Multiple myeloma cells' capacity to decompose H2O2 determines lenalidomide sensitivity.

Lenalidomide is an immunomodulatory drug (IMiDs) with clinical efficacy in multiple myeloma (MM) and other late B-cell neoplasms. Although cereblon (CRBN) is an essential requirement for IMiD action, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated sensitivity or resistance remain unknown. Here, we report that IMiDs work primarily via inhibition of peroxidase-mediated intracellular H2O2 decomposition in MM cells. MM cells with lower H2O2-decomposition capacity were more vulnerable to lenalidomide-induced H2O2 accumulation and associated cytotoxicity. CRBN-dependent degradation of IKZF1 and IKZF3 was a consequence of H2O2-mediated oxidative stress. Lenalidomide increased intracellular H2O2 levels by inhibiting thioredoxin reductase (TrxR) in cells expressing CRBN, causing accumulation of immunoglobulin light-chain dimers, significantly increasing endoplasmic reticulum stress and inducing cytotoxicity by activation of BH3-only protein Bim in MM. Other direct inhibitors of TrxR and thioredoxin (Trx) caused similar cytotoxicity, but in a CRBN-independent fashion. Our findings could help identify patients most likely to benefit from IMiDs and suggest direct TrxR or Trx inhibitors for MM therapy.

Evaluation of Revised International Staging System (R-ISS) for transplant-eligible multiple myeloma patients.

The International Myeloma Working Group has proposed the Revised International Staging System (R-ISS) for risk stratification of multiple myeloma (MM) patients. There are a limited number of studies that have validated this risk model in the autologous stem cell transplant (ASCT) setting. In this retrospective study, we evaluated the applicability and value for predicting survival of the R-ISS model in 134 MM patients treated with new agents and ASCT at the Mayo Clinic in Arizona and the University Hospital of Salamanca in Spain. The patients were reclassified at diagnosis according to the R-ISS: 44 patients (33%) had stage I, 75 (56%) had stage II, and 15 (11%) had stage III. After a median follow-up of 60 months, R-ISS assessed at diagnosis was an independent predictor for overall survival (OS) after ASCT, with median OS not reached, 111 and 37 months for R-ISS I, II and III, respectively (P < 0.001). We also found that patients belonging to R-ISS II and having high-risk chromosomal abnormalities (CA) had a significant shorter median OS than those with R-ISS II without CA: 70 vs. 111 months, respectively. Therefore, this study lends further support for the R-ISS as a reliable prognostic tool for estimating survival in transplant myeloma patients and suggests the importance of high-risk CA in the R-ISS II group.

Prevalence of BCL-2/J(H) Translocation in Healthy African Americans.

The translocation t(14;18)(q32;q21) (BCL-2/J(H)) is present in over 80 % of all follicular lymphomas and is detectable in peripheral blood lymphocytes (PBL) of healthy individuals. The prevalence of this translocation has not been studied in African Americans (AAs). Given the higher incidence of follicular lymphomas in whites compared to AAs in the United States (USA), we hypothesized that the translocation prevalence in the blood of AAs would be lower. DNA was isolated from PBL from blood samples collected from participants from FL. Polymerase chain reaction was performed on the BCL-2/J(H) major (MBC) and minor breakpoint cluster (mBC) regions. Eight of the 77 (10.4 %) blood samples from AA participants were positive for MBC (95 % CI, 4.6-19.5 %), and three (3.9 %) were positive for mBC (95 % CI, 0.81-10.97 %) of BCL-2/J(H), with a total of 11 (14.3 %) participants with positive samples (95 % CI, 7.35-24.13 %). In 167 white patient samples, 22 (13.2 %; 95 % CI, 8.44-19.26 %) were positive for MBC, and five (3.0 %; 95 % CI, 0.98-6.85 %) were positive for mBC, with a total of 25 (15 %) participants with positive samples (CI, 9.93-21.30 %). The prevalence of t(14;18)(q32;q21) is not significantly different among AAs and whites from the USA. The lower prevalence of follicular lymphomas in AAs compared with whites is likely a result of differences in secondary molecular alterations involved in follicular lymphoma development. This study is the first report of prevalence of t(14;18) in an AA cohort.

Uncovering the biology of multiple myeloma among African Americans: a comprehensive genomics approach.

Epidemiological data have suggested that African American (AA) persons are twice as likely to be diagnosed with multiple myeloma (MM) compared with European American (EA) persons. Here, we have analyzed a set of cytogenetic and genomic data derived from AA and EA MM patients. We have compared the frequency of IgH translocations in a series of data from 115 AA patients from 3 studies and 353 EA patients from the Eastern Cooperative Oncology Group (ECOG) studies E4A03 and E9487. We have also interrogated tumors from 45 AA and 196 EA MM patients for somatic copy number abnormalities associated with poor outcome. In addition, 35 AA and 178 EA patients were investigated for a transcriptional profile associated with high-risk disease. Overall, based on this cohort, genetic profiles were similar except for a significantly lower frequency of IgH translocations (40% vs 52%; P = .032) in AA patients. Frequency differences of somatic copy number aberrations were not significant after correction for multiple testing. There was also no significant difference in the frequency of high-risk disease based on gene expression profiling. Our study represents the first comprehensive comparisons of the frequency and distribution of molecular alterations in MM tumors between AA and EA patients. ECOG E4A03 is registered with ClinicalTrials.gov, number NCT00098475. ECOG E9487 is a companion validation set to the ECOG study E9486 and is registered with the National Institutes of Health, National Cancer Institute, Clinical Trials (PDQ), number EST-9486.

Impact of gene expression profiling-based risk stratification in patients with myeloma receiving initial therapy with lenalidomide and dexamethasone.

Detection of specific chromosomal abnormalities by FISH and metaphase cytogenetics allows risk stratification in multiple myeloma; however, gene expression profiling (GEP) based signatures may enable more specific risk categorization. We examined the utility of 2 GEP-based risk stratification systems among patients undergoing initial therapy with lenalidomide in the context of a phase 3 trial. Among 45 patients studied at baseline, 7 (16%) and 10 (22%), respectively, were high-risk using the GEP70 and GEP15 signatures. The median overall survival for the GEP70 high-risk group was 19 months versus not reached for the rest (hazard ratio = 14.1). Although the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk patients. The C-statistic for the GEP70, GEP15, and FISH based risk stratification systems was 0.74, 0.7, and 0.7, respectively. Here we demonstrate the prognostic value for GEP risk stratification in a group of patients primarily treated with novel agents. This trial was registered at www.clinicaltrials.gov as #NCT00098475.

Impact of high-risk classification by FISH: an eastern cooperative oncology group (ECOG) study E4A03.

Lenalidomide with dexamethasone is a standard induction treatment regimen for newly diagnosed myeloma (although a Federal Drug Administration indication is still absent). In the context of the Phase 3 clinical trial E4A03 (lenalidomide plus dexamethasone in low or high doses), we queried whether a fluorescence in situ hybridization (FISH)-based genetic classification into high risk (HR) and standard risk (SR) multiple myeloma (MM) would remain clinically significant. Of 445 E4A03 patients, 126 had FISH analysis; 21 were classified HR with t(4;14), t(14;16), or 17p13 deletions. Median survival follow-up approached 3 years. Patients with FISH data tended to be younger and healthier compared to the rest of the study population and, consequently, had superior overall survival (OS) results. Within the FISH cohort, shorter OS in the HR versus SR group (P = 0·004) corresponded to a hazard ratio of 3·48 [95% confidence interval: (1·42-8·53)], an effect also observed in multivariate analysis. Two-year OS rates were 91% for SR MM and 76% for HR MM. There was also evidence of interaction between risk status and treatment (P = 0·026). HR patients were less likely to attain good partial response (SR 46% and HR 30%, Odds Ratio = 2·0 [0·7-5·6]), but overall response rates were not different. FISH-based risk classification retained prognostic significance in patients receiving lenalidomide-based induction.

6q deletion discriminates Waldenström macroglobulinemia from IgM monoclonal gammopathy of undetermined significance.

IgM monoclonal gammopathy of undetermined significance (IgM MGUS) and Waldenström macroglobulinemia (WM) are sometimes clinically difficult to distinguish. In our previous study, deletion of the long arm of chromosome 6 (6q) was found in about half of WM patients. To further clarify the area of minimal deletion at 6q (6q-) and to address the issue of whether 6q- occurs in IgM MGUS, 12 IgM MGUS and 38 WM patients were studied by fluorescence in situ hybridization using probes targeting different chromosomal segments of 6q. No 6q deletions were found in IgM MGUS samples. Of 38 successfully studied WM patients, 21 (55%) showed a deletion of 6q. The area of minimal deletion was between 6q23 and 6q24.3, but the deletion usually encompassed a large fragment of the 6q arm. These results indicate that 6q- can distinguish WM from IgM MGUS and is likely to be a secondary event.

Biological and prognostic significance of interphase fluorescence in situ hybridization detection of chromosome 13 abnormalities (delta13) in multiple myeloma: an eastern cooperative oncology group study.

Chromosome 13 abnormalities (Delta13) have been associated with an unfavorable prognosis in patients with multiple myeloma (MM). The significance of this has been unresolved because of diverse methods of detection and heterogeneous groups of patients. We conducted a study of Delta13 in patients entered into the Eastern Cooperative Oncology Group trial E9486/E9487. Patients with newly diagnosed MM (median follow-up of survivors >100 months) were studied for Delta13, using bone marrow samples obtained at study enrollment. We used interphase fluorescence in situ hybridization with the probes LSI13 (Rb)/D13S319 with simultaneous immunofluorescence detection of bone marrow plasma cells (PCs). We detected Delta13 in 176 of 325 (54%) evaluable patients. Patients with Delta13 were more likely to have a serum monoclonal protein at a concentration < or =1 g/dl (22 versus 13%; P = 0.04), light-chain-only MM (19.3 versus 10.8%; P = 0.04), gamma light chain (42 versus 28%; P = 0.027), stage III (56 versus 42%; P = 0.014), and be female (60 versus 50%; P = 0.087). The PC labeling index and Delta13 correlated (P = 0.03). Patients with Delta13 were less likely to respond to treatment (74 versus 63%; P = 0.041) and had a significantly shorter median overall survival (34.9 versus 51 months; P = 0.021). The association of Delta13 and survival remained an independent prognostic variable in a regression model. Among patients with Delta13, those receiving IFN had a worse overall survival that those not receiving the medication (P = 0.03). The presence of Delta13 is an important and independent adverse prognostic factor in newly diagnosed MM and is associated with specific biological features.

Deletions of 17p13.1 and 13q14 are uncommon in Waldenström macroglobulinemia clonal cells and mostly seen at the time of disease progression.

Waldenström macroglobulinemia (WM) is a plasma cell dyscrasia characterized by a monoclonal IgM paraproteinemia. Deletions of 17p13.1 and 13q14 are associated with tumor progression and worsened outcome in multiple myeloma (MM), and we thus investigated WM patients for their presence. Patients (n = 40) were required to have a > or = 1.5 g/dl serum IgM paraproteinemia and a monoclonal lymphoplasmacytic infiltrate. We used interphase fluorescence in situ hybridization (FISH) with probes that localized to 17p13.1(LSI p53/CEP 17) and 13q14 (D13S319 and LSI 13 Rb). Of 40 successfully studied patients for 17p13.1(p53) deletions, 6 were abnormal, consistent with hemizygous deletion (15%). Of 37 cases successfully studied for the 13q14 deletions, 6 were also abnormal with one pair of signals deleted (16%). Patients with deletions were more likely to be later in the course of the disease. No obvious clinical associations were noted with the exception that patients with 17p13.1(p53) deletions had a higher percent involvement of clonal cells in the bone marrow. Deletions of these two regions are uncommon in WM, being more common in the late stages of the disease, thus unlikely playing a role in primary disease pathogenesis.

Clinical implications of t(11;14)(q13;q32), t(4;14)(p16.3;q32), and -17p13 in myeloma patients treated with high-dose therapy.

Fluorescence in situ hybridization (FISH) is more sensitive than conventional cytogenetics for recognizing chromosomal changes. Several FISH-detected abnormalities have been associated with inferior prognosis, including deletion of chromosomes 17 and 13 (Delta13) and t(4;14)(p16.3;q32). We analyzed the prognostic value of FISH testing in 238 patients who received high-dose therapy between January 1990 and September 2001. All patients had pretransplantation cytoplasmic immunoglobulin FISH done on cytospin slides from bone marrow aspirates for t(11;14), t(4;14), and -17(p13.1) (TP53). Time to progression and overall survival were significantly shorter for patients with t(4;14) and those with -17(p13.1) but were not affected by t(11;14). Overall survival was significantly shorter for patients with both t(4;14) and Delta13 abnormalities than for those with Delta13 alone (26.8 vs 18.8 months). In a multivariable analysis of the effect of Delta13 and t(4;14), the risk ratio for t(4;14) was greater than for Delta13 (2.6 vs 1.5). For high-dose therapy patients, -17(p13) and t(4;14) have clinical importance for estimating time to progression and overall survival. The presence of t(4;14) identifies a subset of patients whose time to progression is only 8.2 months. These patients receive minimal benefit from autologous stem cell transplantation and are candidates for novel therapeutic approaches.

A validated FISH trisomy index demonstrates the hyperdiploid and nonhyperdiploid dichotomy in MGUS.

Two major genetic categories of multiple myeloma (MM) exist. Hyperdiploid MM (48 to 74 chromosomes, median 53 chromosomes) is associated with trisomies especially of chromosomes 3, 7, 9, 11, 15, and 19, whereas the nonhyperdiploid (< 48 chromosomes or more than 74 chromosomes) MM is associated with primary translocations such as t(11;14), t(4;14), and t(14;16). Whether this dichotomy exists in monoclonal gammopathy of undetermined significance (MGUS) is uncertain due to limitations of current methods in the study of ploidy. This is especially true in MGUS where the number of clonal plasma cells is small. In this study, we derived a fluorescent in situ hybridization (FISH)-based trisomy index from pooled cytogenetic data (karyotype analysis) from 2 large cohorts of patients with MM with abnormal karyotype, and then validated it in 2 independent cohorts of patients who had known ploidy status either by karyotyping or DNA content measurement using flow cytometry. Using the criteria of 2 or more trisomies from a 3-chromosome combination, hyperdiploid myeloma can be detected with high specificity. Applying this index on 28 patients with smoldering multiple myeloma (SMM) or MGUS (11 SMM, 17 MGUS) who had normal karyotype, 11 cases of hyperdiploid SMM/MGUS were detected. This percentage (40%) is remarkably similar to the percentage of hyperdiploid MM reported in the literature, suggesting that hyperdiploid MM may originate early during disease evolution.

Clinical and biologic implications of recurrent genomic aberrations in myeloma.

Nonrandom recurrent chromosomal abnormalities are ubiquitous in multiple myeloma (MM) and include, among others, translocations of the immunoglobulin heavy chain locus (IgH). IgH translocations in MM result in the up-regulation of oncogenes, and include more commonly t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23). Based on the recurrent nature of these translocations and their finding since the early stages of the plasma cell (PC) disorders, we hypothesized that they would confer biologic and clinical variability. In addition, deletions of 13q14 and 17p13 have also been associated with a shortened survival. We used cytoplasmic Ig-enhanced interphase fluorescent in situ hybridization to detect deletions (13q14 and 17p13.1), and translocations involving IgH in 351 patients treated with conventional chemotherapy entered into the Eastern Cooperative Oncology Group clinical trial E9486/9487. Translocations were frequently unbalanced with loss of one of the derivative chromosomes. The presence of t(4; 14)(p16;q32) (n = 42; 26 vs 45 months, P <.001), t(14;16)(q32;q23) (n = 15; 16 vs 41 months, P =.003), - 17p13 (n = 37; 23 vs 44 months, P =.005), and - 13q14 (n = 176; 35 vs 51 months, P =.028) were associated with shorter survival. A stratification of patients into 3 distinct categories allowed for prognostication: poor prognosis group (t(4;14)(p16;q32), t(14; 16)(q32;q23), and - 17p13), intermediate prognosis (- 13q14), and good prognosis group (all others), with median survivals of 24.7, 42.3, and 50.5 months, respectively (P <.001). This molecular cytogenetic classification identifies patients into poor, intermediate, and good risk categories. More importantly it provides further compelling evidence that MM is composed of subgroups of patients categorized according to their underlying genomic aberrations.

Waldenström macroglobulinemia neoplastic cells lack immunoglobulin heavy chain locus translocations but have frequent 6q deletions.

Lymphoplasmacytic lymphoma (LPL) is characterized by t(9;14)(p13;q32) in 50% of patients who lack paraproteinemia. Waldenström macroglobulinemia (WM), which has an immunoglobulin M (IgM) paraproteinemia, is classified as an LPL. Rare reports have suggested that WM sometimes is associated with 14q23 translocations, deletions of 6q, and t(11;18)(q21;q21). We tested for these abnormalities in the clonal cells of WM patients. We selected patients with clinicopathologic diagnosis of WM (all had IgM levels greater than 1.5 g/dL). Southern blot assay was used to detect legitimate and illegitimate IgH switch rearrangements. In addition to conventional cytogenetic (CC) and multicolor metaphase fluorescence in situ hybridization (M-FISH) analyses, we used interphase FISH to screen for t(9;14)(p13;q32) and other IgH translocations, t(11;18)(q21;q21), and 6q21 deletions. Genomic stability was also assessed using chromosome enumeration probes for chromosomes 7, 9, 11, 12, 15, and 17 in 15 patients. There was no evidence of either legitimate or illegitimate IgH rearrangements by Southern blot assay (n = 12). CC (n = 37), M-FISH (n = 5), and interphase FISH (n = 42) failed to identify IgH or t(11;18) translocations. Although tumor cells from most patients were diploid for the chromosomes studied, deletions of 6q21 were observed in 42% of patients. In contrast to LPL tumors that are not associated with paraproteinemia and that have frequent t(9;14)(p13;q32) translocations, IgH translocations are not found in WM, a form of LPL tumor distinguished by IgM paraproteinemia. However, WM tumor cells, which appear to be diploid or near diploid, often have deletions of 6q21.

Myeloma and the t(11;14)(q13;q32); evidence for a biologically defined unique subset of patients.

The t(11;14)(q13;q32) results in up-regulation of cyclin D1 and is the most common translocation detected in multiple myeloma, where it is also associated with a lymphoplasmacytic morphology. We performed an interphase fluorescent in situ hybridization (FISH) study to determine the clinical and biologic significance of the abnormality when testing a large cohort of myeloma patients. Bone marrow slides from multiple myeloma patients entered into the Eastern Cooperative Oncology Group phase III clinical trial E9486 and associated laboratory correlative study E9487 were analyzed using interphase FISH combined with immune-fluorescent (cytoplasmic immunoglobulin-FISH) detection of clonal plasma cells. We used FISH probes that hybridize to the 14q32 and 11q13 chromosomal loci. The t(11;14)(q13;q32) was correlated with known biologic and prognostic factors. Of 336 evaluable patients, 53 (16%) had abnormal FISH patterns compatible with the t(11;14)(q13;q32). These patients appeared to be more likely to have a serum monoclonal protein of less than 10 g/L (1 g/dL) (28% vs 15%, P =.029) and a lower plasma cell labeling index (P =.09). More strikingly, patients were less likely to be hyperdiploid by DNA content analysis (n = 251, 14% vs 62%, P <.001). Patients with the t(11;14)(q13;q32) appeared to have better survival and response to treatment, although this did not reach statistical significance. Multiple myeloma with the t(11;14)(q13;q32) is a unique subset of patients, not only characterized by cyclin D1 up-regulation and a lymphoplasmacytic morphology, but is also more frequently associated with small serum monoclonal proteins and is much less likely to be hyperdiploid. These patients do not have a worsened prognosis as previously thought.