Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3.

Dysregulation of oncogenes by translocation to the IgH locus (14q32) is a seminal event in the pathogenesis of B-cell tumours. In multiple myeloma (MM), translocations to the IgH locus have been reported at an incidence of 20-60%. For most translocations, the partner chromosome is unknown (14q+); for the others, a diverse array of chromosomal partners have been identified, with 11q13 (cyclin D1) the only chromosome that is frequently involved. Recently, we developed a Southern-blot assay that detects translocation breakpoint fragments in most MM tumours, including those with no translocation detected by conventional karyotyping. In a continuing analysis of translocation in 21 myeloma cell lines and primary tumours, we show that the novel, karyotypically silent translocation t(4;14)(p16.3;q32.3) is present in five lines and at least three of ten primary tumours. The chromosome-4 breakpoints are clustered in a 70-kb region centromeric to the fibroblast growth factor receptor 3 gene (FGFR3), the apparent dysregulated oncogene. Two lines and one primary tumour with this translocation selectively express an FGFR3 allele containing activating mutations identified previously in thanatophoric dwarfism. We propose that after the t(4;14) translocation, somatic mutation during tumour progression frequently generates in FGFR3 protein that is active in the absence of ligand.

A practical guide to defining high-risk myeloma for clinical trials, patient counseling and choice of therapy.

Clinical outcomes for multiple myeloma (MM) are highly heterogeneous and it is now clear that pivotal genetic events are the primary harbingers of such variation. These findings have broad implications for counseling, choice of therapy and the design and interpretation of clinical investigation. Indeed, as in acute leukemias and non-hodgkins lymphoma, we believe it is no longer acceptable to consider MM a single disease entity. As such, the accurate diagnosis of MM subtypes and the adoption of common criteria for the identification and stratification of MM patients has become critical. Herein, we provide a consensus high-risk definition and offer practical guidelines for the adoption of routine diagnostic testing. Although acknowledging that more refined classifications will continue to be developed, we propose that the definition of high-risk disease (any of the t(4;14), t(14;16), t(14;20), deletion 17q13, aneuploidy or deletion chromosome 13 by metaphase cytogenetics, or plasma cell labeling index >3.0) be adopted. This classification will identify most of the 25% of MM patients for whom current therapies are inadequate and for whom investigational regimens should be vigorously pursued. Conversely, the 75% of patients remaining have more favorable outcomes using existing - albeit non-curative - therapeutic options.

Pomalidomide-dexamethasone in refractory multiple myeloma: long-term follow-up of a multi-cohort phase II clinical trial.

Despite therapeutic advances, multiple myeloma remains incurable, with limited options for patients with refractory disease. We conducted a large, multi-cohort clinical trial testing various doses and treatment schedules of pomalidomide and dexamethasone (Pom/dex) in patients with refractory multiple myeloma. Overall, 345 patients were enrolled to six cohorts based on number and type of prior lines of therapy, pomalidomide dose and schedule. Median prior lines of therapy were three with near universal prior exposure to proteasome inhibitors and/or immunomodulatory drugs. A confirmed response rate of 35% was noted for all cohorts (range 23-65%) with higher responses in cohorts with fewer prior lines of therapy. Median time to confirmed response was ⩽2 months and the longest progression-free survival and overall survival seen in any cohort were 13.1 and 47.9 months, respectively. Observed adverse reactions were as expected, with myelosuppression and fatigue being the most common hematologic and non-hematologic adverse events (AEs), respectively. Longer durations of treatment and response, higher response rates and fewer AEs were noted with the 2 mg pomalidomide dose. This is the longest follow-up data for Pom/dex in refractory multiple myeloma and will help shape the real-world utilization of this regimen.

Genetic events in the pathogenesis of multiple myeloma.

The genetics of myeloma has been increasingly elucidated in recent years. Recurrent genetic events, and also biologically distinct and clinically relevant genetic subtypes of myeloma have been defined. This has facilitated our understanding of the molecular pathogenesis of the disease. In addition, some genetic abnormalities have proved to be highly reproducible prognostic factors. With the expanding therapeutic armamentarium, it is time to include genetic assessment as part of clinical evaluation of myeloma patients to guide management. In this review we examine the role of various genetic abnormalities in the molecular pathogenesis of myeloma, and the use of such abnormalities in disease classification, prognosis and clinical management.

Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations.

Chromosomal hyperdiploidy is the defining genetic signature in 40-50% of myeloma (MM) patients. We characterize hyperdiploid-MM (H-MM) in terms of its clinical and prognostic features in a cohort of 220 H-MM patients entered into clinical trials. Hyperdiploid-myeloma is associated with male sex, kappa immunoglobulin subtype, symptomatic bone disease and better survival compared to nonhyperdiploid-MM (median overall survival 48 vs 35 months, log-rank P = 0.023), despite similar response to treatment. Among 108 H-MM cases with FISH studies for common genetic abnormalities, survival is negatively affected by the existence of immunoglobulin heavy chain (IgH) translocations, especially those involving unknown partners, while the presence of chromosome 13 deletion by FISH did not significantly affect survival (median overall survival 50 vs 47 months, log-rank P = 0.47). Hyperdiploid-myeloma is therefore a unique genetic subtype of MM associated with improved outcome with distinct clinical features. The existence of IgH translocations but not chromosome 13 deletion by FISH negatively impacts survival and may allow further risk stratification of this population of MM patients.

Prognostic value of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma.

A specific role for increased level of expression of CKS1B, as a consequence of chromosome 1q21 copy number gain, has been postulated as both pathogenic, as well as a powerful clinical prognostic factor in multiple myeloma (MM). The purpose of this study is to determine the clinical associations and prognostic impact of copy number gain at chromosome 1q21 (with a bacteria artificial chromosome clone containing CKS1B) and CKS1B gene level of expression in MM. We studied the chromosome region 1q21 for copy number change in a cohort of myeloma patients treated by high-dose therapy with stem-cell rescue (HDT) (n = 159). A separate cohort of patients, treated by HDT was studied for CKS1B messenger RNA expression by gene expression profiling (n = 67). 1q21 gain was then correlated with clinical parameters and survival. Gain of 1q21 copy number was detected in about a third of MM and was associated with more proliferative disease and poor-risk cytogenetic categories such as t(4;14), and chromosome 13 deletion. Both 1q21 gain and increase gene expression level were significantly associated with reduced survival. However, neither is an independent prognostic marker in MM on multivariate Cox proportional hazard analysis.

Changes in uninvolved immunoglobulins during induction therapy for newly diagnosed multiple myeloma.

Little is known about the impact of multiple myeloma (MM) treatment on uninvolved immunoglobulins (Ig). We identified 448 patients who received high-dose dexamethasone (HD-DEX), lenalidomide and dexamethasone (RD), bortezomib and dexamethasone (VD), bortezomib, cyclophosphamide and dexamethasone (VCD) or bortezomib, lenalidomide and dexamethasone (VRD) for newly diagnosed MM at our institution between 2000 and 2013, and who had available data on absolute lymphocyte count (ALC) and quantitative uninvolved Ig at baseline and at the end of four cycles of therapy. Changes in ALC and uninvolved Ig were significantly different across treatments, with VCD and HD-DEX producing reductions in uninvolved Ig, and RD, VD and VRD leading to increases in uninvolved Ig. In addition, treatment with RD, VD and VRD was independently associated with higher odds of achieving a ⩾25% increase in or normalization of the primary uninvolved Ig on multivariate analysis. Although achievement of a humoral response in the primary uninvolved Ig was associated with a higher odds of achieving VGPR or better after four cycles of therapy, it was not associated with improved overall survival. These data highlight the different mechanisms of action of MM drugs and point toward a possible role for the use of VCD in treating antibody-mediated autoimmune disease.

Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Ploidy status rarely changes in myeloma patients at disease progression.

Hyperdiploid and non-hyperdiploid multiple myeloma represents distinct biological entities characterized by different patterns of genetic changes. We sought to determine whether ploidy category (non-hyperdiploid versus hyperdiploid) remains stable over time from diagnosis to progression. Of the 43 patients studied (39 by flow cytometry DNA index and 4 by a FISH-based index), only five (12%) altered their ploidy status at progression. In three of these patients, the change may possibly be attributable to technical artifacts because of the low absolute change in DNA index. For those who retain their ploidy subtypes, the DNA index change minimally (3.75+/-4.87%). It would appear that the initiating genetic events underlying hyperdiploid and non-hyperdiploid MM that marks them out as distinct entities continue to dominate and persist during disease evolution and progression.

Selective expression of one c-myc allele in two human myeloma cell lines.

Chromosomal translocations or DNA rearrangements affecting c-myc occur in almost all murine plasmacytoma and human Burkitt's lymphoma tumors and are associated with a high incidence of exon 2 missense mutations and selective expression of the affected allele. Screening nine multiple myeloma cell lines, we identified no exon 2 missense mutations but did identify two lines with single, silent mutations in exon 1 and exon 2, respectively. Each of these informative multiple myeloma cell lines selectively expresses only one c-myc allele despite the apparent absence of chromosomal translocations or DNA rearrangements affecting c-myc.

WWOX, the FRA16D gene, behaves as a suppressor of tumor growth.

We recently reported the cloning of WWOX, a gene that maps to the common fragile site FRA16D region in chromosome 16q23.3-24.1. It was observed that the genomic area spanned by WWOX is affected by chromosomal translocations and homozygous deletions. Furthermore, the high incidence of allelic loss in breast, ovarian, prostate, and other cancers affecting this region suggests that WWOX is a candidate tumor suppressor gene. Expression of WWOX is highly variable in breast cancer cell lines, with some cases showing low or undetectable levels of expression. In this report, we demonstrate that ectopic WWOX expression strongly inhibits anchorage-independent growth in soft agar of breast cancer cell lines MDA-MB-435 and T47D. Additionally, we observed that WWOX induces a dramatic inhibition of tumorigenicity of MDA-MB-435 breast cancer cells when tested in vivo. We also detected the common occurrence of aberrant WWOX transcripts with deletions of exons 5-8 or 6-8 in various carcinoma cell lines, multiple myeloma cell lines, and primary breast tumors. These aberrant mRNA forms were not detected in normal tissues. Interestingly, we further observed that proteins encoded by such aberrant transcripts display an abnormal nuclear localization in contrast to the wild-type WWOX protein that localizes to the Golgi system. Our data indicate that WWOX behaves as a potent suppressor of tumor growth and suggest that abnormalities affecting this gene at the genomic and transcriptional level may be of relevance in carcinogenesis.

Carfilzomib and the cardiorenal system in myeloma: an endothelial effect?

Carfilzomib (Cfz) has been associated with an ~5% incidence of unexplained and unpredictable cardiovascular toxicity in clinical trials. We therefore implemented a detailed, prospective, clinical cardiac and renal evaluation of 62 Cfz-treated myeloma patients, including serial blood pressure (BP), creatinine, troponin, NT-proBNP and pre- and post-treatment echocardiograms, including ejection fraction (EF), average global longitudinal strain and compliance. Pre-treatment elevations in NT-proBNP and BP, as well as abnormal cardiac strain were common. A rise in NT-proBNP occurred frequently post-treatment often without corresponding cardiopulmonary symptoms. A rise in creatinine was common, lessened with hydration and often reversible. All patients had a normal EF pre-treatment. Five patients experienced a significant cardiac event (four decline in EF and one myocardial infarction), of which 2 (3.2%) were considered probably attributable to Cfz. None were rechallenged with Cfz. The ideal strategy for identifying patients at risk for cardiac events, and parameters by which to monitor for early toxicity have not been established; however, it appears baseline echocardiographic testing is not consistently predictive of toxicity. The toxicities observed suggest an endothelial mechanism and further clinical trials are needed to determine whether or not this represents a class effect or is Cfz specific.

Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients.

We employed a customized Multiple Myeloma (MM)-specific Mutation Panel (M(3)P) to screen a homogenous cohort of 142 untreated MM patients for relevant mutations in a selection of disease-specific genes. M(3)Pv2.0 includes 77 genes selected for being either actionable targets, potentially related to drug-response or part of known key pathways in MM biology. We identified mutations in potentially actionable genes in 49% of patients and provided prognostic evidence of STAT3 mutations. This panel may serve as a practical alternative to more comprehensive sequencing approaches, providing genomic information in a timely and cost-effective manner, thus allowing clinically oriented variant screening in MM.

Evolving changes in disease biomarkers and risk of early progression in smoldering multiple myeloma.

We studied 190 patients with smoldering multiple myeloma (SMM) at our institution between 1973 and 2014. Evolving change in monoclonal protein level (eMP) was defined as ⩾10% increase in serum monoclonal protein (M) and/or immunoglobulin (Ig) (M/Ig) within the first 6 months of diagnosis (only if M-protein ⩾3 g/dl) and/or ⩾25% increase in M/Ig within the first 12 months, with a minimum required increase of 0.5 g/dl in M-protein and/or 500 mg/dl in Ig. Evolving change in hemoglobin (eHb) was defined as ⩾0.5 g/dl decrease within 12 months of diagnosis. A total of 134 patients (70.5%) progressed to MM over a median follow-up of 10.4 years. On multivariable analysis adjusting for factors known to predict for progression to MM, bone marrow plasma cells ⩾20% (odds ratio (OR)=3.37 (1.30-8.77), P=0.013), eMP (OR=8.20 (3.19-21.05), P<0.001) and eHb (OR=5.86 (2.12-16.21), P=0.001) were independent predictors of progression within 2 years of SMM diagnosis. A risk model comprising these variables was constructed, with median time to progression of 12.3, 5.1, 2.0 and 1.0 years among patients with 0-3 risk factors respectively. The 2-year progression risk was 81.5% in individuals who demonstrated both eMP and eHb, and 90.5% in those with all three risk factors.

Chromosome translocations in multiple myeloma.

Multiple myeloma (MM), a malignant tumor of somatically mutated, isotype-switched plasma cells (PC), usually arises from a common benign PC tumor called Monoclonal Gammopathy of Undetermined Significance (MGUS). MM progresses within the bone marrow, and then to an extramedullary stage from which MM cell lines are generated. The incidence of IgH translocations increases with the stage of disease: 50% in MGUS, 60-65% in intramedullarly MM, 70-80% in extramedullary MM, and >90% in MM cell lines. Primary, simple reciprocal IgH translocations, which are present in both MGUS and MM, involve many partners and provide an early immortalizing event. Four chromosomal partners appear to account for the majority of primary IgH translocations: 11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (FGFR3 and MMSET), and 16q23 (c-maf). They are mediated primarily by errors in IgH switch recombination and less often by errors in somatic hypermutation, with the former dissociating the intronic and 3' enhancer(s), so that potential oncogenes can be dysregulated on each derivative chromosome (e.g., FGFR3 on der14 and MMSET on der4). Secondary translocations, which sometimes do not involve Ig loci, are more complex, and are not mediated by errors in B cell specific DNA modification mechanisms. They involve other chromosomal partners, notably 8q24 (c-myc), and are associated with tumor progression. Consistent with MM being the malignant counterpart of a long-lived PC, oncogenes dysregulated by primary IgH translocations in MM do not appear to confer an anti-apoptotic effect, but instead increase proliferation and/or inhibit differentiation. The fact that so many different primary transforming events give rise to tumors with the same phenotype suggests that there is only a single fate available for the transformed cell.

Sequence and expression of a murine cDNA encoding PC326, a novel gene expressed in plasmacytomas but not normal plasma cells.

Using a subtractive cDNA approach we have identified a gene, PC326, expressed in 13 of 14 murine plasmacytoma cell lines, but not in any B- or pre-B-lymphoma cell lines. It expresses 4.6-kb and 5.2-kb mRNAs that encode a 747 amino acid protein containing two highly acidic domains flanking a novel, moderately acidic 20 amino acid sequence that is repeated 7.5 times. Sequence comparison identifies an additional 43 amino acid domain that is homologous to a repeated sequence found in the members of the beta-transducin gene family. The PC326 mRNA is detectable in testis but in no other murine tissues, including plasma cells induced by lipopolysaccharide stimulation of splenocytes. Somatic cell hybrids derived from plasmacytomas and fibroblast or T-cell lines have a fibroblastic or T-cell phenotype respectively. Unlike B-cell-specific genes (e.g. immunoglobulin), the expression of which is extinguished in these hybrids, PC326 mRNA appears to be irreversibly turned on in these hybrids. Since PC326 is not expressed in normal plasma cells, it appears that its expression is a cause or consequence of the tumorigenic process that generates murine plasmacytomas.

Identification of consensus genes expressed in plasmacytomas but not B lymphomas.

We have combined subtractive cDNA and PCR technologies to construct and analyze a plasmacytoma minus a highly differentiated B lymphoma subtractive cDNA library. We detected no plasmacytoma-specific clones by hybridization with differential cDNA probes or the subtractive insert. However, random selection of 115 clones has identified 16 quantitatively subtractive and 39 qualitatively subtractive clones. From these clones we have identified 8 potentially interesting genes. One quantitatively subtractive clone (clone 315) identifies an mRNA that is expressed in most plasmacytoma cell lines, but is expressed at an approximately 10-fold lower level in B and pre-B lymphoma cell lines; preliminary evidence suggests that the expression of this gene is increased by IL-6. From the 31 unrelated qualitatively subtractive clones, we have identified two classes of genes that are expressed in one or none of 8 B lymphomas examined: 1) those expressed in most plasmacytoma and pre-B lymphoma cell lines (clone 70 and clone 260); and 2) those expressed in most plasmacytoma cell lines, but not in any of the ten pre-B lymphomas examined (clones 251, 289A, 289B, 326, 291).

Genes expressed selectively in plasmacytomas: markers of differentiation and transformation.

We have analyzed a murine plasmacytoma minus highly differentiated B lymphoma subtractive cDNA library and identified eight genes that are expressed in most plasmacytomas but at a much lower level, or not at all, in most B lymphomas. Four of the genes are markers of the terminal differentiation of B lymphocytes into plasma cells: placental alkaline phosphatase, also expressed in pre-B lymphomas xlr-3, a new X-linked member of the xlr multi-gene family EGP314, a pan-epithelial glycoprotein with sequence features of an adhesion molecule PC315, a gene that is up-regulated by IL6, but without obvious sequence homologies. Two of the genes are not clearly related to normal B cell differentiation, appearing to be associated with malignant transformation of plasma cells: PC326 is a new member of the beta-transducin mosaic protein gene family. It is an X-linked gene, expressed at a very low level in testis, but in no other normal tissue, including LPS- or IL6-induced plasma cells. It has a high level of expression (apparently dysregulated) in most (> 85%) mineral oil induced plasmacytomas. However the likelihood that PC326 is expressed decreases as the tumor latency decreases when different retroviral agents are used to accelerate mineral oil induced plasmacytomagenesis. This suggests that PC326 expression may be a late event in a multi-step process of tumorigenesis. PC251 a new member of the hematopoietic growth factor receptor family, most homologous to IL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Dysregulation of c-myc in multiple myeloma.

Translocation of c-myc to IgH switch regions, or less frequently to one of the IgL loci, is essentially an invariant event in murine plasmacytomas. This results in dysregulation of c-myc, manifested by selective expression of the translocated allele. Human multiple myeloma (MM) has a similarly high incidence of translocations involving IgH switch regions, but c-myc is infrequently involved as a partner in these translocations. However, in screening a panel of 20 MM cell lines, we identified six lines containing two genetically distinguishable c-myc alleles. For these six informative lines (and the corresponding tumor for one line) there is selective expression of one c-myc allele despite the apparent absence of translocation, DNA rearrangement, or amplification involving c-myc. This result suggests frequent tumor specific cis-dysregulation of c-myc in MM by a presently unknown mechanism.

IgH translocations in multiple myeloma: a nearly universal event that rarely involves c-myc.

Dysregulation of c-myc by translocation to the switch regions of the IgH locus occurs in most murine plasmacytomas. Translocations involving 14q32 have been reported in 20-40% of abnormal karyotypes from human multiple myeloma (MM), and involve a variety of loci. Using cytogenetics, FISH and a Southern blot assay, we analyzed 21 MM cells lines and one plasma cell leukemia and identified evidence of a 14q32 translocation in 20/22 samples. The partner loci involved are 11q13 in 6 (associated with cyclin D1 expression), 4p16 in 6 (associated with FGFR3 expression), unidentified in 3 and 1p13, 6, 8q24, 12q24, 16q23, and 21q22 once each. We conclude that conventional karyotypes underestimate the frequency of 14q32 translocations in MM, where they appear to be a nearly universal event. The translocations most frequently involve IgH switch regions, and include two recurrent partner loci (11q13 and 4p16) and a promiscuous array of other partner loci. Although c-myc appears to be cis-dysregulated frequently in MM, it is only rarely translocated to the IgH locus.