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The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 μM to more than 200 μM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 μM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo., (© 2014 by The American Society of Hematology.)

Purpose: Although the translocation t(4;14) is supposed to be a primary event in multiple myeloma, we have been surprised to observe that in large relapse series of patients, the t(4;14) can be observed only in subpopulations of plasma cells, in contrast to what is seen at diagnosis. This observation raised the question of possible subclones harboring the translocation that would be observable only at the time of relapse., Experimental Design: To address this issue, we analyzed by FISH a cohort of 306 patients for whom we had at least two samples obtained at different disease phases., Results: We observed a "gain" of the t(4;14) in 14 patients, and conversely, a "loss" of the translocation in 11 patients. Two hypotheses were raised: either an acquisition of the translocation during evolution or the existence of small t(4;14)-positive subclones at the time of diagnosis. To address this question, we had the opportunity to analyze two patients at the time of diagnosis by RT-PCR (reverse transcription-polymerase chain reaction) to look for the chimeric Eμ-MMSET transcript, and one patient positive at diagnosis, but negative at relapse. The samples were positive, supporting the second hypothesis. Furthermore, the IGH sequences of two patients who "lose" the t(4;14) were identical at diagnosis and relapse, confirming the existence of a common ancestral clone., Conclusion: Thus, the conclusion of this study is that the t(4;14) is not a primary event in multiple myeloma and that it can be present in silent subclones at diagnosis, but also at relapse., (©2013 AACR.)

Recent studies have provided direct evidence for genetic variegation in subclones for various cancer types. However, little is known about subclonal evolutionary processes according to treatment and subsequent relapse in multiple myeloma (MM). This issue was addressed in a cohort of 24 MM patients treated either with conventional chemotherapy or with the proteasome inhibitor, bortezomib. As MM is a highly heterogeneous disease associated with a large number of chromosomal abnormalities, a subset of secondary genetic events that seem to reflect progression, 1q21 gain, NF-κB-activating mutations, RB1 and TP53 deletions, was examined. By using high-resolution single-nucleotide polymorphism arrays, subclones were identified with nonlinear complex evolutionary histories. Such reordering of the spectrum of genetic lesions, identified in a third of MM patients during therapy, is likely to reflect the selection of genetically distinct subclones, not initially competitive against the dominant population but which survived chemotherapy, thrived and acquired new anomalies. In addition, the emergence of minor subclones at relapse appeared to be significantly associated with bortezomib treatment. These data support the idea that new strategies for future clinical trials in MM should combine targeted therapy and subpopulations' control to eradicate all myeloma subclones in order to obtain long-term remission.

Deletion of the 17p13 chromosomal region [del(17p)] is associated with a poor outcome in multiple myeloma. Most of the studies have targeted the TP53 gene for deletion analyses, although no study showed that this gene is the deletion target. In order to address this issue, we sequenced the TP53 gene in 92 patients with multiple myeloma at diagnosis, 54 with a del(17p) and 38 lacking del(17p). At least one mutation was found in 20 patients, all of them presenting a del(17p). The analysis of the mutation location showed that virtually all of them occurred in highly conserved domains involved in the DNA-protein interactions. In conclusion, we showed that 37% of the myeloma patients with del(17p) present a TP53 mutation versus 0% in patients lacking the del(17p). The prognostic significance of these mutations remains to be evaluated.

Purpose: Survival of patients with multiple myeloma is highly heterogeneous, from periods of a few weeks to more than 10 years. We used gene expression profiles of myeloma cells obtained at diagnosis to identify broadly applicable prognostic markers., Patients and Methods: In a training set of 182 patients, we used supervised methods to identify individual genes associated with length of survival. A survival model was built from these genes. The validity of our model was assessed in our test set of 68 patients and in three independent cohorts comprising 853 patients with multiple myeloma., Results: The 15 strongest genes associated with the length of survival were used to calculate a risk score and to stratify patients into low-risk and high-risk groups. The survival-predictor score was significantly associated with survival in both the training and test sets and in the external validation cohorts. The Kaplan-Meier estimates of rates of survival at 3 years were 90.5% (95% CI, 85.6% to 95.3%) and 47.4% (95% CI, 33.5% to 60.1%), respectively, in our patients having a low risk or high risk independently of traditional prognostic factors. High-risk patients constituted a homogeneous biologic entity characterized by the overexpression of genes involved in cell cycle progression and its surveillance, whereas low-risk patients were heterogeneous and displayed hyperdiploid signatures., Conclusion: Gene expression-based survival prediction and molecular features associated with high-risk patients may be useful for developing prognostic markers and may provide basis to treat these patients with new targeted antimitotics.

Purpose: Recent development of interphase fluorescence in situ hybridization (FISH) allows analysis on non-proliferant plasma cells. We describe the most frequent genetic abnormalities in multiple myeloma and their prognostic value., Current Knowledge and Key Points: Most frequent genetic abnormalities are illegitimate rearrangements involving the IGH gene at 14q32 (60% of patients), hyperdiploidy (50 to 60% of patients), chromosome 13 deletion (40 to -50% of patients), chromosome 1q gain (30 to -40% of patients) chromosome 17 deletion (10% of patients). Some of these genetics abnormalities are observed in monoclonal gammopathy of undetermined significance (MGUS), a pre-malignant state. t(4;14) and t(14;16) translocations and chromosome 17 deletion negatively impact the overall survival. Patients with these genomic aberrations should be treated with specific treatment., Future Prospects and Projects: Identification of genetic abnormalities is important for evaluation of prognosis and treatment protocol in multiple myeloma.

Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma. However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma). We validated a novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients. The method was very specific and sensitive for the detection of hyperdiploidy. Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%). Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.

In the past decade, many progresses have been made in our knowledge of the genetics of multiple myeloma. The use of molecular cytogenetic techniques has led to the identification of several recurrent (cyto)genetic abnormalities, representing either prognostic markers, or novel therapeutic targets. More global analyses of this genetic heterogeneity using expression array technologies should further extend our understanding of the disease, hopefully enabling some improvements in the treatment of the patients. The goal of this minireview is to summarize these recent advances.

Although most multiple myeloma (MM) cases are characterized by the detection of a monoclonal immunoglobulin in the serum, about 15% of the patients present only immunoglobulin light chains, detected either in the urine or serum or both. These patients are designated as having light-chain (LC) MM. Using fiber-fluorescent in situ hybridization, and in contrast to patients and myeloma cell lines secreting heavy chains (who presented a legitimate functional IgH rearrangement in every case), LC MM never displayed a functional IgH recombination. Interestingly, most LC MM cases presented one IgH allele with a germline configuration (including the DJ region), the second allele being usually involved in an illegitimate recombination. Of note, most of these translocations occurred close to (or at) switch regions, even though in some cases, breakpoints involving nonswitch regions were observed. Thus, this study clearly showed that LC MM is due to the absence of legitimate IgH rearrangement at the DNA level, reflecting possible abnormalities in the IgH gene recombinations during B-cell maturation. Furthermore, it showed that this defect did not prevent the activation of the switch process because most of 14q32 translocations observed in LC MM occurred at switch regions.

Even though the diagnosis of Waldenstrom's macroglobulinemia WM is usually clear, the differential diagnosis with IgM multiple myeloma (MM) might be possible. IgM MM is usually characterized by the accumulation of small mature plasma cells within the bone marrow, and the detection of a monoclonal IgM in the serum. However, in contrast with classical MM, IgM MM is rarely associated with these patients' extensive osteolytic lesions. We analyzed eight cases of IgM MM. None presented with extensive bone lesions. All cases were characterized by the presence of small mature plasma cells within the bone marrow. Molecular cytogenetic analysis revealed a t(11;14) in seven of the eight cases. In contrast, a similar analysis in 17 WM cases failed to detect any t(11;14) cases. We performed further fluorescence in situ hybridization (FISH) experiments, focused on the 14q32 region, and especially on the IgH gene. In contrast to MM (in which illegitimate IgH rearrangements are common), we did not detect any abnormality in the WM cases. In conclusion, even though the cells of origin in WM and MM are mature heavily mutated cells, they differ by the IgH gene rearrangements. Especially in IgM MM, the search for t(11;14) might be useful in difficult cases to discriminate with WM., (Copyright 2003 Elsevier Inc. All rights reserved.)

In an attempt to address the issue of cytogenetic features of multiple myeloma (MM) variants, we have analyzed a series of 8 IgM, 9 IgD, 2 IgE, and 14 nonsecretory (NS) MM cases using fluorescence in situ hybridization. A very high incidence (83%) of t(11;14)(q13;q32) was detected in the IgM (7 of 8), IgE (2 of 2), and NS (11 of 14) MM cases, but not in the IgD cases (2 of 9). Of note, no t(4;14) was observed in this cohort of patients. This increased incidence of t(11;14) was associated with 2 dominant features in these variants, namely, a "lymphoplasmacytic" presentation mainly in IgM MM and a lower secreting capacity in the others, 2 features previously associated with t(11;14). Of major interest, t(11;14) was never observed in Waldenström macroglobulinemia or in IgG/IgA "lymphoplasmacytic" lymphomas. Thus, for unknown reasons, t(11;14) is the hallmark of IgM, IgE, and NS MM, (but not IgD MM), with a 5-fold increase of its incidence compared to that of IgG and IgA MM.

Abstract: Purpose: Recent development of interphase fluorescence in situ hybridization (FISH) allows analysis on non-proliferant plasma cells. We describe the most frequent genetic abnormalities in multiple myeloma and their prognostic value. Current knowledge and key points: Most frequent genetic abnormalities are illegitimate rearrangements involving the IGH gene at 14q32 (60% of patients), hyperdiploidy (50 to 60% of patients), chromosome 13 deletion (40 to –50% of patients), chromosome 1q gain (30 to –40% of patients) chromosome 17 deletion (10% of patients). Some of these genetics abnormalities are observed in monoclonal gammopathy of undetermined significance (MGUS) a pre-malignant state. t(4;14) and t(14;16) translocations and chromosome 17 deletion negatively impact the overall survival. Patients with these genomic aberrations should be treated with specific treatment. Future prospects and projects: Identification of genetic abnormalities is important for evaluation of prognosis and treatment protocol in multiple myeloma. [Copyright &y& Elsevier]