Chromosomal Translocation In Multiple Myeloma Research Articles

Different Translocations of Multiple Myeloma on Fluorescent In Situ Hybridization (FISH) with Clinical Correlation.

To evaluate the association of chromosomal translocations in multiple myeloma (MM) detected by Fluorescent In Situ Hybridization (FISH) and its clinical characteristics. Cross-sectional study. Place and Duration of the Study: Department of Haematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from February to August 2022. A total of 40 cases of MM were included. All cases were diagnosed using international myeloma working group (IMWG) criteria. Clinical presentations like bone pain, backache, fatigue, pallor, and weight loss were noted. The workup for myeloma-defining events was done. FISH analysis was done for t (4;14), t (11,14), t (14;16), t (14;20), and del 17p. Data were analysed using the chi-square test. A p-value ≤0.05 was considered statistically significant. Out of 40 patients, 8 (20%) were females and 32 (80%) were males. The highest frequency of cases were noted among males in the age group >60 years and females in the age group 40-60 years. FISH for t (4;14) was positive in 22 (55%) patients, for t (11;14) was positive in 4 (10%) patients, for t (14;16) was positive in 3 (7.5%) patients, and for t (14;20) was positive in 3 (7.5%) patients, while for del17p was positive in 8 (20%) patients. Cases with t (4;14), t (11;14), and t (14;20) had bone pain, fatigue, and backache as the most common presentations. Among the various parameters studied, lytic lesions, beta-2 microglobulin, spike protein, deranged haemoglobin, TLC, ESR, albumin, and creatinine were significant risk factors in patients who were tested positive for various mutations. The FISH technique has brought an immense uprising in the genetic analysis of MM. Among translocations, t (4;14) and del17p are associated with poor clinical outcomes and prognosis. If the diagnosis of MM is delayed, then an increase in morbidity and mortality can occur. Multiple myeloma, FISH, Translocations.

Current Testing Practices for t(11;14) Rearrangements in Patients with Newly Diagnosed Multiple Myeloma in the United States

Background: Despite an improvement in the awareness and use of precision medicine and molecular testing in hematological cancers, there are still significant disparities in biomarker testing. This ultimately leads to inequality in molecular testing access and can lead to suboptimal medical care for some patients, who do not receive the most appropriate and effective therapy. Venetoclax is a selective BCL-2 inhibitor and has shown promising antimyeloma activity in patients with t(11;14) multiple myeloma (MM). t(11;14) is the most frequent chromosomal translocation in MM reported in approximately 15-20% of newly diagnosed MM patients (NDMM) (Kumar et al. Blood 2017). In this study, we assessed current t(11;14)(q13;q32) testing practices in patients with NDMM in the United States (US) to gain insights on challenges for biomarker testing and equitable access to precision medicine. Methods: A retrospective, real-world evidence study was conducted between January and December 2021 using the Diaceutics DXRX Data Repository database. DXRX is a patient-level claims- and laboratory-linked dataset covering more than 4000 laboratories and 360 million patient lives between 2011 and 2022 in the US. Eligible patients were identified using International Classification of Disease (ICD) codes for NDMM (ICD-10 code C90.0). A 2-year lookback was utilized to ensure patients were not in remission or relapse (ICD-10 codes C90.01, C90.02). Percentages were used to describe study population characteristics. t(11;14) and plasma cell enrichment (PCE) testing rates were estimated using descriptive statistics for the entire cohort as well as for race/ethnicity subgroups. PCE testing was considered as it contributes to increasing sensitivity of testing. Results: We identified 27,586 eligible patients with NDMM. The median age at diagnosis was 70 years (interquartile range: 65-75) and 45% were female. Among the eligible patients, 69% (n=19,034) specifically received t(11;14) testing, within 1 month from diagnosis to testing. Fluorescence in situ hybridization (FISH; dual color, dual fusion probe and immunoglobin [IG] break apart) was the most commonly utilized technology (89%; n=16,940) for detecting t(11;14); next-generation sequencing panels were limited (<1%) in utilization. Confirmatory reflex testing with a t(11;14) dual probe following positive IG break apart was not performed in 17% (n=3235) of patients. In patients who were tested with the t(11;14) dual probe FISH assay, less than half (40.9%) were tested for t(11;14) with PCE. Results of the FISH assay were available for 4941 patients. A lower positivity rate was observed with non-PCE testing (13.1%; n=647) compared with those utilizing PCE (16.8%; n=830), p=0.494. Testing rates of t(11;14) in an academic setting were 72% (n=10,659) vs 64% (n=8374) in the community setting, p<0.0001. PCE was performed on only 24.9% (n=2093) of patients in a community setting vs 61% (n=6502) of samples in an academic setting, p<0.0001. For the subgroup analysis, race/ethnicity data were available for 9245 patients with NDMM; 75.7% were White, 15.9% were Black, 2.6% were Asian, 2% were Hispanic, and 3.2% were classified as other. t(11;14) testing rates were higher for patients who were White (72.8%) compared with the minority groups (66.9% for patients who were Black, 68.8% for patients who were Asian, and 65.7% for patients who were Hispanic). The t(11;14) testing setting for minority group patients was community (62%) vs academic (38%). Conclusions: Notable variations in t(11;14) testing patterns were observed among patients with NDMM in different care settings and racial/ethnic groups. There is a clear unmet need for standardization across academic and community settings to mitigate suboptimal testing methodologies that could impact treatment decision-making and patient outcomes. From our study, there is a clear need to improve t(11;14) testing rates as the clinical utility changes from a prognostic to predictive biomarker with the availability of personalized healthcare options. Education of PCE utilization for physicians, especially community-based pathologists and laboratories, will be essential to reduce false negatives and inequalities in optimal testing, especially PCE in ethnic minorities in the US.

Metabolic Effects of Recurrent Genetic Aberrations in Multiple Myeloma.

Simple SummaryCancer is associated with metabolic changes related to increased cell proliferation and growth. These cancer-related metabolic features are largely dictated by specific oncogenes that are activated by chromosomal aberrations and epigenetic alterations in cancer cells. Multiple myeloma is an incurable plasma cell malignancy, which is characterized by recurrent chromosomal aberrations that drive the expression of established oncogenes such as MYC, Cyclin D1, FGFR3/MMSET and MAF/MAFB. In this review, we discuss the specific metabolic features of multiple myeloma plasma cells, and focus on the metabolic consequences of recurrent chromosomal aberrations, thereby providing an outline for the metabolic alterations that characterize multiple myeloma. Oncogene activation and malignant transformation exerts energetic, biosynthetic and redox demands on cancer cells due to increased proliferation, cell growth and tumor microenvironment adaptation. As such, altered metabolism is a hallmark of cancer, which is characterized by the reprogramming of multiple metabolic pathways. Multiple myeloma (MM) is a genetically heterogeneous disease that arises from terminally differentiated B cells. MM is characterized by reciprocal chromosomal translocations that often involve the immunoglobulin loci and a restricted set of partner loci, and complex chromosomal rearrangements that are associated with disease progression. Recurrent chromosomal aberrations in MM result in the aberrant expression of MYC, cyclin D1, FGFR3/MMSET and MAF/MAFB. In recent years, the intricate mechanisms that drive cancer cell metabolism and the many metabolic functions of the aforementioned MM-associated oncogenes have been investigated. Here, we discuss the metabolic consequences of recurrent chromosomal translocations in MM and provide a framework for the identification of metabolic changes that characterize MM cells.

The snoRNA target of t(4;14) in multiple myeloma regulates ribosome biogenesis.

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

Impact of t(11;14) on the Outcome of Autologous Transplantation in Multiple Myeloma: A Matched-Pair Analysis

Abstract:Background:The translocation t(11;14)(q13;q32) is the most common chromosomal translocation in multiple myeloma (MM) with a reported frequency of 15-20%. Most, but not all, reports in the literature have identified this translocation with a relatively favorable outcome. In a smaller cohort, we had previously reported the impact of t(11;14) on the outcomes of MM patients who underwent autologous hematopoietic transplantation (auto-HCT) at our institution1. In this study, we present an updated analysis with a larger cohort of t(11;14) patients, and compared their outcomes to a propensity-matched control group of MM patients with normal diploid cytogenetics and no high-risk abnormalities detected by Fluorescence in Situ Hybridization (FISH).Methods and patients:A total of 1365 MM patients underwent auto-HCT at our institution from 2007 to 2015. We identified 95 patients with t(11;14) by FISH before auto-HCT. Out of these 95 patients, 44 had t(11;14) alone, 26 had co-existent high-risk abnormalities and remaining 25 had standard-risk abnormalities. The control group included 287 MM patients with normal diploid cytogenetics and no abnormalities detected by FISH. From the above two cohorts, using a 1:1 propensity score-matched analysis without replacement, we identified matched controls for 79 patients with t(11;14). Clinical response, relapse, and progression were defined by the International Myeloma Working group criteria. The FISH technique was performed using a LSI IGH/CCND1 XT dual color, dual fusion translocation probe from Abbott Molecular, Inc. The normal cut off for IGH/MYEOV/CCND1 rearrangement using LSI IGH/CCND1 XT probe established at 95 (P<0.05) confidence level in the our Cytogenetics Laboratory is 0.4% for 1R1G2F; 1.1% for 2R2G1F; 3.4% for 3R2G; 7.4% for 2R3G and 0% for 1R3F signal patterns.Results:Table 1 includes the baseline characteristics of the matched doublets. Patients in both groups were well matched for age, ISS stage, serum creatinine, response to induction therapy, induction and preparative regimens, and maintenance therapy. The median follow-up time for the cohort was 48.1 months (range: 0.8-124.6). The overall response rate (CR+VGPR+PR) after auto-HCT was 77/79 (97%) and 78/79 (99%) patients in the t(11;14) and the control group, respectively (P = 1.00). Twenty eight (35%) and 37 (47%) patients achieved a CR in the t(11;14) and the control group, respectively. VGPR rate in the t(11;14) and the control group was 35 (44%) and 31 (39%), respectively. The median PFS for the t(11;14) and the control groups were 33.3 (95%CI: 25.8-not reached) and 39.7 (95%CI: 36.6-not reached) months, respectively (p = 0.24, stratified log-rank test). The median OS has not been reached for both groups (p=0.35). The 4-year PFS rates in the t(11;14) and the control groups were 42% (95%CI: 31%-57%) and 50% (95%CI: 38%-65%), respectively (Fig. A). The 4-year OS rates in the t(11;14) and the control groups were 76% (95%CI: 65%-90%) and 87% (95%CI: 79%-97%), respectively (Fig. B).Conclusions:On a propensity score matching analysis, multiple myeloma patients with translocation t(11;14) had similar response rates, PFS and OS to auto-HCT as standard-risk patients with normal cytogenetics or FISH studies.

Distinct and shared three-dimensional chromosome organization patterns in lymphocytes, monoclonal gammopathy of undetermined significance and multiple myeloma.

The consistent appearance of specific chromosomal translocations in multiple myeloma has suggested that the positioning of chromosomes in the interphase nucleus might play a role in the occurrence of particular chromosomal rearrangements associated with malignant transformation. Using fluorescence in situ hybridization, we have determined the positions of selected chromosome pairs (18 and 19, 9 and 22, 4 and 14, 14 and 16, 11 and 14) in interphase nuclei of myeloma cells compared to normal lymphocytes of treatment‐naïve patients. All chromosome pairs were arranged in a nonrandom pattern. Chromosomes commonly involved in myeloma‐associated translocations (4 and 14, 14 and 16, 11 and 14) were found in close spatial proximity, and this is correlated with the occurrence of overlapping chromosome territories. The spatial distribution of chromosomes may increase the possibility of chromosomal translocations in multiple myeloma.

IRF-4 Suppresses BCR/ABL Transformation of Myeloid Cells in a DNA Binding-independent Manner

Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4-interacting protein found in the cytoplasm. However, IRF-4 tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain.

IRF-4 Suppresses BCR/ABL Transformation of Myeloid Cells in a DNA-Binding Independent Manner

Abstract 1404Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. The finding of IRF-4 functioning as a tumor suppressor raises caution for developing therapies aiming to downregulate IRF-4. Further studies on the mechanisms by which IRF-4 functions as an oncoprotein and as a tumor suppressor are necessary to develop improved therapies for malignancies involving IRF-4. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4 interacting protein found in the cytoplasm. However, IRF-4's tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain and suggest that the oncogenic and tumor suppressor functions of IRF-4 involve distinct pathways. Disclosures:No relevant conflicts of interest to declare.

Cooperation between deficiencies of IRF-4 and IRF-8 promotes both myeloid and lymphoid tumorigenesis

AbstractInterferon regulatory factor 4 (IRF-4) plays important functions in B- and T-cell development and immune response regulation and was originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. Although IRF-4 is expressed in myeloid cells, its function in that lineage is not known. The closely related family member IRF-8 is a critical regulator of myelopoiesis, which when deleted in mice results in a syndrome highly similar to human chronic myelogenous leukemia. In early lymphoid development, we have shown previously that IRF-4 and IRF-8 can function redundantly. We therefore investigated the effects of a combined loss of IRF-4 and IRF-8 on hematologic tumorigenesis. We found that mice deficient in both IRF-4 and IRF-8 develop from a very early age a more aggressive chronic myelogenous leukemia-like disease than mice deficient in IRF-8 alone, correlating with a greater expansion of granulocyte-monocyte progenitors. Although these results demonstrate, for the first time, that IRF-4 can function as tumor suppressor in myeloid cells, interestingly, all mice deficient in both IRF-4 and IRF-8 eventually develop and die of a B-lymphoblastic leukemia/lymphoma. Combined losses of IRF-4 and IRF-8 therefore can cooperate in the development of both myeloid and lymphoid tumors.

IRF-4 functions as a tumor suppressor in early B-cell development

Interferon regulatory factor-4 (IRF-4) is a hematopoietic cell-restricted transcription factor important for hematopoietic development and immune response regulation. It was also originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. In contrast to its oncogenic function in late stages of B lymphopoiesis, expression of IRF-4 is down-regulated in certain myeloid and early B-lymphoid malignancies. In this study, we found that the IRF-4 protein levels are increased in lymphoblastic cells transformed by the BCR/ABL oncogene in response to BCR/ABL tyrosine kinase inhibitor imatinib. We further found that IRF-4 deficiency enhances BCR/ABL transformation of B-lymphoid progenitors in vitro and accelerates disease progression of BCR/ABL-induced acute B-lymphoblastic leukemia (B-ALL) in mice, whereas forced expression of IRF-4 potently suppresses BCR/ABL transformation of B-lymphoid progenitors in vitro and BCR/ABL-induced B-ALL in vivo. Further analysis showed that IRF-4 inhibits growth of BCR/ABL+ B lymphoblasts primarily through negative regulation of cell-cycle progression. These results demonstrate that IRF-4 functions as tumor suppressor in early B-cell development and may allow elucidation of new molecular pathways significant to the lymphoid leukemogenesis by BCR/ABL. The context dependent roles of IRF-4 in oncogenesis should be an important consideration in developing cancer therapies targeting IRF-4.

IRF-4 Functions as a Tumor Suppressor in Early Stages of B Cell Development.

Interferon regulatory factor 4 (IRF-4) is a hematopoietic cell restricted transcription factor important for hematopoietic development and immune response regulation. IRF-4 is essential for mature B and T lymphocyte development, dendritic cell differentiation and some macrophage functions. It was also originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. Recent studies show that expression of IRF-4 is essential for the maintenance of multiple myeloma cells, making IRF-4 an attractive target for the development of cancer therapies. In addition to its role in late stages of lymphocyte development, IRF-4 functions redundantly with its closely related family member, IRF-8, to promote differentiation at the pre-B to immature B transition, apparently by repressing the pre-B cell receptor signaling and promoting light chain gene rearrangement. In contrast to its oncogenic properties, expression of IRF-4 is downregulated in BCR/ABL positive acute B-lymphoblastic leukemia (B-ALL) and is increased in response to imatinib, a selective BCR/ABL tyrosine kinase inhibitor. To assess the role of IRF-4 in the pathogenesis of BCR/ABL-positive B-ALL, we examined the effect of both loss of and forced expression of IRF-4 in BCR/ABL leukemogenesis using a mouse bone marrow transduction and transplantation model system. We found that BCR/ABL induced B-ALL significantly faster in IRF-4-deficient cells than control cells. On the other hand, forced expression of IRF-4 potently suppressed BCR/ABL transformation of bone marrow derived B lymphoid precursors in vitro and BCR/ABL induced B-ALL in vivo. These results provide evidence that IRF-4 functions as tumor suppressor in early B cell development and may allow elucidation of new molecular pathways significant to the pathogenesis of BCR/ABL positive B-ALL. In a parallel study, we have shown that IRF-4 functions as a tumor suppressor in myeloid lineage. The context dependent roles of IRF-4 in oncogenesis should raise caution in the development of cancer therapies targeting IRF-4. Further dissecting the mechanisms by which IRF-4 functions as an oncogene or a tumor suppressor gene is crucial for developing therapeutic strategies precisely targeting the IRF-4 oncogenic pathway as well as for developing therapeutic strategies taking advantage of the IRF-4 tumor suppressor pathway.

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

Common chromosomal fragile site FRA16D mutation in cancer cells

Neither the molecular basis for common fragile site DNA instability nor the contribution of this form of chromosomal instability to cancer is clearly understood. Fragile site FRA16D (16q23.2) is within regions of frequent loss-of-heterozygosity (LOH) in breast and prostate cancers, is associated with homozygous deletions in various adenocarcinomas and t(14;16) chromosomal translocations in multiple myeloma. The FOR (WWOX) gene spans FRA16D and encodes a partner of p53 that also has a role in apoptosis. Previously untested 53 cancer cell lines were screened for deletions within the FOR/WWOX gene. Deletions were detected in Co115, KM12C and KM12SM. Homozygous deletions in these and two previously identified tumour cell lines were intragenic on both alleles, indicating a distinct mutation mechanism from that causing LOH. Identical FRA16D deletions in two cell lines (one derived from the primary carcinoma and the other from a secondary metastasis) demonstrate that FRA16D DNA instability can be an early, transient event. Sequence analysis across one deletion locates one endpoint within a polymorphic AT-dinucleotide repeat and the other adjacent to an AT-rich mini-satellite repeat implicating AT-rich repeats in FRA16D DNA instability. Another deletion is associated with de novo repetition of the 9 bp AT-rich sequence at one of the deletion endpoints. FRA16D deleted cells retain cytogenetic fragile site expression indicating that the deletions are susceptible sites for breakage rather than regions that confer fragility. Most cell lines with FRA16D homozygous deletions also have FRA3B deletions, therefore common fragile sites represent highly susceptible genome-wide targets for a distinct form of mutation.

MUM1/IRF4 expression is an unfavorable prognostic factor in B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL).

B‐Cell chronic lymphocytic leukemia (B‐CLL)/small lymphocytic lymphoma (SLL) consists of heterogeneous diseases that are distinguished by morphological, immunophenotypic and molecular features. MUM1 (multiple myeloma oncogene 1) is a protooncogene that is deregulated as a result of (6;14)(p25;q32) chromosomal translocation in multiple myeloma, and is also expressed in a variety of malignant lymphoma entities. We examined the expression of MUM1 in B‐CLL/SLL, and found that 2 of 4 B‐CLL‐derived cell lines and 14 of 29 patients' specimens expressed MUM1 by immunohistochemical analysis. MUM1 expression was not associated with CD38 expression, somatic hypermutation of immunoglobulin heavy chain gene variable region (IgVH), or any other clinical characteristics of the patients. Interestingly, the patients who were positive for MUM1 showed shorter overall survival tunes than those who were negative for MUM1 (50% survival: 22 months vs. 82 months) (P=0.0008, log‐rank test). Multivariate analysis by Cox's proportional‐hazards regression model showed that MUM1 expression and unmutated IgVH status were independent unfavorable prognostic factors in patients with B‐CLL/SLL. These findings suggest that MUM1 expression is a useful prognostic factor in B‐CLL/SLL. The biological role and mechanism of action of MUM1 in B‐CLL/SLL need to be clarified for the development of therapies for patients with the poor prognostic subtype.

Detection of MUM1/IRF4-IgH fusion in multiple myeloma.

MUM1 (multiple myeloma oncogene 1)/IRF4 (interferon regulatory factor 4) gene has been identified as an oncogene transcriptionally activated by t(6;14)(p25;q32) chromosomal translocation in multiple myeloma (MM). The significance of this alteration in MM remains unknown, as it is not detectable by means of conventional cytogenetic analysis. To address this issue, we established diagnostic procedures based on pulsed-field gel electrophoresis (PFGE) analysis and double color fluorescence in situ hybridization (DCFISH) using DNA probes derived from the MUM1 and the immunoglobulin heavy chain (IgH) gene loci. Among a panel of 17 MM cell lines, three (17.6%) showed fusions between these two loci, which resulted in the juxtaposition of the MUM1 to the IgH 3' alpha-enhancer region by virtue of t(6;14) or insertion of the IgH sequences into the vicinity of the MUM1 gene and in the concomitant overexpression of the MUM1 mRNA. With similar results, fusions between MUM1 and IgH loci were observed by means of interphase DCFISH in eight (21.1%) out of the 38 MM cases, although no definite relationships between MUM1 status and specific clinical findings could be established.

Clinical Significance of the Translocation (11;14)(q13;q32) in Multiple Myeloma

The most common chromosomal translocation in multiple myeloma (MM) is t(11;14)(q13;q32). Here, we describe the clinical characteristics of patients with MM who have this translocation. We have identified 24 patients at our institution who had t(11;14)(q13;q32) as determined by standard cytogenetic analysis (CC). Seven patients had the translocation detected at the time of original diagnosis and 17 at the time of relapse. Median survival in all patients after original diagnosis was 43 months; median survival after the translocation was detected was 11.9 months. Four patients had a clinical diagnosis of plasma cell leukemia. Most patients had an elevated β2-microglobulin (13/20 had ≥4 μg/ml). The bone marrow (BM) labeling index (LI) of patients, at the time of translocation detection, was elevated in most (median 1.4%, 17/23 patients had BMLI ≥1%). Of the 24 patients, 19 (79%) died of disease progression and 5 (21 %) were alive with disease at last follow-up. Lytic lesions, bone pain, or compression fractures eventually developed in all patients. Patients with MM who have t(11;14)(q13;q32) detected by standard cytogenetics seem to have an aggressive clinical course.

A Novel Chromosomal Translocation t(4; 14)(p16.3; q32) in Multiple Myeloma Involves the Fibroblast Growth-Factor Receptor 3 Gene

AbstractChromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), the most consistent chromosomal abnormality is the 14q+ marker, which originates in one third of cases through a t(11; 14)(q13; q32) chromosomal translocation; in the remaining cases, the identity of the partner chromosomes has not been well established. We used a Southern blot approach based on the linkage analysis of the joining (J) and the constant (C) μ, α, and γ regions to detect cases bearing IGH switch-mediated chromosomal translocations. We evaluated DNA of 88 nonkaryotyped patients with MM (78 cases) or plasma cell leukemia (PCL) (10 cases) and found the presence of “illegitimate” rearranged IGH fragments (no comigration between the J and C regions) in 21 cases. To confirm this analysis, we cloned the illegitimate rearranged fragments from three samples, and the molecular and fluorescent in situ hybridization (FISH) analyses indicated the presence of chromosomal translocations juxtaposing a switch IGH region to sequences from chromosomes 11q13 (one PCL case) or 4p16.3 (two MM cases). Interestingly, the breakpoints on 4p16.3 occurred about 14 kb apart in a genomic region located approximately 50 kb centromeric to the fibroblast growth-factor receptor 3 (FGFR3) gene. Moreover, Southern blot analysis using 4p16.3 genomic probes detected a rearrangement in an additional MM tumor. FISH analysis of the MM-derived KMS-11 cell line, reported to be associated with a t(4; 14)(p16.3; q32), showed that the FGFR3 gene was translocated on 14q32. High levels of FGFR3 mRNA expression were observed in the cloned MM tumors and KMS-11 cell line, but not in the cases that were apparently negative for this lesion. Furthermore, a point mutation at codon 373 in the transmembrane domain of the FGFR3 gene resulting in an amino acid substitution (Tyr → Cys) was detected in the KMS-11 cell line. These findings indicate that the t(4; 14)(p16.3; q32) represents a novel, recurrent chromosomal translocation in MM, and suggest that the FGFR3 gene may be the target of this abnormality and thus contribute to tumorigenesis in MM.

Deregulation of MUM1/IRF4 by chromosomal translocation in multiple myeloma.

The pathogenesis of multiple myeloma (MM), an incurable tumour causing the deregulated proliferation of terminally differentiated B cells, is unknown. Chromosomal translocations (14q1) affecting band 14q32 and unidentified partner chromosomes are common in this tumour, suggesting that they may cause the activation of novel oncogenes. By cloning the chromosomal breakpoints in an MM cell line, we show that the 14q+ translocation represents a t(6;14)x(p25;q32) and that this aberration is recurrent in MM, as it was found in two of eleven MM cell lines. The translocation juxtaposes the immunoglobulin heavy-chain (IgH) locus to MUM1 (multiple myeloma oncogene 1)/IRF4 gene, a member of the interferon regulatory factor (IRF) family known to be active in the control of B-cell proliferation and differentiation. As a result, the MUM1/IRF4 gene is overexpressed--an event that may contribute to tumorigenesis, a MUM1/IRF4 has oncogenic activity in vitro. These findings identify a novel genetic alteration associated with MM, with implications for the pathogenesis and diagnostics of this tumour.

The Ig Heavy Chain Gene Is Frequently Involved in Chromosomal Translocations in Multiple Myeloma and Plasma Cell Leukemia as Detected by In Situ Hybridization

AbstractChromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing γ constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11; 14)(q13.3; q32.33) was detected in 5 patients, t(8; 14)(q24.1; q32.33) in 2, t(14; 18)(q32.33; q21.3) in 2, and t(7; 14)(q32.1; q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7; 14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.

The Ig Heavy Chain Gene Is Frequently Involved in Chromosomal Translocations in Multiple Myeloma and Plasma Cell Leukemia as Detected by In Situ Hybridization

Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing γ constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11; 14)(q13.3; q32.33) was detected in 5 patients, t(8; 14)(q24.1; q32.33) in 2, t(14; 18)(q32.33; q21.3) in 2, and t(7; 14)(q32.1; q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7; 14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.