RNA-seq is currently considered the most powerful, robust and adaptable technique for measuring gene expression and transcription activation at genome-wide level. As the analysis of RNA-seq data is ...complex, it has prompted a large amount of research on algorithms and methods. This has resulted in a substantial increase in the number of options available at each step of the analysis. Consequently, there is no clear consensus about the most appropriate algorithms and pipelines that should be used to analyse RNA-seq data. In the present study, 192 pipelines using alternative methods were applied to 18 samples from two human cell lines and the performance of the results was evaluated. Raw gene expression signal was quantified by non-parametric statistics to measure precision and accuracy. Differential gene expression performance was estimated by testing 17 differential expression methods. The procedures were validated by qRT-PCR in the same samples. This study weighs up the advantages and disadvantages of the tested algorithms and pipelines providing a comprehensive guide to the different methods and procedures applied to the analysis of RNA-seq data, both for the quantification of the raw expression signal and for the differential gene expression.
The p53 pathway is inactivated in the majority of human cancers. Although this perturbation frequently occurs through the mutation or deletion of p53 itself, there are other mechanisms that can ...attenuate the pathway and contribute to tumorigenesis. For example, overexpression of important p53 negative regulators, such as murine double minute 2 (MDM2) or murine double minute 4 (MDM4), epigenetic deregulation, or even alterations in
mRNA splicing. In this work, we will review the different mechanisms of p53 pathway inhibition in cancer with special focus on multiple myeloma (MM), the second most common hematological malignancy, with low incidence of p53 mutations/deletions but growing evidence of indirect p53 pathway deregulation. Translational implications for MM and cancer prognosis and treatment are also reviewed.
Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired ...and hyperactive double strand break (DSB) repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR)-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ), and Rad51, involved in homologous recombination (HR). Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ) were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Standard first-line treatment for elderly multiple myeloma (MM) patients ineligible for stem cell transplantation is melphalan plus prednisone (MP). However, complete responses (CRs) are rare. ...Bortezomib is active in patients with relapsed MM, including elderly patients. This phase 1/2 trial in 60 untreated MM patients aged at least 65 years (half older than 75 years) was designed to determine dosing, safety, and efficacy of bortezomib plus MP (VMP). VMP response rate was 89%, including 32% immunofixation-negative CRs, of whom half of the IF– CR patients analyzed achieved immunophenotypic remission (no detectable plasma cells at 10–4 to 10–5 sensitivity). VMP appeared to overcome the poor prognosis conferred by retinoblastoma gene deletion and IgH translocations. Results compare favorably with our historical control data for MP—notably, response rate (89% versus 42%), event-free survival at 16 months (83% versus 51%), and survival at 16 months (90% versus 62%). Side effects were predictable and manageable; principal toxicities were hematologic, gastrointestinal, and peripheral neuropathy and were more evident during early cycles and in patients aged 75 years or more. In conclusion, in elderly patients ineligible for transplantation, the combination of bortezomib plus MP appears significantly superior to MP, producing very high CR rates, including immunophenotypic CRs, even in patients with poor prognostic features.
Multiple myeloma (MM) is a heterogeneous disease with certain genetic features eg, t(4;14), del17p associated with worse outcome. The introduction of thalidomide, lenalidomide, and bortezomib has ...dramatically improved the outlook for patients with MM, but their relative benefit (or harm) for different genetic patient subgroups remains unclear. Unfortunately, the small number of patients in each subgroup frequently limits the analysis of high-risk patients enrolled in clinical trials. Strategies that result in survival of high-risk genetic subgroups approximating that of patients lacking high-risk features are said to overcome the poor prognostic impact of these high-risk features. This outcome has been difficult to achieve, and studies in this regard have so far been limited by inadequate sample size. In contrast, strategies that compare the survival of high-risk genetic subgroups randomized to different treatment arms can identify approaches that improve survival. This type of analysis is clinically useful, even if the absolute gains do not improve outcomes to levels seen in patients without high-risk cytogenetics. Reviewing available data in high-risk MM from this perspective, it appears that bortezomib has frequently been associated with improved survival, whereas thalidomide maintenance has sometimes been associated with a shorter survival.
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.
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.
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.
This study demonstrates that the progression from MGUS to SMM, and eventually to MM, involves a clonal expansion of genetically abnormal PC.
Some genetic abnormalities of multiple myeloma (MM) detected more than two decades ago remain major prognostic factors. In recent years, the introduction of cutting-edge genomic methodologies has ...enabled the extensive deciphering of genomic events in MM. Although none of the alterations newly discovered have significantly improved the stratification of the outcome of patients with MM, some of them, point mutations in particular, are promising targets for the development of personalized medicine. This review summarizes the main genetic abnormalities described in MM together with their prognostic impact, and the therapeutic approaches potentially aimed at abrogating the undesirable pathogenic effect of each alteration.
The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia ...(PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.
Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited.
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.
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.
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.
Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Purpose To perform a critical analysis on the impact of depth of response in newly diagnosed multiple myeloma (MM). Patients and Methods Data were analyzed from 609 patients who were enrolled in the ...GEM (Grupo Español de Mieloma) 2000 and GEM2005MENOS65 studies for transplant-eligible MM and the GEM2010MAS65 clinical trial for elderly patients with MM who had minimal residual disease (MRD) assessments 9 months after study enrollment. Median follow-up of the series was 71 months. Results Achievement of complete remission (CR) in the absence of MRD negativity was not associated with prolonged progression-free survival (PFS) and overall survival (OS) compared with near-CR or partial response (median PFS, 27, 27, and 29 months, respectively; median OS, 59, 64, and 65 months, respectively). MRD-negative status was strongly associated with prolonged PFS (median, 63 months; P < .001) and OS (median not reached; P < .001) overall and in subgroups defined by prior transplantation, disease stage, and cytogenetics, with prognostic superiority of MRD negativity versus CR particularly evident in patients with high-risk cytogenetics. Accordingly, Harrell C statistics showed higher discrimination for both PFS and OS in Cox models that included MRD (as opposed to CR) for response assessment. Superior MRD-negative rates after different induction regimens anticipated prolonged PFS. Among 34 MRD-negative patients with MM and a phenotypic pattern of bone marrow involvement similar to monoclonal gammopathy of undetermined significance at diagnosis, the probability of "operational cure" was high; median PFS was 12 years, and the 10-year OS rate was 94%. Conclusion Our results demonstrate that MRD-negative status surpasses the prognostic value of CR achievement for PFS and OS across the disease spectrum, regardless of the type of treatment or patient risk group. MRD negativity should be considered as one of the most relevant end points for transplant-eligible and elderly fit patients with MM.
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