Cytogenetic Profile of De Novo B lineage Acute Lymphoblastic Leukemia: Determination of Frequency, Distribution Pattern and Identification of Rare and Novel Chromosomal Aberrations in Indian Patients.

BACKGROUND
Chromosomal aberrations identified in acute lymphoblastic leukemia (ALL) have an important role in disease diagnosis, prognosis and management. Information on karyotype and associated clinical parameters are essential to physicians for planning cancer control interventions in different geographical regions.


MATERIALS AND METHODS
In this study, we present the overall frequency and distribution patterns of chromosomal aberrations in both children and adult de novo B lineage ALL Indian patients using conventional cytogenetics, interphase FISH and multiplex RT-PCR.


RESULTS
Among the 215 subjects, cytogenetic results were achieved in 172 (80%) patients; normal karyotype represented 37.2% and abnormal 62.8% with a distribution as follows: 15.3% hypodiploidy; 10.3% hyperdiploidy; 15.8% t(9;22); 9.8% t(1;19); 3.7% t(12;21); 2.8% t(4;11); 2.8% complex karyotypes. Apart from these, we observed several novel, rare and common chromosomal rearrangements. Also, FISH studies using LSI extra-signal dual-color probes revealed additional structural or numerical changes.


CONCLUSIONS
These results demonstrate cytogenetic heterogeneity of ALL and confirm that the incidence of chromosomal abnormalities varies considerably. To the best of our knowledge, this is one of the largest reported series of cytogenetic investigations in Indian B-lineage ALL cases. In addition, ongoing cytogenetic studies are warranted in larger groups of B-lineage ALL cases to identify newly acquired chromosomal abnormalities that may contribute to disease diagnosis and management.

The frequencies of genetic aberrations differ distinctly in childhood and adult ALL. In childhood ALL, the impact of cytogenetic factors like specific translocations and DNA ploidy is well defined, while the prognostic significance of karyotype in adult ALL is less clear, partially as the disease is less frequent. Furthermore, the relative distribution of molecular subtypes in ALL is not uniform in different geographic regions. Thus, we examined the cytogenetic profile of newly diagnosed B-lineage ALL cases to determine the frequency and subtypes of cytogenetic aberrations from an Indian perspective, and their correlation with clinical parameters. An attempt was also made to compare the major aberrations (enlisted by WHO) in childhood and adult Indian ALL patients with Asian and global reports.

Patients
The present study included 215 de novo B-lineage ALL subjects referred from July 2011-October 2013 and were subjected to cytogenetic and/or molecular analysis of chromosomal rearrangements conducted at SRL Ltd., Mumbai, India. The study was in accordance with the declaration of Helsinki and approved by the Institutional ethics committee. Written consent was obtained from all the subjects.

Cytogenetic analysis
We karyotyped bone marrow aspirate or whole blood cells withdrawn at diagnosis as per standard unstimulated direct (0 h, 3 h) and short term (24 h, 48 h) cell culture technique previously described (Bhandari et al., 2014). In brief, cells were cultured in RPMI (Sigma, Schnelldorf, Germany) medium supplemented with 20% FBS (Gibco, Grand Island, NY) at 37°C. After incubation at respective time intervals, the cells were harvested i.e. cell-growth was arrested by exposure to colchicine (HiMedia, 4mg/ ml) for 45 minutes, then hypotonised by 0.075 M KCl for 20 minutes, then fixed with chilled Carnoy's fixative (methanol: glacial acetic acid 3:1). Fixed cells were dropped on chilled frosted slides, then aged overnight at 60°C and stained for GTG banding at resolution of about 400-band level. At least 20 metaphase plates were screened, and 4-5 well spread metaphases were photographed and karyotyped using Ikaros Software (MetaSystems GmbH, Germany). The karyotype was interpreted according to ISCN 2013(ISCN 2013 guidelines.

Fluorescence In Situ Hybridization (FISH) Analysis
FISH was performed on selected cases to refine complex or ill-defined rearrangements identified by chromosome analysis and to confirm recurrent /cryptic chromosomal aberrations such as BCR-ABL, ETV6-RUNX1, and MLL rearrangements, using LSI Vysis Probes (Abbott Molecular, Des Plaines, IL). The slides were hybridized overnight according to Vysis protocol and analyzed using Isis Software (MetaSystems GmbH, Germany).

Molecular Analysis for Detection of Gene Fusion Transcripts
To confirm /reveal recurrent or cryptic chromosomal rearrangements such as t(9;22)(q34;q11), t(1;19) (q23;p13), t(4;11)(q21;q23) and t(12;21)(p13;q22) in cases with apparently normal karyotype or with insufficient metaphases, multiplex nested reverse transcriptase PCR with minor modification (Pallisgaard et al., 1998) was performed. Briefly, fifty nanograms of total RNA extracted using QIAamp RNA blood mini kit (Qiagen, Hilden, Germany) used to prepare cDNA using random hexamer primer (Fermentas, Hanover, MD) and Sensiscript RT Kit (Qiagen). This was followed by two rounds of nested RT-PCR for detection of the fusion genes. Appropriate internal control was also analysed to check the integrity of RNA.

Statistical analysis
The χ 2 test and Fisher's exact test were used to compare the categorical data between adult and pediatric ALL patients. Z-test was performed to compare the frequencies of different abnormalities with available published reports of different countries. Two-side P-values <0.05 were considered statistically significant.
Although several molecular cytogenetic techniques allow detection of chromosomal aberrations, conventional karyotyping still is the gold standard as it provides a global overview of the genome and a baseline to trace the evolution of the disease. To the best of our knowledge, this is one of the largest reported series of cytogenetic investigation in Indian B-lineage ALL cases presenting prevalence of each cytogenetic subtype listed in WHO classification.

Distributions of chromosomal rearrangements
In the current study, we compared frequencies of major cytogenetic subtypes between different groups and our series for pediatric and adult ALL, respectively (Tables  4 and 5). Considering the potential ethnic background difference between Western and Indian populations, such kind of investigation is important for Indian ALL patients.
As seen in Tables 4 and 5, Hypodiploidy was the most common abnormality with a high frequency in our pediatric (15.4% vs 1-6.5%, all P<0.05) and adult (15.2% vs 1.9-3.9%, all P<0.05) series. Several reports from France (15.4%, 10%) show concordance with our adult results (GFCH, 1996;Braekeleer et al., 2010) (Table 5). Interestingly, Amare et al. From India reported further enhanced incidences that were two fold higher or even greater (44.4% in adults and 38.4% in pediatrics) . These findings specify the fact of relatively high frequency of hypodiploidy in India suggesting that geographical and ethnic factors cannot be excluded. The scientific significance of hypodiploidy in ALL is its association with various clinical outcomes. A progressively worse outcome is reported with decreasing chromosome number, with an approximate 6-year EFS chromosomal changes (Brigaudeau C and Bilhou-Nabera C, 1998). In the present study, we observed terminal deletion at 6q15 along with del(12)(p11.2); and interstitial deletion at 6q13q21as part of a complex karyotype. Deletion of chromosome arm 3p25/p26 and 9p/q is frequent in hematologic malignancies (Johansson et al., 1997). Furthermore, 9p abnormalities may hold significant negative impact on survival in adult BCP-ALL patients (Nahi et al., 2008). In this study, we observed deletion at 3p25-26 in a hypodiploid infant, in a complex karyotype and as a sole abnormality; while deletion at 9p/q occurred in a complex karyotype, with XXY karyotype and as a sole anomaly. Isochromosomes are relatively unusual and those readily identified in 1-4% childhood ALL include i(6p), i(7q), i(9q), and i(17q). Both i(9q) and i(7) occur in ALL either as a sole anomaly or in combination with non-random translocations like t(1;19), t(9;22) and (4;11) (Pui et al., 1992).
In conclusion, this study reveals that chromosomal aberrations are frequent in de novo Indian B-lineage ALL patients. Furthermore their frequency and distribution differs between childhood and adult patients, and also differs between Indian and Western settings. Hence the potential impact of these differences on clinical behavior and treatment outcome merits further investigation. Our study bears important epidemiological data and confirms previous reports demonstrating the identity of unusual complex clonal chromosome aberrations in B-lineage ALL, suggesting that it is heterogeneous in its pathogenesis. Similarities and dissimilarities of the present findings with those of other researchers may be attributed to the influence of differential environmental exposure to unknown carcinogenic agents, differences in geographical distribution and ethnic origins.