The genomic profiling of high-risk smoldering myeloma patients treated with an intensive strategy unveils potential markers of resistance and progression.

Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.

Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

PURPOSE: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use. BACKGROUND: MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases. METHODS AND MATERIALS: In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0.01, 0.05 and 0.1 µg mL(-1) ) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use. RESULTS: Our results show that the highest number of metaphases was obtained when MSC were incubated with 0.05 µg mL(-1) of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use. CONCLUSION: We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes.

Managing hematological cancer patients during the COVID-19 pandemic: an ESMO-EHA Interdisciplinary Expert Consensus.

BACKGROUND: The COVID-19 pandemic has created enormous challenges for the clinical management of patients with hematological malignancies (HMs), raising questions about the optimal care of this patient group. METHODS: This consensus manuscript aims at discussing clinical evidence and providing expert advice on statements related to the management of HMs in the COVID-19 pandemic. For this purpose, an international consortium was established including a steering committee, which prepared six working packages addressing significant clinical questions from the COVID-19 diagnosis, treatment, and mitigation strategies to specific HMs management in the pandemic. During a virtual consensus meeting, including global experts and lead by the European Society for Medical Oncology and the European Hematology Association, statements were discussed and voted upon. When a consensus could not be reached, the panel revised statements to develop consensual clinical guidance. RESULTS AND CONCLUSION: The expert panel agreed on 33 statements, reflecting a consensus, which will guide clinical decision making for patients with hematological neoplasms during the COVID-19 pandemic.

Frequency of HLA-A, -B and -DRB1 specificities and haplotypic associations in the population of Castilla y León (northwest-central Spain).

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y León and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y León is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.

The combination of thalidomide, cyclophosphamide and dexamethasone is potentially useful in highly resistant Hodgkin's lymphoma.

Few diseases have a prognosis worse than Hodgkin's lymphoma (HL), patients relapsing after autologous or allogeneic stem cell transplantation. Here, we report two highly refractory patients with HL who successfully responded to a combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex). Despite the use of a very large number of different drugs (>5 different schemes) including high-dose therapy and autologous and allogeneic stem cell transplantation, both patients proved to be suffering from a highly resistant disease. Fortunately, they finally responded to the ThaCyDex combination, achieving sustained complete remission that would support the running of a trial within this setting.

Bisphosphonate-related osteonecrosis: genetic and acquired risk factors.

The objectives of this study were to review epidemiological, clinical and biological aspects associated with the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ) in multiple myeloma (MM) patients, with special emphasis on the genetic aspects. A detailed review of previously described risk factors as well as recent genetic findings mostly comprises this work. The most recent meeting abstracts and relevant articles published in journals covered by the Science Citation Index and Medline are also examined. The review pays special attention to the genetic component of BRONJ. A total of 15 series and 14 guidelines or revisions were selected to fit the aims of the review. Gene variability was reviewed in depth to give a clinical illustration on the genetic aspects of BRONJ. Crude prevalence and 5-year cumulative incidence were considered as the most important end points for predictive purposes. Several acquired factors were recognized as predictors for BRONJ in MM, especially intravenous bisphosphonates, dental trauma and advanced age. Among genetic factors, polymorphisms on CYP2C8 gene arise as a promising risk factor. Bisphosphonate-related osteonecrosis of the jaw can be predicted with a conjunction of genetic and environmental risk factors.

Low expression of ZHX2, but not RCBTB2 or RAN, is associated with poor outcome in multiple myeloma.

RAN, ZHX2 and RCBTB2 (CHC1L) expression was evaluated by quantitative real time reverse transcription polymerase chain reaction in plasma cells from 85 monoclonal gammopathies: 58 symptomatic multiple myeloma (MM) (52 untreated, six relapsed), eight smouldering MM, five monoclonal gammopathy of undetermined significance, four plasma cell leukaemias and 10 myeloid cell lines. ZHX2 was weakly expressed in high-risk/proliferative disease compared to low-risk or indolent disease. High ZHX2 expression was associated with better response and longer survival after high-dose therapy. RCBTB2 expression was weaker in hyperdiploid versus non-hyperdiploid cases while RAN was more expressed in symptomatic MM and cell lines.

Pemetrexed acts as an antimyeloma agent by provoking cell cycle blockade and apoptosis.

Multiple myeloma (MM) is a malignancy of terminally differentiated B lymphocytes, characterized by accumulation of a monotypic plasma cell population in the bone marrow, monoclonal immunoglobulin in serum and/or urine and osteolytic lesions. Despite recent advances in the treatment, MM remains an incurable disease. This calls for an effort to develop novel therapeutics in order to eradicate the disease. Here we have evaluated the potential antimyeloma action of Pemetrexed, an antifolate drug that has shown promising results in other neoplastic diseases. Pemetrexed had a potent antimyeloma effect on cell lines sensitive and resistant to conventional therapeutic agents, and was also efficient on fresh cells from patients and in a murine MM model. Furthermore, Pemetrexed abrogated the protective action on MM cell death of several growth factors produced by the bone marrow microenvironment. Mechanistic studies indicated that Pemetrexed provoked this action by a combined effect that included cell cycle blockade, probably by p21 upregulation, and induction of apoptosis through caspase-dependent and -independent mechanisms. These data, together with the fact that Pemetrexed is already licensed for the therapy of other neoplastic diseases, opens the possibility for the inclusion of Pemetrexed in the therapeutic armamentarium to battle MM.

Gene expression profiling of B lymphocytes and plasma cells from Waldenström's macroglobulinemia: comparison with expression patterns of the same cell counterparts from chronic lymphocytic leukemia, multiple myeloma and normal individuals.

The tumoral clone of Waldenström's macroglobulinemia (WM) shows a wide morphological heterogeneity, which ranges from B lymphocytes (BL) to plasma cells (PC). By means of genome-wide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell counterparts from chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes downregulated in WM-BL were IL4R, which plays a relevant role in CLL B-cell survival, and BACH2, which participates in the development of class-switched PC. Interestingly, one of the upregulated genes in WM-BL was IL6. A set of four genes was able to discriminate clonal BL from WM and CLL: LEF1 (WNT/beta-catenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5, which was overexpressed in WM-PC, and IRF4 and BLIMP1, which were underexpressed. In addition, three of the target genes activated by PAX5 - CD79, BLNK and SYK - were upregulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell counterpart.

Prognostic and biological implications of genetic abnormalities in multiple myeloma undergoing autologous stem cell transplantation: t(4;14) is the most relevant adverse prognostic factor, whereas RB deletion as a unique abnormality is not associated with adverse prognosis.

Fluorescence in situ hybridization (FISH) has become a powerful technique for prognostic assessment in multiple myeloma (MM). However, the existence of associations between cytogenetic abnormalities compels us to re-assess the value of each abnormality. A total of 260 patients with MM at the time of diagnosis, enrolled in the GEM-2000 Spanish transplant protocol, have been analyzed by FISH in order to ascertain the independent influence on myeloma prognosis of IGH translocations, as well as RB and P53 deletions. Survival analyses showed that patients with t(4;14), RB or P53 deletions had a significantly shorter survival than patients without these abnormalities. However, patients with RB deletions without other abnormalities in FISH analysis, displayed a similar outcome to those patients without genetic changes by FISH (46 vs 54 months, P=0.3). In the multivariate analysis the presence of t(4;14), RB deletion associated with other abnormalities, age >60 years, high proportion of S-phase cells and advanced stage of the disease according to the International Staging System retained their independent prognostic influence. In summary, RB deletion as a sole abnormality does not lead to a shortening in the survival of MM patients, whereas t(4;14) confers the worst prognosis in MM patients treated with high-dose chemotherapy.

Prognostic value of antigen expression in multiple myeloma: a PETHEMA/GEM study on 1265 patients enrolled in four consecutive clinical trials.

Persistence of minimal residual disease (MRD) after treatment for myeloma predicts inferior outcomes, but within MRD-positive patients there is great heterogeneity with both early and very late relapses. Among different MRD techniques, flow cytometry provides additional information about antigen expression on tumor cells, which could potentially contribute to stratify MRD-positive patients. We investigated the prognostic value of those antigens required to monitor MRD in 1265 newly diagnosed patients enrolled in the GEM2000, GEM2005MENOS65, GEM2005MAS65 and GEM2010MAS65 protocols. Overall, CD19pos, CD27neg, CD38lo, CD45pos, CD81pos, CD117neg and CD138lo expression predicted inferior outcomes. Through principal component analysis, we found that simultaneous CD38lowCD81posCD117neg expression emerged as the most powerful combination with independent prognostic value for progression-free survival (HR:1.69; P=0.002). This unique phenotypic profile retained prognostic value among MRD-positive patients. We then used next-generation flow to determine antigen stability throughout the course of the disease, and found that the expression of antigens required to monitor MRD is mostly stable from diagnosis to MRD stages, except for CD81 whose expression progressively increased from baseline to chemoresistant tumor cells (14 vs 28%). Altogether, we showed that the phenotypic profile of tumor cells provides additional prognostic information, and could be used to further predict risk of relapse among MRD-positive patients.

Bortezomib in multiple myeloma.

Multiple myeloma (MM) remains an incurable disease, and novel agents are therefore needed to improve outcome. Bortezomib is the first proteasome inhibitor to be approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicinal Products for the treatment of refractory/relapsed MM. Bortezomib has demonstrated significant anti-myeloma activity as a single agent in refractory/relapsed MM. When used in combination with other agents, responses have suggested the possibility of chemosensitization and synergy. All these facts have been the rationale for the use of bortezomib-based regimens as upfront treatment in young and elderly newly diagnosed MM patients. Furthermore, bortezomib does not appear to have an adverse effect on subsequent stem-cell collection. Bortezomib is well tolerated; most side-effects are only mild to moderate and manageable. Practical management of these side-effects is given so that they can be recognized and minimized by dose modification or concomitant therapy.

Hyperhomocysteinemia is a risk factor of recurrent coronary event in young patients irrespective to the MTHFR C677T polymorphism.

Despite the well-known pro-coagulant effect of hyperhomocysteinemia, data is limited regarding the result on recurrent coronary event (RCE) in young people. One hundred and forty patients <55 years old with a first acute coronary syndrome (ACS) were prospectively followed for a mean (+/-S.D.) follow-up of 49+/-14 months in order to investigate the relationship between homocysteine levels (tHcy) at admission and the incidence of RCE. The tHcy values were divided into quartiles to examine their relationship with end points. Furthermore, we determined the effect of C677T methylene tetrahydrofolate reductase (MTHFR) polymorphism, as well as other risk factors for developing a RCE. The median plasma homocysteine concentration was 9.6 mumol/L (interquartile range, 3.7). In the screening of MTHFR C677T polymorphism in patients with ACS, the T allele frequency was 0.4 and the genotype frequency distributions were in Hardy-Weinberg equilibrium. At time of final evaluation, 49 (35%) of the 140 valuable patients had developed a RCE. Increasing numbers of RCE were observed for increasing quartiles of tHcy according to Kaplan-Meier survival (Log-rank test=0.0092). The MTHFR C677T polymorphism was not associated with an increased incidence of RCE. In multivariate analysis, the variables independently associated with a higher risk of RCE were age older than 45 years [HR=2.7; (95% CI, 1.3-6.1); p=0.030], body mass index more than 25 [HR=2.6; (95% CI, 1.1-5.9); p=0.034] and tHcy levels into quartile 4 (tHcy>12.37 mumol/L) [HR=2.5; (95% CI, 1.1-4.7); p=0.04]. Elevated plasma homocysteine level at admission is an independent risk factor for RCE after the first episode of ACS in young patients irrespective of the status of MTHFR C677T.

In leukapheresis products from non-Hodgkin's lymphoma patients, the immature hematopoietic progenitors show higher CD90 and CD34 antigenic expression.

Damage to the stem cell progenitors caused by the chemotherapy received in patients diagnosed with non-Hodgkin's lymphoma (NHL) may be an important factor limiting progenitor cell mobilization. The aim of the present analysis was to evaluate the effect of the chemotherapy on the different progenitor cell subpopulations obtained in the leukapheresis. For this purpose, a combination of immunophenotype and functional assays has been performed in 26 mobilized peripheral blood (PB) samples from NHL patients and 36 healthy donors. The different progenitor subpopulations analyzed by flow cytometry significantly correlated with the corresponding populations assessed by functional assays in both healthy donors and NHL patients (p<0.05, r>0.5). The number of committed CFU-GM was similar in both groups (p=0.246), but we found significant decrease in the number of BFU-E and more immature progenitors in PB from NHL patients as compared to donors (p<0.05). Moreover, the number of total CFU was significantly lower in NHL patients (p=0.007). Accordingly, CD34+ cells (p=0.018) and CD34+ subpopulations was decreased in NHL patients. Nevertheless, CD90 and CD34 intensity was significantly higher within CD34+ cells from NHL patients as compared to donors. However, although numerically reduced non-committed CD34+ cells are more immature in chemotherapy mobilized NHL patients. In summary, our results show that all NHL hematopoietic progenitors, analyzed by both immunophenotypical and functional approaches, are impaired in leukapheresis products.

Molecular biology of myeloma.

Multiple myeloma (MM) is a B-cell malignancy characterised by the accumulation of clonal plasma cells (PC) in the bone marrow (BM). The molecular bases for this incurable disease have been widely investigated in the last years, and the development of modern genomic technologies has contributed to the understanding of the pathogenesis of MM. The molecular mechanisms that explain the cellular origin of myeloma cells, the cytogenetic abnormalities and their clinical implications, and the biological information provided by gene expression profiling analysis are reviewed in this paper. In addition, a molecular classification of MM in seven groups based on the relationship between gene expression profiling, chromosomal translocations and prognostic outcome is also presented. And finally, the recent hypothesis of a potential unifying event in the pathogenesis of MM, supported by cyclin D deregulation in virtually all MM tumours, will be summarised.

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.

Differentiation stage of myeloma plasma cells: biological and clinical significance.

The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n=20) that the CD19-CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19-CD81+ and CD19-CD81- BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19-CD81-) clones, 38% intermediate-differentiated (CD19-CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P=0.005) and overall survival (HR: 2.1; P=0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n=40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles.

A randomized phase III study of carfilzomib vs low-dose corticosteroids with optional cyclophosphamide in relapsed and refractory multiple myeloma (FOCUS).

This randomized, phase III, open-label, multicenter study compared carfilzomib monotherapy against low-dose corticosteroids and optional cyclophosphamide in relapsed and refractory multiple myeloma (RRMM). Relapsed and refractory multiple myeloma patients were randomized (1:1) to receive carfilzomib (10-min intravenous infusion; 20 mg/m2 on days 1 and 2 of cycle 1; 27 mg/m2 thereafter) or a control regimen of low-dose corticosteroids (84 mg of dexamethasone or equivalent corticosteroid) with optional cyclophosphamide (1400 mg) for 28-day cycles. The primary endpoint was overall survival (OS). Three-hundred and fifteen patients were randomized to carfilzomib (n=157) or control (n=158). Both groups had a median of five prior regimens. In the control group, 95% of patients received cyclophosphamide. Median OS was 10.2 (95% confidence interval (CI) 8.4-14.4) vs 10.0 months (95% CI 7.7-12.0) with carfilzomib vs control (hazard ratio=0.975; 95% CI 0.760-1.249; P=0.4172). Progression-free survival was similar between groups; overall response rate was higher with carfilzomib (19.1 vs 11.4%). The most common grade ⩾3 adverse events were anemia (25.5 vs 30.7%), thrombocytopenia (24.2 vs 22.2%) and neutropenia (7.6 vs 12.4%) with carfilzomib vs control. Median OS for single-agent carfilzomib was similar to that for an active doublet control regimen in heavily pretreated RRMM patients.

Prognostic factors for donor lymphocyte infusions following non-myeloablative allogeneic stem cell transplantation in multiple myeloma.

In this retrospective study, we evaluated donor lymphocyte infusions given for relapsed (n=48) or persistent (n=15) myeloma following non-myeloablative allogeneic stem cell transplantation (Allo-SCT). Twenty-four of 63 patients (38.1%) responded: 12 patients (19.0%) with a partial response (PR) and 12 patients (19.0%) with a complete response (CR). Overall survival after donor lymphocyte infusions (DLI) was 23.6 months (1.0-50.7+). Median overall survival for non-responding patients was 23.6 months and has not been reached for the patients responding to DLI. In responders, progression-free survival after DLI was 27.8 months (1.2-46.2+). Patients with a PR had a median progression-free survival of 7.0 months, whereas patients with a CR to DLI had a median progression-free survival of 27.8 months. Major toxicities were acute graft-versus-host disease (GVHD) (38.1%) and chronic GVHD (42.9%). Seven patients (11.1%) died from treatment-related mortality. The only significant prognostic factors for response to DLI were the occurrence of acute and chronic GVHD. There was a trend towards significance for time between transplantation and DLI, and response. Donor lymphocyte infusion following non-myeloablative Allo-SCT is a valuable strategy for relapsed or persistent disease.