children (23.53%). The incidence of 11q23 and t (1;19) were also high than those reported in the previous studies. There was a difference in the prevalence of abnormalities in the current study, maybe due to the geographical variation and/or small sample size.

Introduction:-
Acute lymphoblastic leukemia (ALL) is a clonal proliferation of lymphoid precursor cells in the bone marrow. ALL is the most common malignancy diagnosed in children, accounting for one-fourth of all childhood cancers. In India, the incidence of childhood cancer ranges from 38 to 124 per million children per year, where 60 to 85% of all leukemia reported are ALL (Arora et al., 2009).
ALL is a genetic disease because most patients harbor acquired genetic mutations that results in increased proliferation, prolonged survival and/or impaired differentiation of lymphoid progenitor cells. In majority of the patients with ALL the genetic alterations are present in the form of numerical and/or structural chromosomal aberrations (Mrozek et al., 2009). The chromosomal aberrations play a major role in the prognosis and treatment outcome of ALL (Sabir et al., 2012), hence detection of these abnormalities is necessary. The detection can be done by conventional cytogenetic method like karyotyping and molecular cytogenetic techniques like Fluorescent in-situ hybridization (FISH). These methods are helpful in assessing the classification, risk stratification and prediction of outcome of ALL patients (Goud et al., 2015).

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Conventional G-banding is commonly used to detect chromosomal aberrations (Chang et al., 2006). However, this method cannot be used for identification of certain translocations like t(12;21)/ ETV-RUNX1, which requires FISH for detection (Spathas et al., 1999). Hence, chromosomal analysis with FISH is more useful in diagnosis of certain cryptic rearrangements (Nordgren et al., 2002). In this study interphase-FISH was used as a complementary method along with G-banding for some ALL patients to confirm cytogenetic findings and for the poor quality samples or slides with no metaphases. The combination of both the method can improve the detection of genetic abnormalities (Goud et al., 2015).
The distribution of subtypes of ALL varies among population, maybe due to racial, geographical and genetic variations (Perez-Vera et al., 2001). The prevalence of ALL is more in male to that of the female and the male to female ratio in most resource rich countries is around 1. In the current study, we present the cytogenetic findings of 35 B-ALL patients and compare the distribution of numerical abnormalities and four most commonly occurring fusion oncogenes between children and adults. The aim of this study is to define the frequency and types of chromosomal translocations and fusion oncogenes in ALL patients using G-banding and FISH technique and compare our findings with the data reported in the previous studies.

Materials and methods:-
Patients: 35 patients of both gender, aged between 5 months and 76 years, suspected of B-lineage ALL, were included in this study. The study was conducted between 2015 and 2017 and patients were analyzed by conventional cytogenetic and molecular analysis technique in our centre.

Cytogenetic analysis:
The bone marrow samples were cultured in 5ml of MarrowMax Media (Gibco-Invitrogen-USA) at 37°C for 12-14 hours. After incubation, the cells were treated with 200µl of colchicine (Sigma-Aldrich, Germany) for 1 hour to arrest the cells at metaphase stage followed by harvesting the cells with hypotonic solution (0.075 M KCL) at 37°C for 20 minutes. Then, the cells were fixed using 3-4ml of Carnoy's fixative (methanol: glacial acetic acid 3:1) at 4°C for 2 hours. Fixed cell suspension was dropped over pre-chilled slides and slides were aged overnight at 64°C followed by staining using the standard G-banding technique (Seabright, 1971). At least 20 metaphases were analyzed using Zeiss Axio Imager Z2 microscope and image was captured using Ikaros karyotyping software (Metasystems, Germany). Chromosome identification and karyotype designation were made according to International System of Human Cytogenetic Nomenclature (ISCN, 2009).

Fluorescent in-situ hybridization (FISH) analysis:
In this method, the cells were directly harvested with hypotonic solution (0.075 M KCL) at 37°C for 20 minutes. It was followed by fixation and slide preparation similar to cytogenetic protocol. Then, 10µl of dual colour LSI/CEP probes (Vysis) were applied on the selected areas of slide and the slides were hybridized overnight at 37°C. After hybridization, the cells were counterstained with DAPI (4, 6-diamidino 2-phenylindole) and at least 100 interphase nuclei were examined under Zeiss Axio Imager Z2 with appropriate dual filters. The images were captured using the Isis Software (MetaSystems, Germany).

Discussion:-
In the present study, we determine the frequency and types of chromosomal aberrations in 35 patients (17 children and 18 adults), suspected of having B-ALL and compare our findings with other relevant reports. We used conventional karyotyping and FISH techniques to detect the numerical abnormalities such as hypodiploidy and hyperdiploidy and the prevalence of four most common oncogenes i.e. BCR-ABL/t(9;22), ETV-RUNX1/t(12;21), TCF3-PBX1/t(1;19) and 11q23 rearrangement in the patients with B-ALL.
In this study, the bone marrow culture of 14.28% (5/35) cases yielded no metaphase or the quality of chromosome was too poor to be analyzed. This result is comparable to the findings in the earlier studies (Safaei et al., 2013). The incidence of normal karyotype in the previous studies is reported as 48.32% (Siddaiahgari et al., 2015) and 38.30% (Safaei et al., 2013) while it was 31.42% in our study maybe, due to the improvement in the culture techniques and introduction of interphase FISH technique, which led to higher detection of chromosomal abnormalities as in many other studies which used FISH (Goud et al., 2015).

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The study concludes that, there is difference in the prevalence of numerical abnormalities and the fusion genes in the studied population, which could be due to the geographical variations and/or other unknown factors. The larger sample size could throw better light in this respect.