Safe and prolonged survival with long-term exposure to pomalidomide in relapsed/refractory myeloma.

BACKGROUND: The IFM2009-02 trial studied pomalidomide (4 mg daily, 21/28 versus 28/28) and dexamethasone in very advanced relapsed or refractory multiple myeloma (RRMM). We observed that 40% of patients had a prolonged progression-free survival (PFS) and subsequently overall survival (OS). We sought to analyze the characteristics of these patients and study the effect of long exposure to pomalidomide. DESIGN: We separated the studied population into two groups: 3 months to 1 year (<1 year) and more than 1 year (≥1 year) of treatment with pomalidomide and dexamethasone based on clinical judgment and historical control studies. We then analyzed the characteristics of patients according to duration of treatment. RESULTS: The overall response rate (ORR) for the <1-year group was 43%, the median PFS 4.6 months [95% confidence interval (95% CI) 3.8-6.4] with only 6% at 12 months, and the median OS was 15 months (11.7-20.3) and 40% at 18 months. For the ≥1-year group, the response rate and survival were strikingly different, ORR at 83%, median PFS 20.7 months (14.7-35.4), median OS not reached, and 91% at 18 months. CONCLUSION: Pomalidomide and dexamethasone favored prolonged and safe exposure to treatment in 40% of heavily treated and end-stage RRMM, a paradigm shift in the natural history of RRMM characterized with a succession of shorter disease-free intervals and ultimately shorter survival. Although an optimization of pomalidomide-dexamethasone regimen is warranted in advanced RRMM, we claim that pomalidomide has proven once more to change the natural history of myeloma in this series, which should be confirmed in a larger study.

Molecular cytogenetic aberrations in Tunisian patients with multiple myeloma identified by cIg-FISH in fixed bone marrow cells.

Cytogenetic studies in multiple myeloma (MM) are hampered by the hypo-proliferative nature of plasma cells. In order to circumvent this problem, we have used a combination of immunolabeling of cytoplasmic Ig light chains (λ or κ) and FISH (cIg-FISH), which allowed a comprehensive detection of the most common and/or recurrent molecular cytogenetic aberrations on fixed bone marrow cells of 70 Tunisian patients. Translocations involving the chromosome 14q32 region were observed in 32 cases (45.7%), including 18 cases with a t(11;14), 8 cases with a t(4;14), and 2 cases with a t(14;16). Deletions of the 13q14 region (D13S319/RB1) were detected in 18.6%, and deletions of the 17p13 region (TP53) in 5.7% of the cases, respectively. Of all patients with a D13S319/RB1 deletion, 61.5% also carried a 14q32 translocation, whereas TP53 deletions were associated with a t(11;14) in 2 cases (50%) and a D13S319 deletion in 1 case (25%). Our results suggest that there is a correlation between the presence of 14q32 translocations and chromosome 13q14 deletions in MM patients and that cIg-FISH is more sensitive as compared to conventional karyotyping in detecting molecular cytogenetic abnormalities in this disease.

Heterogeneity of t(4;14) in multiple myeloma. Long-term follow-up of 100 cases treated with tandem transplantation in IFM99 trials.

One hundred de novo multiple myeloma patients with t(4;14) treated with double intensive therapy according to IFM99 protocols were retrospectively analyzed. The median overall survival (OS) and event-free survival (EFS) were 41.4 and 21 months, respectively, as compared to 65 and 37 for patients included in the IFM99 trials without t(4;14) (P<10(-7)). We identified a subgroup of patients presenting at diagnosis with both low beta(2)-microglobulin <4 mg/l and high hemoglobin (Hb) >/=10 g/l (46% of the cases) with a median OS of 54.6 months and a median EFS of 26 months, respectively, which benefits from high-dose therapy (HDT); conversely patients with one or both adverse prognostic factor (high beta(2)-microglobulin and/or low Hb) had a poor outcome. The achievement of either complete response or very good partial response after HDT was also a powerful independent prognostic factor for both OS and EFS.

Heavy+light chain monitoring correlates with clinical outcome in multiple myeloma patients.

Novel anti-myeloma agents have improved patient response rates, which are historically based on reductions of the M-protein. These methods can be inaccurate for quantifying M-proteins at low concentrations. We compared the consistency and clinical impact of response assignment by electrophoretic and heavy+light chain (HLC) immunoassays post-consolidation in 463 newly diagnosed patients. The two methods gave similar assignments in patients with partial (PR; 79% agreement) or complete response (⩾CR; 92%). However, in patients achieving very good PR (VGPR) there was poor concordance between methods (45%). Median progression-free survival (PFS) for standard VGPR patients was 34.5 months; HLC responses stratified these patients further into PR, VGPR and ⩾CR, with median PFS of 21.3, 28.9 months and not reached, respectively; P<0.001. At this time, abnormal HLC ratios had better concordance with multiparametric flow cytometry (sensitivity 10-4) (37 and 34% positive, respectively), compared to immunofixation (62% positive). In addition, HLC-pair suppression was identified in 38% of patients and associated with shorter PFS (30.6 months vs not reached; P<0.001). We conclude that HLC monitoring could augment electrophoretic assessments in patients achieving VGPR. The prognostic significance of HLC responses might partly depend on the patients' ability to recover their immune system, as determined by normalisation of HLC measurements.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

[Genetic abnormalities in multiple myeloma: role in oncogenesis and impact on survival]. / Anomalies génétiques dans le myélome: rôle dans l'oncogenèse et implications pronostiques.

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.

Expressed fusion gene landscape and its impact in multiple myeloma.

Multiple myeloma is a plasma cell malignancy characterized by recurrent IgH translocations and well described genomic heterogeneity. Although transcriptome profiles in multiple myeloma has been described, landscape of expressed fusion genes and their clinical impact remains unknown. To provide a comprehensive and detailed fusion gene cartography and suggest new mechanisms of tumorigenesis in multiple myeloma, we performed RNA sequencing in a cohort of 255 newly diagnosed and homogeneously treated multiple myeloma patients with long follow-up. Here, we report that patients have on average 5.5 expressed fusion genes. Kappa and lambda light chains and IgH genes are main partners in a third of all fusion genes. We also identify recurrent fusion genes that significantly impact both progression-free and overall survival and may act as drivers of the disease. Lastly, we find a correlation between the number of fusions, the age of patients and the clinical outcome, strongly suggesting that genomic instability drives prognosis of the disease.

Prevention and management of adverse events of Novel agents in multiple myeloma: A consensus of the european myeloma network.

During the last few years, several new drugs have been introduced for treatment of patients with multiple myeloma, which have significantly improved treatment outcome. All of these novel substances differ at least in part in their mode of action from similar drugs of the same drug class, or are representatives of new drugs classes, and as such present with very specific side effect profiles. In this review, we summarize these adverse events, provide information on their prevention, and give practical guidance for monitoring of patients and for management of adverse events.Leukemia accepted article preview online, 18 December 2017. doi:10.1038/leu.2017.353.

Genetic heterogeneity in multiple 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.

Ploidy, as detected by fluorescence in situ hybridization, defines different subgroups 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.

Mcl-1 is overexpressed in multiple myeloma and associated with relapse and shorter survival.

We and others have shown that Mcl-1 was essential for the survival of human myeloma cells in vitro. Furthermore, this antiapoptotic protein is upregulated by interleukin-6, which plays a critical role in multiple myeloma (MM). For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC), that is, myeloma cells from 51 patients with MM and 21 human myeloma cell lines (HMCL) using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. In total, 52% of patients with MM at diagnosis (P=0.017) and 81% at relapse (P=0.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only HMCL but not reactive plasmacytoses have abnormal Mcl-1 expression, although both PC expansions share similar high proliferation rates. Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. Finally, the level of Mcl-1 expression is related to disease severity, the highest values at diagnosis being associated with the shortest event-free survival (P=0.002). In conclusion, Mcl-1, which has been shown to be essential for the survival of human myeloma cells in vitro, is overexpressed in vivo in MM in relation with relapse and shorter survival. Mcl-1 represents a potential therapeutical target in MM.

14q32 translocations and monosomy 13 observed in monoclonal gammopathy of undetermined significance delineate a multistep process for the oncogenesis of multiple myeloma. Intergroupe Francophone du Myélome.

Clonal plasma cells in monoclonal gammopathy of undetermined significance (MGUS) have been shown to bear copy number chromosome changes. To extend our knowledge of MGUS to structural chromosomal abnormalities, we have performed fluorescence in situ hybridization experiments with probes directed to the 14q32 and 13q14 chromosomal regions in 100 patients with either MGUS or smoldering multiple myeloma (SMM). 14q32 abnormalities were observed in at least 46% of patients with MGUS/SMM, with these abnormalities being present in the majority of clonal plasma cells. Whereas t(11;14)(q13;q32) occurs in 15% of MGUS/SMM patients, an incidence similar to that of overt multiple myeloma (MM) patients, translocation t(4;14)(p16;q32) is observed in only 2% of these cases [P = 0.002 for difference with t(11;14)], as compared with 12% in MM patients (P = 0.013). Monoallelic deletions of the 13q14 region were found in 21% of patients, with two types of situations. In half of the evaluable patients, and especially in patients with SMM, the deletion is present in the majority of clonal plasma cells, as in MM, whereas in the other half of the evaluable patients (essentially in MGUS patients), it is observed in subclones only. These data enable us to elaborate a plasma cell oncogenesis model from MGUS to MM.

High incidence of translocations t(11;14)(q13;q32) and t(4;14)(p16;q32) in patients with plasma cell malignancies.

Abnormalities involving the 14q32 region are recurrent chromosomal changes in plasma cell malignancies. Recent preliminary molecular analyses found IGH rearrangements in almost 100% of human myeloma cell lines and in 75% of patients. However, no systematic study analyzing the nature of the partner chromosomal regions have been reported thus far. To define the exact incidence of illegitimate IGH rearrangements and the respective incidence of partner genes cloned to date, we analyzed 141 patients with either multiple myeloma (MM, n = 127) or primary plasma cell leukemia (PCL, n = 14) using fluorescence in situ hybridization. The overall incidence of illegitimate recombinations was 57% (80 of 141 patients). Analysis of this incidence according to Durie and Salmon stage, patients' status, i.e., MM versus primary PCL and diagnosis versus relapse, immunoglobulin type and subtype, and beta2-microglobulin value, did not show any correlation. To analyze the nature of the partner chromosomal region, we selected probes specific for the following genes: FGFR3 (4p16), MYC (8q24), CCND1 (11q13), MAF (16q23), and BCL2 (18q21). These probes, combined with differentially labeled 14q32 probes, were used for dual-color fluorescence in situ hybridization on interphase plasma cells. Among the 80 patients with illegitimate IGH rearrangement, we identified 23 IGH-CCND1 fusion cases [i.e., t(11;14)], 17 IGH-FGFR3 fusion cases [i.e., t(4;14)], 3 IGH-MYC fusion cases [i.e., t(8;14)], and only one IGH-MAF fusion case. No IGH-BCL2 fusion case was detected. In 37 of 80 patients, none of these partner genes was involved. Analysis of cases with specific translocations according to their bioclinical features at diagnosis did not show any correlation. This study demonstrated that CCND1 and FGFR3 genes are involved together in about 50% of MM and primary PCL patients with illegitimate IGH rearrangements.

Gene signature combinations improve prognostic stratification of multiple myeloma patients.

Multiple myeloma (MM) is a plasma cell neoplasm with significant molecular heterogeneity. Gene expression profiling (GEP) has contributed significantly to our understanding of the underlying biology and has led to several prognostic gene signatures. However, the best way to apply these GEP signatures in clinical practice is unclear. In this study, we investigated the integration of proven prognostic signatures for improved patient risk stratification. Three publicly available MM GEP data sets that encompass newly diagnosed as well as relapsed patients were analyzed using standardized estimation of nine prognostic MM signature indices and simulations of signature index combinations. Cox regression analysis was used to assess the performance of simulated combination indices. Taking the average of multiple GEP signature indices was a simple but highly effective way of integrating multiple GEP signatures. Furthermore, although adding more signatures in general improved performance substantially, we identified a core signature combination, EMC92+HZDCD, as the top-performing prognostic signature combination across all data sets. In this study, we provided a rationale for gene signature integration and a practical strategy to choose an optimal risk score estimation in the presence of multiple prognostic signatures.

A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma.

Fluorescence in situ hybridization on peripheral-blood specimens is a reliable method to evaluate cytogenetic response in chronic myeloid leukemia.

PURPOSE: To evaluate the usefulness of fluorescence in situ hybridization (FISH) on peripheral-blood specimens to evaluate the cytogenetic response to treatment in patients with chronic myeloid leukemia (CML). PATIENTS AND METHODS: In a first attempt, we analyzed 62 bone marrow specimens using interphase FISH and compared the results with those of conventional cytogenetics. In a second step, we analyzed 60 paired sets of bone marrow and peripheral-blood specimens with interphase FISH. RESULTS: The results of interphase FISH agreed with conventional cytogenetics on bone marrow for most patients, and only minor differences were found (r =.98). The comparison of interphase FISH on bone marrow versus peripheral-blood specimens showed a strong correlation between these two specimen sources (r =.97). CONCLUSION: Our results confirmed that FISH is a sensitive technique for the evaluation of response to treatment in patients with CML. Moreover, our study suggests that follow-up of cytogenetic response to therapy can be evaluated on peripheral-blood specimens, thus enabling an easier and more frequent evaluation of patients. The next step will be to evaluate this technique in a large prospective trial to define the prognostic value of complete remissions evaluated by FISH.

N-Myc gene amplification is a major prognostic factor in localized neuroblastoma: results of the French NBL 90 study. Neuroblastoma Study Group of the Société Francaise d'Oncologie Pédiatrique.

PURPOSE: To assess the relevance of N-Myc gene amplification (NMA) as a prognostic factor in localized neuroblastoma (NB) and to evaluate whether less intensive adjuvant treatment is advisable in infants without NMA. PATIENTS AND METHODS: Assessment of NBs included clinical and imaging data to allow tumor-node-metastasis (TNM) staging, biologic determinations (N-Myc gene analysis), and standard histology and work-up to eliminate metastatic spread (metaiodobenzylguanidine [MIBG] scintigraphy and extensive bone marrow staging). Resectability was defined according to imaging findings. Chemotherapy was indicated in children older than 1 year at diagnosis who had postoperative residual disease or lymph node (LN) involvement, in infants with NMA, or as primary treatment in children with an unresectable NB, including dumbbell tumors. Radiotherapy was recommended in children older than 1 who presented with persistent gross residual disease at the end of therapy. RESULTS: Between 1990 and 1994, 316 consecutive children who presented with a localized NB were registered in the NBL 90 study. The median age was 12 months, and 42 patients had dumbbell tumors (13%). NMA was found in 22 of 225 assessable children (10%) and correlated with adverse prognostic indicators such as age older than 1 year, an abdominal primary tumor, a large tumor (T3), and unresectability. Among 186 children who had primary excision, five died of surgery-related complications. Primary chemotherapy was given to 130 patients, which allowed removal of the tumor in all but four. The 5-year overall survival (OS) and event-free survival (EFS) rates were, respectively, 91% and 84% with a median follow-up time of 36 months. The outcome of infants and older children was similar (P = .2). EFS of patients with resectable tumors was slightly better than with unresectable primary tumors (EFS, 89% v 78%; P = .02). In dumbbell NBs, neurologic recovery was achieved in 74% of cases that presented with symptoms, and initial laminectomy was avoided in 75% of children. In a univariate analysis, large tumors, high neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) levels, positive LNs, macroscopic residue, and NMA adversely influenced outcome. In the multivariate analysis, NMA was the most powerful unfavorable predictive indicator: OS and EFS rates for these children were 36% and 32%, compared with 98% and 90% in nonamplified tumors (P < .001). CONCLUSION: Our data confirm the overall good prognosis of localized NBs, even when unresectable. NMA is the most relevant adverse prognostic factor in localized NBs, and more intensive treatment should be investigated in these patients. Prospective studies of other biologic factors are warranted to tailor therapy more accurately. The EFS of children who underwent primary surgery was excellent, and further justifies elimination of adjuvant treatment provided they have no NMA. Despite the elimination of postoperative therapy, infants with non-NMA tumors have an excellent outcome, which suggests that initial chemotherapy can be further reduced in case of unresectable NBs.

t(11;14) and t(4;14) translocations correlated with mature lymphoplasmacytoid and immature morphology, respectively, in multiple myeloma.

Recent studies have shown that two recurrent translocations, t(4;14)(p16;q32) and t(11;14)(q13;q32), define distinct entities with different prognosis in multiple myeloma (MM). We addressed the issue of whether these illegitimate IGH rearrangements could contribute to the morphological heterogeneity of the malignant plasma cells (PC). Bone marrow aspirates of 178 untreated MM cases with successful molecular cytogenetics analysis using fluorescence in situ hybridization were reviewed. PC of 25/48 (52%) patients with t(11;14) exhibited a lymphoplasmacytoid morphology. Moreover, 25/27 (93%) of the cases with this morphological profile bore the t(11;14). In addition, both cytogenetics and morphological subtypes shared higher incidence of nonsecretory MM. In contrast, 17 out of 28 cases (61%) with t(4;14) exhibited PC with diffuse chromatin pattern. Interestingly, both t(4;14) translocation and immature morphology correlated with higher incidence of high tumor mass and chromosome 13 abnormality. In conclusion, our results suggest that a particular morphology can be the signature of chromosomal abnormalities in MM.

Is t(14;18)(q32;q21) a constant finding in follicular lymphoma? An interphase FISH study on 63 patients.

The translocation (14;18)(q32;q21) is the hallmark of follicular lymphoma (FL). However, conventional cytogenetics and PCR techniques fail to detect it in at least 10% of cases. In order to evaluate the true incidence of this translocation in FL, we analyzed 63 patients with FL, and 17 patients with diffuse large cell lymphoma (DLCL) corresponding to suspected FL transformations using interphase fluorescence in situ hybridization (FISH). Colocalized signals related to the translocation were observed in 19-92% of cells (median = 51%), corresponding to positivity over the threshold in all (63/63) cases. Similarly, 16/17 possibly secondary DLCL displayed the translocation. Although some cytogenetic changes might be missed by this FISH assay (such as rare insertion, or translocations with other chromosomal partners), our results stress t(14;18)(q32;q21) as an almost constant finding in FL. Our sensitive interphase FISH assay should be of great value to define FL more accurately, namely in patients included into therapeutic trials. Furthermore, this approach could be of interest in (re)defining some types of FL, especially the grade 3 FL which frequently lack t(14;18).