Cytogenetic Abnormalities in Multiple Myeloma Patients at a Tertiary Healthcare Center in India

Objective: Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell neoplasm. The prognosis of MM patients is dependent on several factors including the patient’s age, the stage of disease and genetic alterations. This study aimed to determine the frequency of common chromosomal abnormalities and their significance in MM patients referred to a tertiary healthcare center in India. Methods: Fluorescence in situ hybridization on interphase nuclei from bone marrow cells using seven MM-specific probes for recurrent aberrations was performed in a total of 215 newly diagnosed patients. Results: Chromosomal abnormalities were detected in 161 (74.9%) MM patients in this study. The most frequent aberration was trisomy(ies) involving only gain of chromosomes in 48 (22.3%) cases. A translocation involving the IGH gene alone or accompanied by trisomy(ies) or by monosomy 13/13q deletion or by both was registered in 80 (37.2%) patients. Atypical patterns such as a deletion of the IGH variable segment (IGHv) on the derivative chromosome 14 or on the native (normal) chromosome 14, biallelic deletion of IGHv, deletion of the IGH constant segment on the rearranged chromosome14 and extra fusions were noticed in 21 (9.8%) patients with an IGH rearrangement. Monosomy 13/deletion 13q was identified singly or as part of a complex karyotype in 74 patients (34.4%). Clonal heterogeneity and additional abnormalities including TP53 deletion and monosomies of chromosomes 4, 9, 14 and 16 were recorded in 18.6% and 16.3% of patients respectively. Patients with abnormalities exhibited plasmacytosis, reduced hemoglobin value and high level of ß2-microglobulin. Conclusions: A lower median age and a low frequency of IGH translocations particularly t(11;14) and chromosome 13 abnormalities suggest ethnic diversity. Further investigations on genetic alterations including IGH deletions will contribute to improved insights into the biology of myeloma disease, risk stratification and patient management.


Introduction
Multiple myeloma (MM) is a heterogeneous hematologic malignancy which is characterized by an uncontrolled proliferation of antibody-secreting plasma cells within the bone marrow (Talley et al., 2015;Fairfield et al., 2016). It usually evolves from the premalignant monoclonal gammopathy of unknown significance (MGUS) and then progresses through smouldering multiple myeloma (SMM) to end-stage MM. In 2014, the International Myeloma Working Group (IMWG) updated the diagnostic criteria for MM to include specific biomarkers (clonal bone marrow plasma cells ≥60%, serum free light chain ratio ≥100, and >1 focal lesion) to existing markers of end-organ damage (hypercalcemia, renal insufficiency, anemia and bone lesions) (Rajkumar et al., 2014). The Durie-Salmon staging system which specified tumor burden for staging MM was substituted or deletion of 13q14), deletion of 17p specifically TP53 and/or chromosome 1 aberrations (deletions in 1p and amplification in 1q) are associated with shorter survival, aggressive disease, and the presence of extramedullary disease (Bergsagel and Kuehl, 2005;Nadiminti et al., 2013;Prideaux et al., 2014;Rajan and Rajkumar, 2015). Refinement of the prognostic markers in MM through advanced genetic technologies will eventually lead to enhancement of the efficacy of the therapeutic approaches (Hanbali et al., 2017;Sommaluan et al., 2017). Conventional G-banded metaphase analysis provides a global view of the chromosomal changes at the cellular level, albeit at a low resolution while interphase FISH improved the detection of these abnormalities by the use of region-specific probes and facilitated analysis on a cell-by-cell basis (Nilsson et al., 2003;Gutierrez et al., 2007). It is now recommended to include conventional cytogenetic analysis together with iFISH in the initial diagnostic work-up for MM patients (Li et al., 2016;Sommaluan et al., 2017). Published reports on cytogenetic abnormalities in MM are scarce in patients of South Asian descent (Amare et al., 2016;Ashok et al., 2017;Sommaluan et al., 2017). The present study was aimed to evaluate the frequency of cytogenetic abnormalities and to determine their clinico-pathological significance in MM patients at a tertiary referral centre in South India.

Patients
A total of 215 (146 male and 69 female) untreated multiple myeloma patients, aged between 30 and 87 years, were referred to the Department of Centralised Molecular Diagnostics, Apollo Hospitals, Chennai-6 during the period July 2012 to December 2015. The criteria for diagnosis of MM was based on revised IMWG guidelines (Rajkumar et al., 2014). This study was approved by the Ethics Committee of Apollo Hospitals, Chennai. After written informed consent was obtained from the patients, detailed case history was collected and heparinized bone marrow samples were collected with the help of clinicians.
For every case, a total of 300 interphase nuclei were scored for each probe under oil immersion lens for the presence of signals using different filters in Olympus BX-51 microscope (Olympus, Tokyo, Japan). The images were captured and analysed with the Applied Spectral Imaging Systems software (FISH View version 6.0). The cut-off level for all the probes was 5% (Yuregir et al., 2009).

Results
The majority of the patients were aged between 50 and 65 years (median 58 years) and the chief complaints were bone pain and fatigue. They showed reduced hemoglobin value and high levels of creatinine and β2-microglobulin. Immunofixation electrophoresis revealed monoclonal IgG in 55.8%, IgA in 19.1% and IgM in 1.9%. Light chain only disease was present in 5.1% of patients (Table 1). At diagnosis 143 patients (66.5%) were in ISS stage III. The majority (77.2%) belonged to the states of Tamil Nadu, West Bengal and Assam while 8.4% were from outside India. Interphase FISH (iFISH) revealed chromosomal abnormalities in 161 (74.9%) MM patients and based on the type of abnormalities these patients were classified into eight groups ( Table 2). The most frequent group was trisomy(ies) which comprised of only a gain of one, two or more chromosomes and was recorded in 48 (22.3%) patients. The most commonly observed gain (trisomy and tetrasomy) (partial or complete) was that of chromosome 9 (63.6%), followed by those of chromosomes 11 (44.6%), 16 (16.4%), 4 (12.7%), 17 (11.8%), 14 (8.2%) and 13 (6.4%) (Figure 1a-h).
A translocation involving IGH locus alone was seen in 14 cases while it was accompanied by monosomy Normal range -WBC 4-11 x10 9 /L; Hb 11-16 g/dL; Platelets 150-450 x10 9 /L; Plasma cells 0-3.5%; Calcium 8.5-10.5 mg/dL; Creatinine 0.9-1.3 mg/ dL; Albumin 3.5-5.2 g/dL; β2-microglobulin 1.1-2.4 mg/L. deletion 13q was noted singly (4.7%) (Figure 2e) or as part of a complex karyotype in 74 patients (34.4%). Atypical FISH signals were observed in 21 (9.8%) patients with an IGH rearrangement. The monoallelic deletion of 5'IGH involving the variable segment (IGHv) on the derivative chromosome 14 was found in 13 MM patients (Figure 2f-g). The deletion of IGHv was biallelic in two cases as demonstrated by O2 pattern with IGH break apart probe ( Figure 2h). The IGH translocation was t(11;14) in seven cases, t(4;14) in one, t(14;16) in two, and an unknown partner in five cases. The deletion of IGHv on the native (or normal) chromosome 14 was seen in a single patient who also exhibited a cell line with the typical t(4;14) translocation (without deletion) ( Figure  3a). Two cases, one with t(4;14) and the other with t(14;?) translocation exhibited a deletion of the 3'IGH constant segment on the derivative chromosome 14 (Figure 3b). It was of interest to observe the coexistence of IGH deletions with chromosomal gains in most of the cases. Five patients including two cases with t(11;14), and one each with t(4;14), t(14;16) and t(14;?) showed monosomy 13/deletion 13q. Extra fusions were seen in three cases (two with t(4;14) and one with t(14;16)), who also showed the classical fusion (Figure 3c-e).
Comparable differences were noticed between patient group positive for abnormalities versus iFISH negative group. Patients with more than one type of abnormality were found to have significantly reduced hemoglobin (Hb), higher percentage of plasma cells and higher ß2microglobulin level (Table 3). Follow-up revealed 12 iFISH negative patients to be in clinical remission at 20.5 months from diagnosis. Similarly, 14 patients belonging to the group trisomies only showed a longer median survival of 20.78 months. The median survival duration in IGH only (n=3), IGH with trisomies (n=7), IGH with monosomy 13/13q-(n=6), and IGH with both abnormalities (n=2) were 19.3, 18.2, 16, and 16.5 months respectively. In contrast, the median survival in the two patients with -13/13q-and nine patients with trisomies and -13/13q-were 7 and 14.1 months respectively denoting a poor prognosis. These findings need to be validated over a larger sample size and longer survival duration.

Discussion
Multiple myeloma is a heterogeneous disease which is characterized by genomic alterations causing dysregulation of intracellular pathways and of importance in MM risk stratification. The clinical manifestations at presentation and a lower median age in comparison with that in developed nations were in accordance with published reports (Kumar et al., 2010). Interphase FISH enables a rapid identification of specific target regions frequently affected in MM in 50-90% of MM cases depending on the probes used (Fonseca et al., 2004;Avet-Loiseau et al., 2007;Kumar et al., 2012;Amare et al., 2016). Trisomy of at least one of the odd-numbered  8.3 (7.4-9.9) 8.9 (6.9-9.4) 8.8 (7.72-9.4) 7.8 (6.5-9.2) 7.9 (7.15-8.8) 8.4 (7.5-9.1) 8.2 (6.9-9.4) 9.35 (7.9 -10.8)  Table 3. Comparison of Clinical Parameters in Myeloma Patients with Chromosome Abnormalities and iFISH Negative Patients chromosomes was observed in 42% patients using centromeric probes for three additional chromosomes 3, 7 and 15 besides chromosome 9 (Rajan and Rajkumar 2015). In contrast only 48 patients (22.3%) exhibited gain of chromosome(s) in this study. Trisomies which confer a good prognosis are the result of a single catastrophic mitotic event and are thought to increase the copy number of gene loci critical for tumor suppression or mediating drug sensitivity (Prideaux et al., 2014). However, the presence of trisomies together with translocations as seen in approximately 10% of patients could ameliorate the adverse outcome (Kumar et al., 2012). This effect could be seen in the few patients who were followed up. The frequencies of the cytogenetically cryptic translocations t(4;14) and t(14;16) were in agreement with the reported frequency of 15-20% and 5-7% respectively (Moreau et al., 2002;Fonseca et al., 2004). However, the translocation t(11;14) was found to occur at a lower frequency (7.4%) in contrast to about 15% described in the literature (Fonseca et al., 2009;Prideaux et al., 2014;Talley et al., 2015). This finding could imply geographic heterogeneity with racial and ethnic diversity (Greenberg et al., 2015;Amare et al., 2016). A majority of the patients with t(4;14) also exhibited monosomy 13/13q-as demonstrated earlier (Avert-Loiseau et al., 2002). The occurrence of only chromosome 13 abnormalities in a subset of patients suggests that this could also be an early event in MM pathogenesis. Chng et al., (2007) observed -13/13q-in about 40-50% of MGUS tumors and in 50% of MM tumors. It has been hypothesized that deletion of chromosome 13q precedes the t(4;14) translocation in the pathogenesis of MM (Kalff and Spencer, 2012).
Interphase FISH also identified submicroscopic deletions adjacent to the breakpoints of rearrangements undetected by conventional cytogenetics. IGH gene deletions including monoallelic deletion of entire IGH locus, monoallelic deletions of variable (IGHv)/ constant (IGHc) regions and biallelic deletion of IGHv have been reported in 14-23% of newly diagnosed patients with MM (Trakhtenbrot et al., 2010;Hwang et al., 2011;He et al., 2015;Smol and Daudignon, 2017). Atypical FISH signal patterns corresponding to deletion of the variable/ constant regions of the gene were observed in 8.4% of our patients (n=18) in the present study. These monoallelic deletions of the IGH variable regions were suggested to be a result of the DNA loss that accompanied somatic V-D-J recombination as a physiological event, not as part of an oncogenic process of B-lineage cells (Wlodarska et al., 2007). In most cases, IGHv deletions were on the normal chromosome 14 (Wlodarska et al., 2007;Hwang et al., 2011). However, the rearranged chromosome 14 was involved in IGH deletions in 15 cases and the normal chromosome 14 in only a single case in the present study. The IGH deletions were reported to most often coexist with 13q deletions in patients with MM (Trakhtenbrot et al., 2010;Hwang et al., 2011;He et al., 2015). Further, IGH deletions with the t(11;14) were found only in MM cases with 13q deletion, while those t(11;14)-positive cases without any IGH deletions exhibited normal 13q (Trakhtenbrot et al., 2010;Hwang et al. 2011). In contrast, He et al., (2015) observed an increased incidence of IGH deletions in patients with 13q deletion and without t(4;14). No such association was observed in this study. The patients carrying IGH deletion showed better overall response rate to induction therapy with bortezomib, epirubicin and dexamethasone but the deletion did not influence prognosis (He et al., 2015). Longitudinal studies in larger cohorts are needed to determine the significance of IGH deletions on prognosis of our MM patients.
Additional abnormalities, namely TP53 deletion and monosomies of chromosomes 4, 9, 14 and 16 were observed in 35 (16.3%) patients in this study. The occurrence of cytogenetic abnormalities such as deletions involving chromosome 17p are typically considered as late events and are suggestive of clonal evolution and disease progression (Rajkumar et al., 2013;Jian et al., 2016). Patients with MM with sole translocation t(11;14) were found to have better overall survival (OS) when compared with patients with translocation t(11;14) combined with the aberrations del(1p), del(13q), del(17p), multiple gains(1q) and del(IGH) (Leiba et al., 2016). A brief follow-up for a period of about two years from the time of initial diagnosis revealed that the patients who exhibited a normal or gain of chromosomes FISH pattern showed improved survival while chromosome 13 abnormalities were an adverse prognostic factor. Further studies are needed to validate the clinical significance of IGH rearrangements especially IGH deletions and abnormalities of chromosome 1 (deletion of 1p and multiple gains of 1q) in multiple myeloma.
In conclusion, this study has demonstrated the importance of interphase FISH analysis in the identification of prognostically significant abnormalities for the implementation of stratified treatment approaches in the management of myeloma patients. Further investigations on geographically diverse populations with long-term follow-up would enable data comparison to confirm ethnic disparity.

Statement conflict of Interest
No relevant conflicts of interest to declare.