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Although follicular lymphoma (FL) patients relapsing within 24 months after first-line treatment (POD24) have a poor prognosis, some cases show notable survival after first relapse (SF1R). We aimed to characterize the POD24 FL population and to identify the main prognostic factors at progression. We selected 162 POD24 patients (80F; median age at first relapse 59 years) from a cohort of 1067 grades 1-3a FL-treated patients. The remaining 905 patients treated with first-line immunochemotherapy and diagnosed during the same period were used to compare outcomes in terms of survival. After a median follow-up of 11.0 years, 96 patients died (10y-SF1R of 40%). Age over 60 years (p 0.001), high lactate dehydrogenase (LDH) (p 0.001), haemoglobin (Hb) less than 120 g/L (p 0.001), advanced stage (p 0.001), high-risk Follicular Lymphoma International Prognostic Index (FLIPI) (p 0.001), histological transformation (HT) (p0.001) and reaching less than complete response (CR) after salvage therapy (p 0.001), predicted poor SF1R at relapse. In multivariate analysis only high-risk FLIPI and HT maintained prognostic significance for SF1R. POD24 patients not transformed and with low/intermediate FLIPI at relapse behaved better than the remaining cases. POD24 patients showed an excess mortality of 38% compared to the general population. Although outcome of POD24 FL patients is poor, a considerable group of them (low/intermediate FLIPI and not transformed at first relapse) behave better.

Loss and/or mutation of the TP53 gene are associated with short survival in multiple myeloma, but the p53 landscape goes far beyond. At least 12 p53 protein isoforms have been identified as a result of a combination of alternative splicing, alternative promoters and/or alternative transcription site starts, which are grouped as α, β, γ, from transactivation domain (TA), long, and short isoforms. Nowadays, there are no studies evaluating the expression of p53 isoforms and its clinical relevance in multiple myeloma (MM). We used capillary nanoimmunoassay to quantify the expression of p53 protein isoforms in CD138-purified samples from 156 patients with newly diagnosed MM who were treated as part of the PETHEMA/GEM2012 clinical trial and investigated their prognostic impact. Quantitative real-time polymerase chain reaction was used to corroborate the results at RNA levels. Low and high levels of expression of short and TAp53β/γ isoforms, respectively, were associated with adverse prognosis in MM patients. Multivariate Cox models identified high levels of TAp53β/γ (hazard ratio [HR], 4.49; p, The Instituto de Salud Carlos III, European Union FEDER funds, Grant Numbers: PI16/01074 and PI19/00674; The Asociación Española Contra el Cáncer, Grant Number: AECC, PROYE20047GUTI; National Science Centre, Poland, Grant Number: UMO-2019/35/B/NZ5/02824; The Consejería de Educación de Castilla y León and FEDER funds; AECC, Grant Number: CLJUN18010DERA.

Purpose: PIM kinases are a family of serine/threonine kinases recently proposed as therapeutic targets in oncology. In the present work, we have investigated the effects of the novel pan-PIM kinase inhibitor, PIM447, on myeloma cells and myeloma-associated bone disease using different preclinical models.Experimental Design: In vitro/ex vivo cytotoxicity of PIM447 was evaluated on myeloma cell lines and patient samples. Synergistic combinations with standard treatments were analyzed with Calcusyn Software. PIM447 effects on bone cells were assessed on osteogenic and osteoclastogenic cultures. The mechanisms of PIM447 were explored by immunoblotting, qPCR, and immunofluorescence. A murine model of disseminated multiple myeloma was employed for in vivo studies.Results: PIM447 is cytotoxic for myeloma cells due to cell-cycle disruption and induction of apoptosis mediated by a decrease in phospho-Bad (Ser112) and c-Myc levels and the inhibition of mTORC1 pathway. Importantly, PIM447 demonstrates a very strong synergy with different standard treatments such as bortezomib + dexamethasone (combination index, CI = 0.002), lenalidomide + dexamethasone (CI = 0.065), and pomalidomide + dexamethasone (CI = 0.077). PIM447 also inhibits in vitro osteoclast formation and resorption, downregulates key molecules involved in these processes, and partially disrupts the F-actin ring, while increasing osteoblast activity and mineralization. Finally, PIM447 significantly reduced the tumor burden and prevented tumor-associated bone loss in a disseminated murine model of human myeloma.Conclusions: Our results demonstrate dual antitumoral and bone-protective effects of PIM447. This fact, together with the very strong synergy exhibited with standard-of-care treatments, supports the future clinical development of this drug in multiple myeloma. Clin Cancer Res; 23(1); 225–38. ©2016 AACR.

Supplementary Figure 1. Evaluation of apoptosis and cell cycle in MM cell lines after PIM447 treatment. -Supplementary Figure 2. PIM447 very strongly synergizes with different antimyeloma agents in the RPMI-8226 cell line. -Supplementary Figure 3. Effect of PIM447 on PIM kinase-related targets in RPMI-8226, NCI-H929 and OPM-2 cells. -Supplementary Figure 4. Correlation analysis between PIM kinase-related targets and sensitivity to PIM447. -Supplementary Figure 5. Oral administration of PIM447 does not significantly affect body weight in a mouse model of disseminated MM.

Purpose: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure.Experimental Design: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays.Results: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride.Conclusions: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies. Clin Cancer Res; 23(21); 6602–15. ©2017 AACR.

Supplementary methods, tables and references.

Supplementary Table S1.

Purpose: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma.Experimental Design: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR.Results: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction.Conclusions: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma. Clin Cancer Res; 22(1); 207–17. ©2015 AACR.

Supplementary Tables Supplementary Table 1. Cytogenetic characteristics of MM samples Supplementary Table 2. Sequences of primers used in the study Supplementary Table 3. CCND2 mRNA FISH probes sequences Supplementary table 4. microRNAs predicted to target CCND1 or/and CCND2 Supplementary table 5. microRNAs expression in MM cell lines. Supplementary Figures Supplementary Figure 1. CCND2 mRNA level 24h after transfection of MM1S, RPMI and H929 cell lines with indicated miRNAs Supplementary Figure 2. Expression of miR-15a and miR-196b in H929 and MM1S cell lines after transfection Supplementary Figure 3. (A) Alignment of 3'RACE PCR product sequence from MM1S cell line with CCND2 mRNA (NM_001759.3). (B) Sequence of the 3'RACE PCR product withhighlighted APA signal. Supplementary Figure 4. CCND2 mRNA expression visualized with FISH probes Supplementary Figure 5. Luciferase activity in HEK923 cells cotransfected with various miRNAs, plasmid encoding GFP and plasmid containing luciferase and 3'UTR of CCND2 short isoform Supplementary Figure 6. Regulation of cyclin D2 level by CCND1/CCND3.

Figure S1. (A) Protein expression of p53 in MM cell lines.; Figure S2. (A) Melphalan resistant MM cell line RPMI-LR5 (panel left) and dexamethasone resistant MM cell line MM1R (panel right) were treated with the indicated doses of amiloride for 24, 48 and 72 h, and cell viability was analyzed by CellTiter-Glo luminescent assays; respectively. Figure S3. (A) MM1S-luc cells were treated for 48 hours with the indicated concentrations of amiloride in the presence or absence of MSCs derived from newly-diagnosed (ND) and relapsed/refractory (RR) MM patients, and proliferation was analyzed by bioluminescence (photons/sec). Figure S4. Amiloride induced apoptosis in MM cells. Figure S5. Amiloride did not induce changes in the cell cycle profile of MM cells. Figure S6. Amiloride deregulated mitochondrial potential in MM cells. Figure S7. Activity of amiloride through caspase-dependent and independent mechanisms. Figure S8. The triple and double combination of dexamethasone and melphalan with amiloride displayed superior anti-MM activity and improved median survival compared with single agents and double combinations in a subcutaneous plasmacytoma model. Figure S9. Experimental design for RNA-Seq assay. Figure S10. Pathway enrichment analysis at gene level and differential transcript isoforms expression. Figure S12. Validation of gene expression. Figure S13. Validation of p53 pathway activation in patient cells.

Purpose: Genetic aberrations detected in multiple myeloma (MM) have also been reported in the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Our aim was to investigate in depth the level of clonal heterogeneity of recurrent genetic abnormalities in these conditions.Experimental Design: Immunoglobulin heavy chain (IGH) translocations, 13q14 and 17p13 deletions, and 1q21 gains using FISH were evaluated in 90 MGUS, 102 high-risk SMM, and 373 MM. To this end, we not only purified plasma cells (PC) for the FISH analysis (purity > 90%), but subsequently, we examined the correlation between the proportion of PC with cytogenetic changes and the number of clonal PC present in the same sample, as measured by multiparametric flow cytometry.Results: We observed a significant difference between the proportion of clonal PC with specific genetic abnormalities in MGUS compared with SMM and in SMM compared with MM. Thus, the median proportion of PC with IGH translocations globally considered, t(11;14) and 13q deletions was significantly lower in MGUS than in SMM, and in SMM than in MM [IGH translocations: 34% vs. 57% vs. 76%; t(11;14): 38% vs. 61% vs. 81%; and 13q deletion: 37% vs. 61% vs. 74% in MGUS, SMM, and MM, respectively]. For t(4;14), the difference was significant in the comparison between MGUS/SMM and MM and for 1q between MGUS and SMM/MM.Conclusions: This study demonstrates that the progression from MGUS to SMM, and eventually to MM, involves a clonal expansion of genetically abnormal PC. Clin Cancer Res; 17(7); 1692–700. ©2011 AACR.

(1) Background: New therapeutic strategies have improved the prognosis of multiple myeloma (MM), changing the accepted view of this disease from being incurable to treatable. (2) Methods: We studied 1001 patients with MM between 1980 and 2020, grouping patients into ten-year periods by diagnosis 1980–1990, 1991–2000, 2001–2010 and 2011–2020. (3) Results: After 65.1 months of follow-up, the median OS of the cohort was 60.3 months, and OS increased significantly over time: 22.4 months in 1980–1990, 37.4 months in 1991–2000, 61.8 months in 2001–2010 and 103.6 months in 2011–2020 (p < 0.001). Using novel agents in the front-line setting for myeloma patients yielded a significantly better OS than in those treated with conventional therapies, especially when combinations of at least two novel agents were used. The median OS of patients treated with the combination of at least two novel agents in induction was significantly prolonged compared to those treated with a single novel agent or conventional therapy in induction: 143.3 vs. 61.0 vs. 42.2 months (p < 0.001). The improvement was apparent in all patients regardless of age at diagnosis. In addition, 132 (13.2%) patients were long-term survivors (median OS ≥ 10 years). Some independent clinical predictors of long-term survival were identified: ECOG < 1, age at diagnosis ≤ 65 years, non-IgA subtype, ISS-1 and standard-risk cytogenetic. Achieving CR and undergoing ASCT were positively associated with >10 years of survival. (4) Conclusions: The combination of novel agents appears to be the main factor for the improvement in survival in MM, which is becoming a chronic and even curable disease in a subtype of patients without high-risk features.

[Context]: Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. [Objective]: To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. [Design]: The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. [Results]: Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. [Conclusions]: Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures. Multiple myeloma (MM) is a plasma-cell dyscrasia characterized by the accumulation of plasma cells in the bone marrow that produces an excess of clonal immunoglobulins (M-protein or monoclonal component).1 New treatment approaches have increased the number of patients achieving complete response (CR),2–5 progressively improving progression-free and overall survival rates in the last 10 years.6–11 Nonetheless, the presence of low levels of drug-resistant cells (known as minimal residual disease, MRD)12–14 that remain undetected by conventional serologic and morphologic methods explains frequent relapses with this disease, which is still considered an incurable illness.Minimal residual disease is currently considered one of the most informative prognostic parameters, since those patients with undetectable disease have shown prolonged survival rates as compared with MRD-positive patients,15–17 and this difference is still significant even when patients achieving only stringent complete response (sCR) are taken into account.18 The International Myeloma Working Group (IMWG) defined MRD positivity as the persistence of clonal malignant plasma cells assessed with a sensitivity of at least 10−5 (1 malignant cell per hundred thousand normal cells)19 ; therefore, MRD should be monitored with only highly sensitive methods. To date, 3 different approaches have been tested for MRD monitoring in hematologic malignancies: immunophenotypic (multiparametric flow cytometry [MFC]),20 molecular (quantitative polymerase chain reaction [PCR], next-generation sequencing [NGS], digital PCR),21–23 and imaging tools (positron emission tomography–computed tomography; magnetic resonance imaging).24,25 However, in MM standardization has been achieved only for MFC26 and NGS.27,28 As a result, the IMWG recommended the use of highly sensitive, standardized flow and sequencing approaches,19 including EuroFlow's next-generation flow (NGF)29 and Adaptive Biotechnologies' ClonoSEQ solutions (Adaptive Biotechnologies, Seattle, Washington). NGF is a 2-tube, 8-color flow assay that allows the simultaneous analysis of 10 million cells, providing a sensitivity of around 2·10−6., This work was partially supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01956, CIBERONC-CB16/12/00233, and “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010). García-Álvarez, Prieto-Conde, and Jiménez were supported by the Fundación Española de Hematología y Hemoterapia (FEHH, cofunded by Fundación Cris in the latter case), Medina by the European Social Fund through the University of Salamanca and the ISCIII (FI19/00320), and Sarasquete by the ISCIII (CPII18/00028). All Spanish funding is cosponsored by the European Union FEDER program.

Peripheral T-cell lymphomas (PTCL) have an aggressive clinical behavior and poor response to treatment; therefore, the search for new drugs is required. We have previously demonstrated the antitumor activity of amiloride in multiple myeloma. This prompted us to investigate the possibility of a cytotoxic effect of amiloride in this kind of T-cell lymphoma.To evaluate the antitumor activity of amiloride in different PTCL using in-vitro assays and identify the possible mechanisms involved.Two anaplastic large-cell lymphoma (ALCL) cell lines, KARPAS-299 and SR-786, and two cutaneous T-cell lymphoma cell lines, HH and HuT-78 (Sézary syndrome), were used. Cell viability and apoptosis were studied after exposure to different increasing concentrations of the drug at 24, 48, and 72 hours by colorimetric MTT and annexin V/propidium iodide double-labeling assays, respectively.Amiloride significantly inhibited cell growth in the PTCL cell lines, depending on increasing drug concentration and time. Inhibition was statistically significant (P0.05) from 0.15 mM for KARPAS-299, SR-786, and HH at 24, 48, and 72 hours and from 0.3 mM for HuT-78 at 48 and 72 hours. KARPAS-299 and SR-786 were the most sensitive cell lines to amiloride exposure, whereas HuT-78 cells were less sensitive, although with the highest concentration of amiloride, its cell viability decreased to levels below 50% and 40% at 48 and 72 hours, respectively. Induction of cell death by apoptosis was detected in all cell lines, with apoptosis levels up to 25% at 24 hours and more than 80% at 48 and 72 hours using intermediate doses of the drug.1) Amiloride significantly decreased the cell viability of PTCL. 2) ALCL cell lines were the most sensitive to amiloride exposure. 3) The cytotoxic effect induced by amiloride in PTCL was mediated by apoptosis. Ongoing Objectives: To evaluate apoptosis as the possible mechanism of cell death induction through the analysis of caspases, synergism with drugs commonly used in the treatment of PTCL, and changes in gene and protein expression induced by amiloride.Regional Health Management of Castilla y León (GRS 2142/A/2020);); Carlos III Health Institute European Union (PI19/00674); AECC (PROYE20047GUTI).

Biallelic inactivation of TP53 has been included in the definition of double-hit (DH) multiple myeloma (MM), which entails an ominous prognosis. However, this condition, or even the presence of high-risk cytogenetic abnormalities, cannot accurately capture the 15%–20% of the MM population with a median overall survival below 24 months. This prompted us to look for other MM patients who might have transcriptional characteristics similar to those with DH-TP53. In the present study, we analysed RNA-seq, whole-genome and whole-exome sequencing data from 660 newly diagnosed MM (NDMM) patients from the MMRF (Multiple Myeloma Research Foundation) CoMMpass study to characterize the transcriptional signature of TP53 double-hit (DH-TP53) MM. We found 78 genes that were exclusively deregulated in DH-TP53 patients. A score based on these genes identified a group of 50 patients who shared the same transcriptional profile (DH-TP53-like group) whose prognosis was particularly unfavourable [median overall survival (OS), This work has been supported by grants from the Instituto de Salud Carlos III (ISCIII) co-funded by European Union FEDER funds (projects PI16/01074 and PI19/00674), the Asociación Española Contra el Cáncer (grant PROYE20047GUTI) and the Gerencia Regional de Salud, Junta de Castilla y León (grants GRS 2058/A/19 and GRS 2331/A/21). Cristina De Ramón, Elizabeta A. Rojas and Ignacio J. Cardona-Benavides have been supported by the Asociación Española Contra el Cáncer (CLJUN18010DERA), the Consejería de Educación de Castilla y León and the Instituto de Salud Carlos III (PFIS-2020: FI20/00226), respectively. The authors thank the MMRF for access to the CoMMpass dataset.

[Background]: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells., [Methods]: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot., [Results]: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found., [Conclusion]: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation., This study was partially supported by the Instituto de Salud Carlos III, co-financed by FEDER, “PI16/01074” and “PI19/00674”; the Gerencia Regional de Salud, Junta de Castilla y León grants “GRS 1833/A/18” and “GRS 2058/A/19″, and the "Asociación Española Contra el Cancer (AECC)”, PROYE20047GUTI. E.A.R. was supported by the Consejería de Educación de Castilla y León and FEDER funds. I.J.C.-B. was supported by a fellowship (contract PFIS-2020: FI20/00226) from the Instituto de Salud Carlos III.

Acute myeloid leukemias (AMLs) are currently genomically characterized by karyotype, fluorescence in situ hybridization (FISH), real-time quantitative PCR, and DNA sequencing. Next-generation sequencing offers the promise of detecting all genomic lesions in a single run. However, technical limitations have hampered the detection of chromosomal rearrangements, so most studies are limited to somatic mutation assessment or require the use of RNA-based strategies. To overcome these limitations, we designed a targeted-DNA capture next-generation sequencing approach associated with easy-to-perform public bioinformatic tools for one-step identification of translocations, inversions, and somatic mutations in AML. Thirty well-characterized newly diagnosed myeloid leukemia patients (27 AML and 3 chronic myeloid leukemia) were tested with the panel. Twenty-three of 24 known rearrangements, as well as one novel fusion gene that could not be detected by karyotype/fluorescence in situ hybridization/real-time quantitative PCR, were detected. This strategy also identified all chromosomal breakpoints as potential targets for future high-sensitive minimal residual disease studies. In addition, mutation analysis revealed the presence of missense protein-coding alterations in at least 1 of the 32 genes evaluated in 21 of 30 patients (70%). This strategy may represent a time- and cost-effective diagnostic method for molecular characterization in AML., Supported by the Fundación Española de Hematología y Hemoterapia (FEHH) (M.I.P.-C. and M.G.-A.); the Spanish Association Against Cancer Scientific Foundation (AECC) (M.C.C.); Miguel Servet program CP13/00080 from the Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades, Spain (M.E.S.); and the “Beca de investigación FEHH-CRIS 2018” (C.J.). Partially supported by ISCIII research grants PI15/01706, CIBERONCCB16/12/00233, and “Una manera de hacer Europa” Innocampus, CEI-2010-1-0010.

Next-generation sequencing (NGS) has greatly improved our ability to detect the genomic aberrations occurring in multiple myeloma (MM); however, its transfer to routine clinical labs and its validation in clinical trials remains to be established. We designed a capture-based NGS targeted panel to identify, in a single assay, known genetic alterations for the prognostic stratification of MM. The NGS panel was designed for the simultaneous study of single nucleotide and copy number variations, insertions and deletions, chromosomal translocations and V(D)J rearrangements. The panel was validated using a cohort of 149 MM patients enrolled in the GEM2012MENOS65 clinical trial. The results showed great global accuracy, with positive and negative predictive values close to 90% when compared with available data from fluorescence in situ hybridization and whole-exome sequencing. While the treatments used in the clinical trial showed high efficacy, patients defined as high-risk by the panel had shorter progression-free survival (p = 0.0015). As expected, the mutational status of TP53 was significant in predicting patient outcomes (p = 0.021). The NGS panel also efficiently detected clonal IGH rearrangements in 81% of patients. In conclusion, molecular karyotyping using a targeted NGS panel can identify relevant prognostic chromosomal abnormalities and translocations for the clinical management of MM patients., The study was supported by the Accelerator Grant: next steps: Early detection and intervention: Understanding the mechanisms of transformation and hidden resistance of incurable hematological malignancies, grants from Instituto de Salud Carlos III PI15/01484 and CRIS Foundation 2020/0063 and 2021/0088.

Multiple myeloma is a treatable, but currently incurable, hematological malignancy of plasma cells characterized by diverse and complex tumor genetics for which precision medicine approaches to treatment are lacking. The MMRF CoMMpass study is a longitudinal, observational clinical study of newly diagnosed multiple myeloma patients where tumor samples are characterized using whole genome, exome, and RNA sequencing at diagnosis and progression, and clinical data is collected every three months. Analyses of the baseline cohort identified genes that are the target of recurrent gain- and loss-of-function events. Consensus clustering identified 8 and 12 unique copy number and expression subtypes of myeloma, respectively, identifying high- risk genetic subtypes and elucidating many of the molecular underpinnings of these unique biological groups. Analysis of serial samples showed 25.5% of patients transition to a high-risk expression subtype at progression. We observed robust expression of immunotherapy targets in this subtype, suggesting a potential therapeutic option.

Background t(11;14) is the most frequently IgH rearrengement in newly diagnosed multiple myeloma (NDMM) with a 15-20% prevalence and is considered a standard-risk cytogenetic abnormality (SR). However, t(11;14) is controversial since recent reports describe inferior outcomes in contrast to other SR patients. We analyze the long-term outcome of t(11;14) compared to the remaining SR population in transplant-eligible patients included in GEM05MENOS65 and GEM2012 spanish trials for NDMM. Methods 386 patients included in GEM05MENOS65 trial were randomized to receive induction with 6 cycles of thalidomide and dexamethasone (TD) vs. 6 cycles of VTD vs. 4 cycles of alternating chemotherapy (VBMCP/VBAD) plus 2 cycles of bortezomib (VBMCP/VBAD/B). 458 patients included in GEM2012 trial received induction with 6 cycles of VRD. Results From a total of 414 SR patients, 84 (20%) were positive for t(11;14) and 328 (80%) were SR non-t(11;14). There were no differences in baseline characteristics among groups. In the VBMCP/VBAD/B arm, there were no significant differences in ORR or CR rate after induction between patients with t(11;14) or other SR (ORR 93 vs. 85%, p=0.4; CR 21 vs. 16%, p=0.6). However, patients with t(11;14) treated with VTD had lower ORR and CR rates after induction than SR patients (ORR 85 vs. 97%, p=0.04; CR 15 vs. 38%, p=0.1). Patients with t(11;14) treated with TD had lower ORR and CR rates although the differences were not statistically significant (ORR 63 vs. 78%, p=0.2, CR 5 vs. 11%, p=0.4). In the VRD arm, patients with t(11;14) had similar ORR and CR compared to other SR (ORR 87 vs. 89%, p=0.8; CR 24 vs. 32%, p=0.3). With a median follow-up of 54.2 months, no differences in median PFS were observed in the VBMCP/VBAD/B arm (44 vs. 46 months; p=0.7). However, shorter PFS was observed in patients with t(11;14) in the TD (30 vs. 46 months; p=0.03) and VTD arms (28 vs. 72 months; p=0.003). With VRD, median PFS was not reached for patients with t(11;14) and SR non-t(11;14) (p=0.8). Among patients with t(11;14), VTD showed shorter median PFS compared to VRD (28 months vs. not reached, p Conclusion Thalidomide-based induction regimens (VTD/TD) are suboptimal in patients with t(11;14) concerning response rates and PFS than SR patients, although this does not impact on OS. In contrast, VRD showed similar ORR and PFS in patients with t(11;14) and SR. Despite this is a non-randomized comparison, VRD appears to be more effective than VTD in this subset of patients. Patients with t(11;14) receiving induction with chemotherapy have the same response and PFS than SR patients but display a shorter OS. These results should be confirmed in larger series.

© The Author(s) 2021., Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma (NHL, 20–30%) after diffuse large B-cell lymphoma (DLBCL). Despite the introduction of rituximab and the high response rate to first-line treatment, approximately 20% of the FL patients relapse or progress within 2 years of receiving first-line therapy. Therefore, the major challenge is finding biomarkers that identify high-risk patients at diagnosis., This work was partially supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01393, PI18/00410, CIBERONC-CB16/12/00233, and “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010)”, the Education Council or Health Council of the Junta de Castilla y León (CAS102P17, GRS 1180/A/15), Spanish Association Against Cancer (AECC; PROYE18020BEA), and Gilead Sciences (GLD17/00334). CJ, MES, and AMe are supported by the ISCII (CD19/00030, CPII18/00028, and FI19/00320). MGA, IPC, and CJ were supported by the Spanish Society of Hematology Foundation (FEHH). All Spanish funding is co-sponsored by the European Union FEDER program.

PETHEMA/GEM Cooperative Group., Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species–mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.

© The Author(s) 2021., Although case-control analyses have suggested an additive value with the association of clarithromycin to continuous lenalidomide and dexamethasone (Rd), there are not phase III trials confirming these results. In this phase III trial, 286 patients with MM ineligible for ASCT received Rd with or without clarithromycin until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). With a median follow-up of 19 months (range, 0–54), no significant differences in the median PFS were observed between the two arms (C-Rd 23 months, Rd 29 months; HR 0.783, p = 0.14), despite a higher rate of complete response (CR) or better in the C-Rd group (22.6% vs 14.4%, p = 0.048). The most common G3–4 adverse events were neutropenia [12% vs 19%] and infections [30% vs 25%], similar between the two arms; however, the percentage of toxic deaths was higher in the C-Rd group (36/50 [72%] vs 22/40 [55%], p = 0.09). The addition of clarithromycin to Rd in untreated transplant ineligible MM patients does not improve PFS despite increasing the ≥CR rate due to the higher number of toxic deaths in the C-Rd arm. Side effects related to overexposure to steroids due to its delayed clearance induced by clarithromycin in this elderly population could explain these results. The trial was registered in clinicaltrials.gov with the name GEM-CLARIDEX: Ld vs BiRd and with the following identifier NCT02575144. The full trial protocol can be accessed from ClinicalTrials.gov. This study received financial support from BMS/Celgene.

The use of non‐pegylated liposomal doxorubicin (Myocet®) in diffuse large B‐cell lymphoma (DLBCL) has been investigated in retrospective and single‐arm prospective studies. This was a prospective phase 2 trial of DLBCL patients ≥60 years old with left ventricular ejection fraction (LVEF) ≥55% randomized to standard R‐CHOP or investigational R‐COMP (with Myocet® instead of conventional doxorubicin). The primary end point was to evaluate the differences in subclinical cardiotoxicity, defined as decrease in LVEF to, The use of non‐pegylated liposomal doxorubicin (Myocet®) in diffuse large B‐cell lymphoma (DLBCL) has been investigated in retrospective and single‐arm prospective studies. This prospective randomized phase 2 trial of RCOMP versus RCHOP in DLBCL patients ≥60 years and normal cardiac function demonstrated that R‐COMP is a feasible immunochemotherapy schedule for DLBCL patients ≥60 years, with similar efficacy to R‐CHOP. However, the use of non‐pegylated doxorubicin instead of conventional doxorubicin was not associated with less early cardiotoxicity, although some reduced cardiac safety signals were observed.

The search for biomarkers based on the mechanism of drug action has not been thoroughly addressed in the therapeutic approaches to multiple myeloma (MM), mainly because of the difficulty in analyzing proteins obtained from purified plasma cells. Here, we investigated the prognostic impact of the expression of 12 proteins involved in the mechanism of action of bortezomib, lenalidomide, and dexamethasone (VRD), quantified by capillary nanoimmunoassay, in CD138-purified samples from 174 patients with newly diagnosed MM treated according to the PETHEMA/GEM2012 study. A high level of expression of 3 out of 5 proteasome components tested (PSMD1, PSMD4, and PSMD10) negatively influenced survival. The 5 analyzed proteins involved in lenalidomide’s mode of action were associated with time to progression (TTP); low levels of cereblon and IRF4 protein and high levels of Ikaros, AGO2, and Aiolos were significantly associated with shorter TTP. Although the glucocorticoid receptor (GCR) level by itself had no significant impact on MM prognosis, a high XPO1 (exportin 1)/GCR ratio was associated with shorter TTP and progression-free survival (PFS). The multivariate Cox model identified high levels of PSMD10 (hazard ratio [HR] TTP, 3.49; P = .036; HR PFS, 5.33; P = .004) and Ikaros (HR TTP, 3.01, P = .014; HR PFS, 2.57; P = .028), and low levels of IRF4 protein expression (HR TTP, 0.33; P = .004; HR PFS, 0.35; P = .004) along with high-risk cytogenetics (HR TTP, 3.13; P < .001; HR PFS, 2.69; P = .002), as independently associated with shorter TTP and PFS. These results highlight the value of assessing proteins related to the mechanism of action of drugs used in MM for predicting treatment outcome., This study was funded by the Instituto de Salud Carlos III, cofinanced by FEDER grant PI16/01074; the Gerencia Regional de Salud, Junta de Castilla y Leon grants GRS1654/A/17, GRS1849/ ´ A/18, and GRS2058/A/19; and a Brian D. Novis, Black Swan Research Initiative research grant from the International Myeloma Foundation. The WES platform was acquired thanks to the INNOCAMPUS program (CEI10-1-0010).