Immunoglobulin Heavy Chain Translocations Research Articles

The biological and clinical impact of deletions before and after large chromosomal gains in multiple myeloma

AbstractAcquisition of a hyperdiploid (HY) karyotype or immunoglobulin heavy chain (IgH) translocations are considered key initiating events in multiple myeloma (MM). To explore if other genomic events can precede these events, we analyzed whole-genome sequencing data from 1173 MM samples. By integrating molecular time and structural variants within early chromosomal duplications, we indeed identified pregain deletions in 9.4% of patients with an HY karyotype without IgH translocations, challenging acquisition of an HY karyotype as the earliest somatic event. Remarkably, these deletions affected tumor suppressor genes (TSGs) and/or oncogenes in 2.4% of patients with an HY karyotype without IgH translocations, supporting their role in MM pathogenesis. Furthermore, our study points to postgain deletions as novel driver mechanisms in MM. Using multiomics approaches to investigate their biologic impact, we found associations with poor clinical outcome in newly diagnosed patients and profound effects on both the oncogene and TSG activity despite the diploid gene status. Overall, this study provides novel insights into the temporal dynamics of genomic alterations in MM.

Abstract P13: Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity

Abstract IG translocations occur in various B-cell neoplasms and result in the deregulation of (onco)genes due to their juxtaposition with regulatory elements in the immunoglobulin loci, predominantly the immunoglobulin heavy chain (IGH) locus (14q32). Some of these IGH translocations are pathognomonic for distinct disease entities, like those involving MYC, BCL2 or CCND1 in Burkitt, Follicular or Mantle Cell Lymphomas, respectively. In Chronic Lymphocytic Leukemia (CLL), the BCL3 gene (19q13), a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family, is a known partner gene in these translocations. Two subsets of IG::BCL3 translocation-positive B-cell neoplasms have been described that exhibit differences in chromosomal aberrations, IGHV mutation status, and histopathology [Martín-Subero et al. 2007]. In CLL, IG::BCL3 translocation is linked to a younger age at diagnosis, a more aggressive clinical course, and an atypical tumor cell phenotype [Michaux et al. 1997; Au et al. 2002]. A total of 89 B-cell neoplasms, primarily diagnosed as CLL, displaying IG::BCL3 translocation detected by fluorescence in situ hybridization (FISH), were examined. Analysis of DNA methylation and copy number aberrations (CNA) was conducted for 86 IGH::BCL3-translocated B-cell neoplasms using 850K EPIC BeadChip array data. Evaluation of chromosomal aberrations typical for CLL was moreover performed by FISH and the IGHV mutation status was determined. IGH::BCL3 breakpoint junctions were sequenced in 23 samples using targeted capture and whole-genome sequencing approaches. RNA expression of BCL3 was assessed using the HTG mRNA pan B-cell panel. In UMAP analysis of DNA methylation, CLLs with IG::BCL3 translocation exhibited segregation from other CLLs, irrespective of IGH translocations. Supervised analysis revealed global hypomethylation of B-cell neoplasms with IG::BCL3 translocation compared to other CLL. A total of 66/69 CLLs with IG::BCL3 translocation showed an unmutated IGHV status (96%). Copy number determination using BeadChip data (n=86) and FISH analysis (n=85) showed a significantly higher incidence of trisomy 12 in IG::BCL3-translocated cases compared to the general CLL population (p=0.011). Breakpoint sequencing indicated IG::BCL3 junctions to be recurrently associated with aberrant class-switch recombination at the IGH locus, involving IGHA (n=11/23), IGHG (n=5/23) and IGHM segments (n=5/23). Breakpoints at the BCL3 locus displayed two groups. 80 cases (all CLL) were located upstream of BCL3 and two other B-cell neoplasms (NHL, DLBCL) were located downstream of BCL3. BCL3 expression analysis in 15 IG::BCL3-translocated CLLs confirmed its transcriptional upregulation compared to the general CLL population. Our genetic and epigenetic analyses support the view that CLL with IG::BCL3 translocation may constitute a genetically and epigenetically distinct subtype of B-cell neoplasms, diverging from the typical CLL. Citation Format: Cosima Drewes, Cristina López, Nnamdi Okeke, Sina Hillebrecht, Petra Schütz, Anja Fischer, Anja Mottok, Susanne Bens, Christof Schneider, Stephan Stilgenbauer, Eugen Tausch, Reiner Siebert. Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P13.

Novel identification of t(14;18)(q32;q21)/IGH::MALT1 in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a clonal B-cell malignancy and remains a chronic disease despite improvements in clinical outcomes since the use of targeted therapies. Both clinical and biological parameters are important for determining prognosis. Unlike other mature B-cell lymphomas, translocations involving the immunoglobulin heavy chain (IGH) locus are uncommon in CLL. There have been few case reports of CLL harbouring t(14;18)/IGH::BCL2 and t(14;19)/IGH::BCL3. Here we describe the first two cases of patients with CLL with documented t(14;18)(q32;q21)/IGH::MALT1. Both cases in this report were associated with lower-risk biological parameters. Thus, FISH testing for MALT1 in cases with unknown IGH translocation partners in the setting of CLL should be implemented in clinical practice to better define such cases.

Revealing Novel Mechanisms Underlying Inactivation of Tumor Suppressor Genes on Duplicated Chromosomes in Multiple Myeloma

INTRODUCTION: Multiple myeloma (MM) evolution is complex and heterogeneous. Hyperdiploidy (HRD) and translocations affecting the immunoglobulin heavy chain (IGH) locus are historically considered initiating genomic events of MM. The potential impact of genomic events acquired before known MM initiating events has never been addressed. METHODS: To investigate whether genomic deletions are acquired before and after known MM initiating events we interrogated whole-genome sequencing (WGS) data from patients with newly diagnosed MM (NDMM, n=319) and relapsed MM (RRMM, n=59). Our newly developed analytical and chronological workflow integrating single nucleotide variants (SNV), structural variants (SV), and copy number variants (CNV) comprises two main steps. First, we estimated the molecular time (i.e. corrected ratio between duplicated and non-duplicated clonal SNV) of large clonal chromosomal duplications and identified the earliest set of chromosomal gains in each patient. Second, within each early large chromosomal gain we identified clonal SV mediating CNV loss. A deletion on a gain can generate three possible scenarios: 1) one of the duplicated alleles is lost after the gain (i.e. post-gain) causing a CNV jump from 3:1 to 2:1 (total alleles : minor alleles); 2) there is a deletion before the duplication (i.e., pre-gain) causing a CNV jump from 3:1 to 1:0. 3) the deletion occurs on the minor, non-duplicated allele, causing a CNV jump from 3:1 to 2:0. Timing the deletion in relation with the chromosomal duplication is impossible in this scenario. RESULTS: Molecular time data were successfully generated for 249/319 (78%) NDMM and 51/59 (86%) RRMM patients. Restricting our analysis to NDMM with HRD without canonical IGH translocations, 16/170 (9%) of patients acquired deletions before the earliest multi-chromosomal gains, suggesting MM precursors can acquire deletions before HRD. Investigating the entire series, post-gain deletions were observed in 126/417 (30%) samples considering both early and late time windows. Leveraging a background model, we demonstrated that pre-gain events involved more tumor-suppressor genes (TSG) than expected by chance, including TCF3, ATM and TRAF3. In contrast, oncogenes were involved less than expected. In post-gain deletions, both oncogenes and TSG were involved more than expected by chance . To validate and assess the impact of deletions on driver gene expression we investigated WGS and RNAsequencing data from the MMRF CoMMpass study (n=752 NDMM). Because of the low coverage not allowing for molecular time estimation, we limited our analysis to HRD without IGH translocations. Pre-gain and post-gain deletions were observed in 47/431 (11%) and 225/431 (52%) of patients, respectively. We defined loss and gain of function events based on whether the expression level of a gene affected by the deletion was in the first or fourth quartile, respectively. Pre-gain deletions were mostly associated with downregulation of TSG expression [n=31 events in 13/431 (3%) patients]. In contrast,post-gain deletions had a more heterogenous impact with loss- and gain-of-function events. Gain-of-function events were driven by two main mechanisms: 1) the deletion joined an oncogene with a distal regulatory region inducing its overexpression [n=44 events in 6/431 (1.3%) patients]; 2) the deletion caused a new and expressed fusion [n=231 events in 109/431 (25%) patients], resulting in either loss- or gain-of-function. Surprisingly, post-gain deletions had also a major impact on TSG expression. In 60 patients (14%), we observed 146 post-gain deletions where the affected tumor suppressor gene (TSG) expression was downregulated to the level of cases with monoallelic and biallelic deletions, even though two alleles were retained. Finally, to validate these findings, we investigated 16 RRMM patients with available WGS, scATAC-seq and scRNA-seq and observed additional evidence of TSG downregulation after both pre- and post-gain deletions. CONCLUSION: Leveraging a large cohort of NDMM we show that somatic deletions can be acquired before HRD trisomies that are assumed to be initiating events. Furthermore, post-gain deletions emerged as a new mechanism inducing TSG down-regulation, despite an apparently diploid gene status.

Characterization of the genetic and epigenetic landscape of B‐cell neoplasms with IG::BCL3‐translocation

Introduction: The BCL3 gene (19q13) encodes a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family. It is recurrently deregulated in B-cell neoplasms by a juxtaposition to regulatory elements of one of the immunoglobulin loci, mostly the immunoglobulin heavy chain (IGH) locus (14q32). Two distinct subsets of IG::BCL3 translocation-positive B-cell neoplasms, differing in the number of chromosomal aberrations, IGHV mutation status and histopathology, have been described [Martín-Subero et al., 2007]. In B-cell chronic lymphocytic leukaemia (CLL), the IG::BCL3 translocation has been associated with younger age at diagnosis, a more aggressive clinical course and an atypical tumour cell phenotype [Michaux et al., 1997; Au et al., 2002]. Methods: A total of 89 B-cell neoplasms with IG::BCL3 translocation detected by fluorescence in situ hybridisation (FISH), including predominately cases diagnosed as CLL, were studied. Breakpoints at the BCL3 and IGH loci were sequenced in a subset of samples using targeted capture and breakpoint-spanning PCR approaches. Chromosomal aberrations detected by FISH as well as the IGHV mutation status were analysed. In addition, DNA methylation and copy number aberration (CNA) analyses were performed for 48 samples with CLL using the 850K EPIC BeadChip array. Results: Targeted capture-based sequencing data showed that in the majority of cases BCL3 translocations are associated with aberrant class-switch recombination at the IGH locus, often involving IGHA segments (n = 4/10). From the 69 IG::BCL3-translocated cases with available IGHV mutation status, a total of 51 (74%) were classified as unmutated and 18 (26%) as mutated. This indicates an overrepresentation of cases with unmutated IGHV status among the IG::BCL3-translocated cases compared to the general CLL population. Based on EPIC BeadChip data (total n = 48) 25 (52%) IG::BCL3-translocated cases carried a trisomy 12, 11 cases (23%) a deletion 13q, 7 cases (15%) a deletion 17p and 3 cases (6%) a deletion 11q. FISH analysis of a partially overlapping set of neoplasms (n = 54) was in line with the EPIC BeadChip data and revealed 17 IG::BCL3-translocated cases (32%) with trisomy 12, 7 cases (13%) with deletion 17p and deletion 13q each, and 4 cases (7%) with deletion 11q. Thus, IG::BCL3-translocated cases showed a striking skewing towards the presence of trisomy 12 compared to the general CLL population (p = 0.011). In the DNA methylation analyses, the IG::BCL3-translocated CLLs segregated separately from other CLLs with and without IGH translocations. Conclusions: Our large multi-OMICs study of IG::BCL3-translocated B-cell neoplasms corroborates previous reports on the pathogenetic heterogeneity of these lymphomas and moreover provides evidence that IG::BCL3-translocated CLL might form a genetically and epigenetically distinct subtype of B-cell neoplasms distinct from common CLL. Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Genomic Landscape of t(11;14) in Multiple Myeloma

Background Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by multistep genetic alterations, including translocations, copy number aberrations (CNA), and mutations. Translocations involving the immunoglobulin heavy chain (IGH) locus contribute to plasma cell immortalization and consequent commitment to the MM disease pathway, however other genetic events largely underlie the complex genomic landscape of MM with molecular and clinical heterogeneity. The t(11;14) translocation is the most common IGH translocation in MM and has been identified as a predictive biomarker of response to venetoclax in relapsed/refractory MM. The objective of this study was to confirm that t(11;14) is stable across the course of disease and across lines of therapy, and to assess the unique genomic landscape of t(11;14) MM. Methods This was a retrospective, single-center, non-interventional study. To confirm t(11;14) stability, longitudinal assessment of 272 patients for t(11;14) status by plasma cell-enriched FISH using the Vysis IGH/CCND1 XT DF FISH probe was performed on bone marrow aspirates (BMAs) collected at diagnosis and relapse. To assess the genomic landscape, 139 patient BMAs were collected from an approved biobank with informed consent and were analyzed by a targeted NGS panel. NGS was performed as described in Bolli et al. (Bolli et al. 2016) using an Agilent capture panel for sequencing on the Illumina NextSeq platform. Raw sequencing data was processed by an in-house bioinformatics pipeline to detect mutations, copy number aberrations and structural variants (SVs). Protein coding mutations with variant allele frequency >10% and minimum sequencing depth >=20X were selected. CNAs were defined as log ratio above 0.3 (copy number gain) or below -0.4 (copy number loss). SVs with 5 or more supporting reads were examined to detect translocations within the IGH locus. Results Among the 272 patients assessed by longitudinal t(11;14) FISH, 118 were t(11;14)+ and 154 were t(11;14)-. All t(11;14)+ patients remained positive from their initial assessment to first and subsequent longitudinal assessments (Figure 1). The t(11;14) FISH fusion (F) patterns were also consistent across longitudinal assessments with 90% of patients possessing and maintaining either a 1F (18%), 2F (69%), or ≥3F (3%) translocation pattern. No t(11;14)- patients acquired the translocation at relapse. Among the 139 samples analyzed for t(11;14), 130 samples had results from both FISH and NGS with a 100% concordance rate; 66 were t(11;14)+ and 64 were t(11;14)-. The mutational landscape in highly recurrent genes was heterogeneous (Figure 2). MAPK pathway mutations were the most common (47/130 pts, 36.2%), with recurrent mutations in BRAF gene more prevalent in t(11;14)- vs t(11;14)+ pts (18.8% vs 6.1%, p<0.05). Conversely, mutations in DIS3 gene were more common in t(11;14)+ vs t(11;14)- pts (21.2% vs 4.7%, p<0.05). DIS3 and BRAF mutations have been associated with poor prognosis in MM. High CNAs are associated with increased genomic instability, resistance to therapy, and decreased survival in MM. Increased CNAs were observed in t(11;14)- vs t(11;14)+ MM (p<0.001). Conclusion This study confirms in a large, longitudinal cohort of patients with MM that t(11;14) is stable from diagnosis to relapse. These analyses also begin to delineate the differentiated genetic architecture of t(11;14) MM, including fewer CNAs associated with increased genomic stability, but increased rates of DIS3 mutations that confer poor prognosis in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Feasibility and clinical utility of comprehensive genomic profiling of hematological malignancies.

Identification of genetic alterations through next‐generation sequencing (NGS) can guide treatment decision‐making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS‐based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0–55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer‐cell lymphoma. These results suggest the clinical utility of NGS‐based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

IGH Translocations in Chinese Patients With Chronic Lymphocytic Leukemia: Clinicopathologic Characteristics and Genetic Profile.

Immunoglobulin heavy chain translocations (IGH-t) have occasionally been reported in Chinese patients with chronic lymphocytic leukemia (CLL). The objective of the present study was to identify the clinicopathologic features of patients with IGH-t CLL and compare them with those of patients with non-IGH-t CLL. We performed fluorescence in situ hybridization (FISH) based on a routine CLL prognostic FISH panel using IGH, IGH-BCL2, BCL3, IGH-CMYC, and BCL6 FISH probes. Furthermore, we retrospectively evaluated the clinical features of 138 newly diagnosed CLL patients via chromosome banding analysis (CBA), FISH, and targeted next-generation sequencing. IGH-t was identified in 25 patients (18.1%). Patients with IGH-t CLL had lower flow scores than those with non-IGH-t CLL. The most frequent translocation was t(14;18) (10 patients), followed by t(14;19) (3 patients), and t(2;14)(p13;q32), t(7;14)(q21.2;q12), t(9;14)(p13;q32) (3 patients). The remaining nine patients included three with abnormal karyotypes without translocation involving 14q32, four with a normal karyotype, and two who failed CBA. The most frequently concomitant FISH-detected aberrations were 13q deletion, followed by +12 and TP53 deletion, while one case involved ATM deletion. Complex karyotypes were detected in five patients with IGH-t CLL, in whom all partner genes were non-BCL2. Available mutational information indicated that KMT2D mutation was the most frequent mutation among tested 70 patients, while TP53 mutation was the most frequent mutation in the IGH-t group. Moreover, the IGH-t group had higher FBXW7 (P=0.014) and ATM (P=0.004) mutations than the non-IGH-t group, and this difference was statistically significant. Our study demonstrates that IGH-t is not uncommon among Chinese CLL patients, and that its partner genes are multiple. The gene mutational profile of the IGH-t group was distinct from that of the non-IGH-t group, and the concomitant chromosomal abnormalities within the IGH-t CLL group differed. Thus, identification of IGH-t and its partner genes in CLL patients may help further refine risk stratification and strengthen the accurate management in CLL patients.

Myeloma Genome Project Panel is a Comprehensive Targeted Genomics Panel for Molecular Profiling of Patients with Multiple Myeloma.

We designed a comprehensive multiple myeloma targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNA). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical FISH (translocations), multiplex ligation probe analysis (MLPA; CNAs), whole-genome sequencing (WGS; CNAs, mutations, translocations), or droplet digital PCR (ddPCR) of known standards (mutations). Canonical immunoglobulin heavy chain translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for 1 patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2 = 0.969. Variant allele frequency (VAF) for 74 mutations were compared between sequencing and ddPCR with concordance of R2 = 0.9849. In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost-effective, comprehensive, clinically actionable, and can be routinely deployed to assist risk stratification at diagnosis or posttreatment to guide sequencing of therapies.

High-Risk Myeloma Is Demarcated By Immunoglobulin Lambda Light Chain Translocations

Multiple myeloma is the second most common hematological malignancy, and is the consequence of uncontrolled proliferation of antibody-secreting B cells, known as plasma cells. Myeloma causes lytic bone lesions and renal failure and is characterized by heterogeneous genetic mutations and clonal selection. Subsequently, understanding the complex mutational repertoire in the context of myeloma prognosis remains challenging. We report the translocation architecture of 826 newly diagnosed myeloma patients as part of the Clinical Outcomes in Multiple Myeloma to Personal Assessment (CoMMpass) study. These data are compared to other genetic mutations, gene expression, and patient outcome to better understand high-risk disease.Whole genome sequencing identified 31% of newly diagnosed myelomas as containing an Immunoglobulin heavy chain (IgH) translocation. The vast majority of IgH translocations occurred with CCND1, WHSC1, MYC, or MAF; all of which were upregulated upon translocation. IgH-CCND1 translocations were more likely to express low levels of IgH (25%), but the majority expressed high levels of IgG1 consistent with other myelomas. Conversely, IgH-WHSC, -MYC, and -MAF translocations were more likely to express IgA (31%) and only IgH-MAF translocated myeloma expressed IgA2. IgH translocations primarily occurred at sites of class-switch recombination except for IgH-MYC translocations that originated from IgH intergenic enhancers suggesting that these occur by a distinct mechanism.MYC translocations occurred in 18% of myelomas and in contrast to IgH, MYC translocation partners were scattered throughout the genome at regions proximal to highly expressed genes. MYC translocations corresponded with MYC amplification which was present in 15% of myelomas, yet half of these contained a translocation. Both types of genetic abnormalities corresponded with increased MYC expression, but neither MYC amplification and/or translocation, nor IgH translocation were prognostic of outcome.Immunoglobulin lambda light chain (IgL) translocations were the most common translocation (7.1%) that did not involve IgH or MYC. Unlike IgH and MYC translocations, IgL translocations were prognostic of poor progression-free and overall survival. IgL translocations occurred at IgL enhancers and most commonly involved MYC, MAP3K14, 6p24.3, CCND (1,2, and 3), and MAF. IgL translocated patients presented myeloma at a similar age and stage and were treated with similar therapies as compared to other patients. IgL translocated myelomas contained a mutational repertoire that mimicked other myelomas and poor prognosis was independent of the IgL translocation partner, suggesting that the prognostic effect was intrinsic to IgL. Interestingly, myelomas with IgL translocations expressed lower levels of inflammatory cytokines including CCL3 and CCL4, suggesting they are less likely to induce anti-tumor innate immune responses. Taken together these data have important insights into myeloma pathology and implications for identifying high-risk myeloma. DisclosuresBoise:Eli Lilly and Company: Research Funding; Abbvie: Consultancy.

Translocation (11;14) and Survival of Patients with AL Amyloidosis and Multiple Myeloma

Translocation (11;14) and survival of patients with AL amyloidosis and multiple myelomaTakao Yogo, Kanji Miyazaki, Kenshi SuzukiDepartment of Hematology, Japanese Red Cross Medical Center, Tokyo, JapanBackgroundAmyloid light-chain (AL) amyloidosis is characterized by the deposition of immunoglobulin light chains as amyloid fibrils. Immunoglobulin heavy chain translocation (11;14) is one of the most prevalent chromosomal abnormality in patients with AL amyloidosis, although its clinical significance is still unknown. In this study, we investigated the clinical significance of t(11;14) with AL amyloidosis by comparing with patients having multiple myeloma bearing the same translocation.MethodsWe reviewed 62 patients with AL amyloidosis and 78 patients with multiple myeloma who were newly diagnosed and examined t(11;14) using fluorescence in situ hybridization (FISH). The study was conducted between May 2012 and December 2016 at the Japanese Red Cross Medical Center.Results FISH-based t(11;14) were detected in 15 (24.2%) patients with AL amyloidosis and 13 (16.7%) with multiple myeloma. In patients with AL amyloidosis, t(11;14) gave poor overall survival (P=0.039), whereas the positivity of t(11;14) gave no significant difference in patients with multiple myeloma (P=0.675). Among AL amyloidosis patients, the 24-month survival rate was 38.6% in the t(11;14)-positive group and 76.7% in the t(11;14)-negative group. Although the percentage of plasma cells in bone marrow is high in the t(11;14)-positive group (7.4% vs. 3.6%, p=0.005), there is no significant difference in the overall survival between patients with more than 10% plasma cells (n=12) and patients with less than 10% plasma cells (n=50). Overall survival was superior in autologous stem-cell transplantation (ASCT) group (n=15) compared with non-ASCT group (n=47) (p=0.029). However, even after taking these factors into consideration, t(11;14) had a negative prognostic impact on the overall survival of patients with AL amyloidosis.Conclusionst(11;14) is associated with adverse outcome in patients with AL amyloidosis. [Display omitted] DisclosuresSuzuki:Bristol-Myers K.K: Honoraria; Novarltis: Honoraria; Celgen: Honoraria; Ono: Honoraria; Fujimoto: Honoraria; Takeda: Honoraria.

Chromatin Accessibility Identifies Regulatory Elements Predictive of Gene Expression and Disease Outcome in Multiple Myeloma.

Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for 1-6 months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA sequencing (RNA-seq) and assay for transpose accessible chromatin sequencing (ATAC-seq). Chromatin accessible regions were used to identify regulatory RNA expression in more than 700 samples from newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass trial (NCT01454297). Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. More than 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with immunoglobulin heavy chain translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.

Role of cytogenetic abnormalities detected by fluorescence in situ hybridization as a prognostic marker: Pathogenesis & clinical course in patients with B-chronic lymphocytic leukaemia.

Background & objectives:B-cell chronic lymphocytic leukaemia (B-CLL) is one of the most common forms of adult leukaemia, with a highly variable clinical course. Specific chromosomal and genetic aberrations are used clinically to predict prognosis, independent from conventional clinical markers. Molecular cytogenetic methods such as fluorescence in situ hybridization (FISH) detect aberrations in up to 80 per cent B-CLL patients. This study was conducted to score the frequencies of recurrent aberrations, i.e., del(13q14), trisomy 12, del(11q22), del(17p13), del(6q21) and IgH (immunoglobulin heavy chain) translocations and to understand their role in prognostication and risk stratification.Methods:FISH studies were performed on bone marrow aspirate or peripheral blood of 280 patients using commercially available disease-specific probe set. The data were correlated with clinical and haematological parameters such as low haemoglobin, splenomegaly and lymphadenopathy.Results:Chromosomal aberrations were detected in 79 per cent of patients, with del(13q14) (57%) as the most common cytogenetic aberration, followed by trisomy 12 (27%), del(11q22) (22%), t(14q32) (19%), del(17p13) (18%) and del(6q21) (9%). Single or in coexistence with other aberration del(13q14) had a favourable outcome in comparison to del(11q22), t(14q32), del(17p13) and del(6q21) which were associated with advanced stages of the disease. Trisomy 12 had a variable clinical course.Interpretation & conclusions:FISH was found to be a sensitive and efficient technique in detecting the prevalence of recurrent cytogenetic abnormalities. Each of these aberrations is an important independent predictor of disease progression and survival which aids in designing risk-adapted treatment strategies for better disease management.

Chronic lymphocytic leukemia patients with IGH translocations are characterized by a distinct genetic landscape with prognostic implications.

Chromosome 14q32 rearrangements/translocations involving the immunoglobulin heavy chain (IGH) are rarely detected in chronic lymphocytic leukemia (CLL). The prognostic significance of the IGH translocation is controversial and its mutational profile remains unknown. Here, we present for the first time a comprehensive next-generation sequencing (NGS) analysis of 46 CLL patients with IGH rearrangement (IGHR-CLLs) and we demonstrate that IGHR-CLLs have a distinct mutational profile with recurrent mutations in NOTCH1, IGLL5, POT1, BCL2, FBXW7, ZMYM3, MGA, BRAF and HIST1H1E genes. Interestingly, BCL2 and FBXW7 mutations were significantly associated with this subgroup and almost half of BCL2, IGLL5 and HISTH1E mutations reported were previously identified in non-Hodgkin lymphomas. Notably, IGH/BCL2 rearrangements were associated with a lower mutation frequency and carried BCL2 and IGLL5 mutations, while the other IGHR-CLLs had mutations in genes related to poor prognosis (NOTCH1, SF3B1 and TP53) and shorter time to first treatment (TFT). Moreover, IGHR-CLLs patients showed a shorter TFT than CLL patients carrying 13q-, normal fluorescence in situ hybridization (FISH) and +12 CLL, being this prognosis particularly poor when NOTCH1, SF3B1, TP53, BIRC3 and BRAF were also mutated. The presence of these mutations not only was an independent risk factor within IGHR-CLLs, but also refined the prognosis of low-risk cytogenetic patients (13q-/normal FISH). Hence, our study demonstrates that IGHR-CLLs have a distinct mutational profile from the majority of CLLs and highlights the relevance of incorporating NGS and the status of IGH by FISH analysis to refine the risk-stratification CLL model.

Abstract 6559: Novel anti-myeloma potential of CAPE analogs: Downregulation of IKZF1-IRF4-MYC axis

Abstract Background: Multiple myeloma (MM) is the second most common hematological malignancy and an incurable bone marrow cancer so far. The hallmark of myeloma is chromosomal translocations that transform plasma cells into malignant myeloma cells. 50-75% of myeloma patients exhibit translocations at the immunoglobulin heavy chain (IgH) locus juxtaposing oncogenes from partner chromosome under the control of strong 3' IgH enhancers. Overexpression of oncogenes such as Cyclin D1, Cyclin D3, interferon regulatory factor (IRF4) etc. occurs, depending on the translocating partner locus. We have earlier reported that elevated expression of octameric binding protein-2 (OCT2), a key transcription factor in IgH translocations as a poor prognostic factor and shown its association with reduced survival in MM patients. IRF4 is another indispensable transcription factor for plasma cell differentiation, and its juxtaposition with IgH locus leads to IRF4 overexpression mediated tumorigenesis. Both OCT2 and IRF4 are regulated by the transcription factor, nuclear factor kappa B (NF-κB). Current chemotherapeutics exhibit several side effects, and face the challenge of chemoresistance, thus warranting the need for novel therapeutics for myeloma. Caffeic acid phenethyl ester (CAPE) is an active principle of propolis from honeybee hives, with potent NF-κB inhibitory activity. Hence, we synthesized and evaluated the structure-activity relationship of twenty-one CAPE analogs for their anti-myeloma potential. Methods: Human myeloma cell lines, lenalidomide, CAPE and its analogs were used for the study. Myeloma cell growth inhibition by CAPE analogs was determined by PrestoBlue cell viability assays. Effect of CAPE analogs on IRF4, OCT2, CyclinD3, caspase-3, Ikaros (IKZF1), MYC levels were evaluated by qRT-PCR and/or immunoblotting methods; apoptosis induction detected by Annexin V-PI flow cytometry assays. Results: Cyclohexylethyl and two phenpropyl ester analogs exhibited high myeloma cell growth inhibition in comparison with CAPE and lenalidomide. IRF4 was significantly downregulated both at mRNA and protein levels by the phenpropyl ester analog that demonstrated a remarkable increase in cleaved caspase-3 levels and apoptotic cell number. For the first time, we have demonstrated the ability of CAPE analogs to decrease OCT2, IKZF1, IRF4 and MYC levels in myeloma cells. Downregulation of the IKZF1-IRF4-MYC axis by CAPE and its key analogs revealed their novel anti-myeloma mechanism of action. Furthermore, the lead analogs showed no adverse cytotoxic effect on normal human mononuclear or naïve B-cells, also exhibited appropriate in silico pharmacokinetic properties and drug-likeness. Conclusion: Thus, our findings suggest the promising application of CAPE analogs to target Ikaros/IRF4 addiction, the so-called Achilles heel of myeloma, thereby leading to better therapeutic outcomes. Citation Format: Alli Murugesan, Gregoire Lasalle-Claux, Lauren Hogan, Elise Vaillancourt, Ayyoub Selka, Katie Luiker, Andy Hong, MinJi Kim, Mohamed Touaibia, Tony Reiman. Novel anti-myeloma potential of CAPE analogs: Downregulation of IKZF1-IRF4-MYC axis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6559.

Clinical and Prognostic Impact of (11;14)(q13;q32) Translocation on Patients with Multiple Myeloma

Introduction. Translocations involving chromosome 14 at band q32, the immunoglobulin heavy chain (IgH) locus, are considered to be the most important initiating events for the development of multiple myeloma (MM). Among the IgH translocations in MM, t(11;14)(q13;q32) is the most frequently reported, and associated with a lymphoplasmacytic morphology. This translocation have been traditionally considered as standard-risk chromosomal abnormality compared to other translocations such as t(4;14) or t(14;16), although some controversies on the prognostic impact of this translocation still remain. This study aimed to clarify the clinical and prognostic impact of t(11;14) in Japanese patients in relation to other clinical variables such as immunophenotype of the tumor cells, other cytogenetic abnormalities, and use of stem cell transplantation (SCT). Patients and methods. Among the 244 consecutive patients with newly diagnosed MM, treated at Kameda Medical Center between April 2009 and July 2019, 234 patients, having cytogenetic analysis data (including t(11;14), t(4;14), t(14;16), and del(17p) by interphase fluorescence in situ hybridization (iFISH)) fully available, were included in this study. Data regarding the patients' clinical and laboratory characteristics, including the International Staging System (ISS), immunophenotype of the tumor cells, baseline circulating plasma cells (CPCs), treatment responses, disease progression, and survival status, were collected. iFISH was performed with CD138-purified bone marrow plasma cells, and the cut off values for translocation were ≥ 10% and for del(17p) ≥ 20%. Using multicolor flow cytometry, surface marker analysis of bone marrow samples and quantification of pre-treatment CPCs on peripheral blood mononuclear cells were simultaneously performed. CPCs were reported as the percentage of total mononuclear cells. Patients were considered to be negative for clonal CPCs at a sensitivity of 10−4 (0.01%) clonal plasma cells for all events evaluated. Results. The incidence of patients harboring t(11;14) was 24.4% (n = 57); t(11;14) was detected significantly high in light-chain-only subtypes (P &lt; 0.001). We compared clinical characteristics of patients carrying t(11;14) with others. Myeloma cells with t(11;14) were associated with negative expression of CD56 (P &lt; 0.001), CD117 (P = 0.046), and CD200 (P = 0.006), and positive expression of CD20 (P = 0.01) and CD81 (P = 0.035). Patients with t(11;14) were associated with positive CPCs (P = 0.011). In order to focus on the impact of t(11;14), we divided the patients into 4 groups: (A) no specific cytogenetic abnormality listed above (n = 137), (B) t(11;14) group (n = 57), (C) t(4;14) or t(14;16) group (n = 29), and (D) del(17p) only (n = 10), and the clinical characteristics and survival of the patients were compared across the three groups (A), (B), and (C). Almost all the patients (&gt; 95%) in this cohort received bortezomib-based therapy. Median progression-free survival (PFS) and overall survival (OS) of patients in (A), (B), and (C) groups were 55.6, 34.2, and 30.2 months (m) (A vs. B, P = 0.036, and A vs. C, P = 0.031), and not reached, 51.2, and 79.8 m (A vs. B, P = 0.11, and A vs. C, P = 1.00), respectively. However, patients harboring t(11;14) were further divided into CD20-positive and -negative groups, the latter having poor prognosis (36.1 vs. 26.7, P = 1.0 for median PFS, and not reached vs. 44.2, P = 0.029 for median OS). Compared to other groups, patients without CD20 expression had significantly shorter OS (vs. A, vs. B, P = 0.024, 0.035, respectively), whereas those with CD20 expression tended to have longer OS, without statistical significance (Figure 1).Univariate analysis revealed ISS stage III, creatinine &gt; 2.0 mg/dL, use of SCT, t(11;14) without CD20 expression, and age ≥ 70 years to be associated with shorter OS, whereas multivariate analysis demonstrated ISS stage III, use of SCT, and t(11;14) without expression CD20 (HR 1.88; 95% CI 1.10-3.21; P = 0.021) to be independent prognostic factors for poor OS. Conclusions. Our findings demonstrated that patients harboring t(11;14) had distinct clinical and immunophenotypic characteristics, two subsets of the disease entities with a clearly different survival according to CD20 expression. Disclosures Matsue: Ono Pharmaceutical: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Novartis Pharma K.K: Honoraria; Celgene: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria.

IgH translocations with undefined partners are associated with superior outcome in multiple myeloma patients

Multiple myeloma (MM) is a genetically complex and heterogeneous disease with multiple genomic events associated with tumor development and progression.Chromosomal translocations into the immunoglobulin heavy-chain (IgH) locus on 14q32 are the main primary events, occurring in about 50% of total MM cases.Currently, there are five main chromosomal translocation partners involving with immunoglobulin heavy-chain (IgH), including t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20). However, IgH translocation with undefined partner genes can still be found in 15% of all newly-diagnosed MM (NDMM) patient, and little is known about their survival.

PF600 IGH TRANSLOCATIONS WITH UNDEFINED PARTNERS ARE ASSOCIATED WITH SUPERIOR OUTCOME IN MULTIPLE MYELOMA PATIENTS

Background:Multiple myeloma (MM) is a genetically complex and heterogeneous disease with multiple genomic events associated with tumor development and progression.Chromosomal translocations into the immunoglobulin heavy‐chain (IgH) locus on 14q32 are the main primary events, occurring in about 50% of total MM cases. Currently, there are five main chromosomal translocation partners involving with immunoglobulin heavy‐chain (IgH), including t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20). However, IgH translocation with undefined partner genes can still be found in 15% of all newly‐diagnosed MM (NDMM) patient, and little is known about their survival.Aims:In the present study, we examined clinical status, as well as genetic features, and their prognostic significance for predicting PFS and OS, in a cohort consisting of 715 patients with NDMM. Specifically, we focused on characterizing the PFS and OS of patients with NDMM and t(14; undefined) translocation.Methods:A prospective non‐randomized clinical study (BDH 2008/02) was conducted at a single center, with a total of 715 NDMM patients included. The median follow‐up time was 45.3 (42.13–48.47) months. Sorted plasma cells(by anti‐CD138‐coated magnetic beads) were analyzed for del(13q14), del(17p), gains of 1q21, IgH split, t(11;14), t(4;14), t(14;16), t(14;20) and t(6;14). K‐M curves for PFS and OS were plotted; univariate analysis and cox proportional hazards model were conducted.Results:There was about 13.6% patients translocation t(14; undefined) positive. Translocations t(4;14) and t(14;16) were classified into one high‐risk group according to IMWG risk stratification. Translocation t(14;20)(0.9%, 3/322) and t(6;14)(0.8%, 2/228) were assigned to the t(14; undefined) group due to their low incidence. Therefore, the whole cohort (n = 715) was divided into four groups: no IgH group (47.7%, 341/715); t(14; undefined) group (13.6%, 97/715); t(11;14) group (17.6%, 126/715); and t(4;14) or t(14;16) group (21.1%, 151/715). The median overall survival (OS) for the four groups was 84.2 [95% confidence interval (CI), 69.7–98.7] months, not reached (NR), 58.7 (95% CI, 41.9–75.5) months, and 44.2 (95% CI, 34.1–54.3) months, respectively, with p values for the t(14; undefined) group compared with no IgH, t(11;14), and t(4;14)/t(14;16) groups of 0.197, 0.022 and 0.001, respectively(Fig 1). In patients receiving bortezomib‐based therapies, the survival prolongation in the no IgH and t(14;undefined) groups was more significant compared with the t(11;14) and t(4;14)/t(14;16) groups. Importantly, in multivariate analysis, translocation t(14;undefined) was an independent predictive factor for OS of MM patients (HR = 0.51, 95% CI 0.30–0.85, p = 0.01). Collectively, our findings indicated the favorable outcome of NDMM in the t(14; undefined) and no IgH groups compared to the t(11;14) and t(4;14)/t(14;16) groups.Summary/Conclusion:In summary, our data confirmed the favorable prognosis of the t(14; undefined) and no IgH groups, especially in the era of novel agents. Most importantly, translocation t(14; undefined) was identified as an independent protective factor for prolonged OS in multivariate analysis. Our study further validated the short survival of patients with t(4;14)/t(14;16), who are generally classified as high‐risk disease; and conversely, indicated that survival of patients with t(11;14) is decreased relative to other patients with standard‐risk disease.image

Cytogenetic intraclonal heterogeneity of plasma cell dyscrasia in AL amyloidosis as compared with multiple myeloma

AbstractAnalysis of intraclonal heterogeneity has yielded insights into the clonal evolution of hematologic malignancies. We compared the clonal and subclonal compositions of the underlying plasma cell dyscrasia in 544 systemic light chain amyloidosis (PC-AL) patients with 519 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or symptomatic MM; ie, PC–non-AL patients). Using interphase fluorescence in situ hybridization, subclones were stringently defined as clone size below two thirds of the largest clone and an absolute difference of ≥30%. Subclones were found less frequently in the PC-AL group, at 199 (36.6%) of 544 as compared with 267 (51.4%) of 519 in the PC–non-AL group (P < .001), and were not associated with the stage of plasma cell dyscrasia in either entity. In both groups, translocation t(11;14), other immunoglobulin heavy chain translocations, and hyperdiploidy were typically found as main clones, whereas gain of 1q21 and deletions of 8p21, 13q14, and 17p13 were frequently found as subclones. There were no shifts in the subclone/main clone ratio depending on the MGUS, SMM, or MM stage of plasma cell dyscrasia. In multivariate analysis, t(11;14) was associated with lower rates of subclone formation and hyperdiploidy with higher rates. PC-AL itself lost statistical significance, demonstrating that the lower subclone frequency in AL is a reflection of its exceptionally high t(11;14) frequency. In summary, the subclone patterns in PC-AL and PC–non-AL are closely related, implying that subclone formation depends on the main cytogenetic categories and is independent of disease entity and stage.