Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n= 49) and CD138+ bone marrow PCs from subjects with MM (n= 16), monoclonal gammopathy of undetermined significance (MGUS) (n= 6), and normal donors (n= 6). We identified overexpression ofmiR-21,miR-106b∼25cluster,miR-181aandbin MM and MGUS samples with respect to healthy PCs. Selective up-regulation ofmiR-32andmiR-17∼92cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs,miR-19aand19b, that are part of themiR-17∼92cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target ofthe miR106b∼25cluster,miR-181aandb, andmiR-32. Xenograft studies using human MM cell lines treated withmiR-19aandb, andmiR-181aandbantagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.

Purpose: We investigated the effect on minimal residual disease, by qualitative and real-time quantitative polymerase chain reaction (RQ-PCR), of a consolidation regimen that included bortezomib, thalidomide, and dexamethasone (VTD) in patients with multiple myeloma (MM) responding to autologous stem-cell transplantation (auto-SCT). Patients and Methods: Patients achieving at least very good partial response who had an available molecular marker based on the immunoglobulin heavy-chain rearrangement received four courses of treatment every month: four infusions per month of bortezomib at 1.6 mg/m², thalidomide at 200 mg/d, and dexamethasone at 20 mg/d on days 1 to 4, 8 to 11, and 15 to 18. Patients were studied with tumor-clone–specific primers by qualitative nested PCR and RQ-PCR. Results: Of 39 patients enrolled, 31 received the four VTD courses. Immunofixation complete responses increased from 15% after auto-SCT to 49% after VTD. Molecular remissions (MRs) were 3% after auto-SCT and 18% after VTD. Median time to maximum response was 3.5 months. So far, no patient in MR has relapsed (median follow-up, 42 months). VTD consolidation induced an additional depletion of 4.14 natural logarithms of tumor burden by RQ-PCR. Patients with a tumor load less than the median value after VTD had outcomes better than those who had tumor loads above the median value after VTD (at median follow-up: progression-free survival, 100% v 57%; P < .001). Conclusion: To the best of our knowledge, this study is the first to document the occurrence of persistent MRs in a proportion of MM patients treated without allogeneic transplantation. Moreover, the major reduction in tumor load recorded by RQ-PCR after VTD suggests that unprecedented levels of tumor cell reduction can be achieved in MM thanks to the new nonchemotherapeutic drugs.

We here describe a novel method for MYD88^L265P mutation detection and minimal residual disease monitoring in Waldenström Macroglobulinemia, by droplet digital PCR, in bone marrow and peripheral blood cells, as well as in circulating cell free DNA. Our method shows a sensitivity of 5.00E-05, by far superior to the widely used allele-specific polymerase chain reaction (1.00E-03). Overall, 291 unsorted samples from 148 patients (133 Waldenstrom 11 IgG-lymphoplasmacytic lymphoma and 4 IgM-monoclonal gammopathy of undetermined significance), 194 baseline and 97 follow-up, were analyzed. 122/128 (95.3%) bone marrow and 47/66 (71.2%) baseline peripheral blood samples scored positive for MYD88^L265P. Moreover, to investigate whether MYD88^L265P by droplet digital PCR could be used for minimal residual disease monitoring, mutation levels were compared with IGH-based minimal residual disease analysis in 10 patients, showing to be as informative as to the classical, standardized but not yet validated in Waldenström Macroglobulinemia, IGH-based minimal residual disease assay (r²=0.64). Finally, MYD88^L265P detection performed by droplet digital PCR on plasmatic circulating tumor DNA from 60 patients showed a good correlation with bone marrow (bone marrow median mutational value 1.92E-02, plasmatic circulating tumor DNA value: 1.4E-02, peripheral blood value: 1.03E-03). This study indicates that droplet digital PCR MYD88^L265P assay is a feasible and sensitive tool for mutational screening and minimal residual disease monitoring in Waldenström Macroglobulinemia. Both unsorted bone marrow and peripheral blood samples can be reliably tested, as well as circulating tumor DNA, that represents an attractive, less invasive alternative to bone marrow for MYD88^L265P detection.