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.
Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may ...represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered at www.clinicaltrials.gov as #NCT01237249.
•We report for the first time the biological features of MRD cells in MM and unravel that clonal selection is already present at the MRD stage.•MRD cells show a singular phenotypic signature that may result from persisting clones with different genetic and gene expression profiles.
Summary
The introduction of Rituximab has improved the outcome and survival rates of Burkitt lymphoma (BL). However, early relapse and refractoriness are current limitations of BL treatment and new ...biological factors affecting the outcome of these patients have not been explored. This study aimed to identify the presence of genomic changes that could predict the response to new therapies in BL. Forty adolescent and adult BL patients treated with the Dose‐Intensive Chemotherapy Including Rituximab (Burkimab) protocol (Spanish Programme for the Study and Treatment of Haematological Malignancies; PETHEMA) were analysed using array‐based comparative genomic hybridization (CGH). In addition, the presence of TP53, TCF3 (E2A), ID3 and GNA13 mutations was assessed by next‐generation sequencing (NGS). Ninety‐seven per cent of the patients harboured genomic imbalances. Losses on 11q, 13q, 15q or 17p were associated with a poor response to Burkimab therapy (P = 0·038), shorter progression‐free survival (PFS; P = 0·007) and overall survival (OS; P = 0·009). The integrative analysis of array‐CGH and NGS showed that 26·3% (5/19) and 36·8% (7/19) of patients carried alterations in the TP53 and TCF3 genes, respectively. TP53 alterations were associated with shorter PFS (P = 0·011) while TCF3 alterations were associated with shorter OS (P = 0·032). Genetic studies could be used for risk stratification of BL patients treated with the Burkimab protocol.
IL-8 promotes cancer cell growth, survival, angiogenesis, and metastasis in several tumors. Herein, we investigated the sources of IL-8 production in multiple myeloma (MM) and its potential roles in ...MM pathogenesis. We found that bone marrow cells from patients with MM secreted higher amounts of IL-8 than healthy donors. IL-8 production was detected in cultures of CD138+ plasma cells and CD138− cells isolated from bone marrows of MM patients, and in three of seven human myeloma cell lines (HMCLs) analyzed. Interactions between MM and stromal cells increased IL-8 secretion by stromal cells through cell-cell adhesion and soluble factors. Interestingly, IL8 expression also increased in HMCLs, stromal cells, and osteoclasts after treatment with the antimyeloma drugs melphalan and bortezomib. In fact, the effect of bortezomib on IL-8 production was higher than that exerted by stromal-MM cell interactions. Addition of exogenous IL-8 did not affect growth of HMCLs, although it protected cells from death induced by serum starvation through a caspase-independent mechanism. Furthermore, IL-8 induced by stromal-MM cell interactions strongly contributed to osteoclast formation in vitro , because osteoclastogenesis was markedly reduced by IL-8–specific neutralizing antibodies. In conclusion, our results implicate IL-8 in myeloma bone disease and point to the potential utility of an anti–IL-8 therapy to prevent unwanted effects of IL-8 up-regulation on survival, angiogenesis, and osteolysis in MM.
This systematic review and meta-analysis aim to provide scientific evidence regarding the effects of training on respiratory muscle training’s impact with the PowerBreath®. A systematic analysis ...based on the PRISMA guides and a conducted research structured around the bases of Web of Science, Scopus, Medline/PubMed, SciELO y Cochrane Library Plus. Six articles published before January 2021 were included. The documentation and quantification of heterogeneity in every meta-analysis were directed through Cochran’s Q test and the statistic I2; additionally, a biased publication analysis was made using funnel plots, whose asymmetry was quantified Egger’s regression. The methodological quality was assessed through McMaster’s. PowerBreath® administering a ≥ 15% resistive load of the maximum inspiratory pressure (PIM) achieves significant improvements (54%) in said pressure within 4 weeks of commencing the inspiratory muscle training. The maximal volume of oxygen (VO2max) considerable enhancements was achieved from the 6 weeks associated with the maximum inspiratory pressure ≥ 21.5% post inspiratory muscle training onwards. Conversely, a significant blood lactate concentration decrement occurred from the 4th week of inspiratory muscle training, after a maximum inspiratory pressure ≥ 6.8% increment. PowerBreath® is a useful device to stimulate sport performance and increase pulmonary function.
Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of ...immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.
Intensive research has been undertaken during the last decade to identify the implication of microRNAs (miRNAs) in the pathogenesis of multiple myeloma (MM). The expression profiling of miRNAs in MM ...has provided relevant information, demonstrating different patterns of miRNA expression depending on the genetic abnormalities of MM and a key role of some miRNAs regulating critical genes associated with MM pathogenesis. However, the underlying causes of abnormal expression of miRNAs in myeloma cells remain mainly elusive. The final expression of the mature miRNAs is subject to multiple regulation mechanisms, such as copy number alterations, CpG methylation or transcription factors, together with impairment in miRNA biogenesis and differences in availability of the mRNA target sequence. In this review, we summarize the available knowledge about the factors involved in the regulation of miRNA expression and functionality in MM.
Upregulation of a cyclin D gene determined by expression microarrays is an almost universal event in multiple myeloma (MM), but this finding has not been properly confirmed at the protein level. For ...this reason, we carried out a quantitative analysis of cyclin D proteins using a capillary electrophoresis nanoimmunoassay in newly diagnosed MM patients. Exclusive expression of cyclin D1 and D2 proteins was detected in 54 of 165 (33%) and 30 of 165 (18%) of the MM patients, respectively. Of note, cyclin D1 or D2 proteins were undetectable in 41% of the samples. High levels of cyclin D1 protein were strongly associated with the presence of t(11;14) or 11q gains. Cyclin D2 protein was detected in all the cases bearing t(14;16), but in only 24% of patients with t(4;14). The presence of cyclin D2 was associated with shorter overall survival (hazard ratio =2.14; P=0.017), although patients expressing cyclin D2 protein, but without 1q gains, had a favorable prognosis. In conclusion, although one of the cyclins D is overexpressed at the mRNA level in almost all MM patients, in approximately half of the patients this does not translate into detectable protein. This suggests that cyclins D could not play an oncogenic role in a proportion of patients with MM (clinicaltrials gov. identifier: NCT01916252).
Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL) is still a challenge.
To characterize the presence of additional DNA copy number ...alterations (CNAs) in children and adults with ALL by whole-genome oligonucleotide array (aCGH) analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults). The NimbleGen CGH 12x135K array (Roche) was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q). CNAs were associated with age, phenotype, genetic subtype and overall survival (OS). In the whole cohort of children, the losses on 14q32.33 (p = 0.019) and 15q13.2 (p = 0.04) were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001) and Xp21.1 (p = 0.029), and the loss of 17p (p = 0.014) were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL.
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