Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells.

Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness.

High miR-34a expression improves response to doxorubicin in diffuse large B-cell lymphoma.

The standard treatment for patients with diffuse large B-cell lymphoma (DLBCL) is the immunochemotherapy-based R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone). Resistance to treatment, intrinsic or acquired, is observed in approximately 40% of patients with DLBCL, who thus require novel interventions to survive. To identify biomarkers for cytotoxic response assessment, microRNAs (miRNAs) associated with doxorubicin sensitivity were determined by combining global miRNA expression profiling with systematic dose-response screens in 15 human DLBCL cell lines. One candidate, miR-34a, was tested in functional in vitro studies and in vivo in a retrospective clinical cohort. High expression of miR-34a was observed in cell lines sensitive to doxorubicin, and upregulation of miR-34a is documented here to increase doxorubicin sensitivity in in vitro lentiviral transduction assays. High expression of miR-34a had a prognostic impact using overall survival as outcome. With risk stratification of DLBCL samples based on resistance gene signatures (REGS), doxorubicin-responsive samples had statistically significant upregulated miR-34a expression. Classification of the DLBCL samples into subset-specific B cell-associated gene signatures (BAGS) revealed differentiation-specific expression of miR-34a. Our data further support FOXP1 as a target of miR-34a, suggesting that downregulation of FOXP1 may sensitize DLBCL cells to doxorubicin. We conclude that miRNAs, in particular miR-34a, may have clinical utility in DLBCL patients as both predictive and prognostic biomarkers.

MicroRNAs as novel biomarkers in diffuse large B-cell lymphoma--a systematic review.

INTRODUCTION: MicroRNAs (miRNAs) are short non-coding RNAs that have the ability to regulate gene expression at the post-transcriptional level. MiRNAs are deregulated in many cancer types, and several miRNAs have been suggested as novel diagnostic and prognostic biomarkers in diffuse large B-cell lymphoma (DLBCL). The objective of this study was to systematically collect and evaluate current knowledge of miRNAs functioning as diagnostic and prognostic biomarkers within DLBCL. METHODS: This review was conducted according to the Preferred Reporting for Systematic Reviews and Meta-analyses guidelines. A systematic search of literature in PubMed and Embase was made and supplemented by screening of reference lists. Only original peer-reviewed studies written in English were included and screened based on miRNA expression, molecular subtypes of DLBCL and patient outcome. RESULTS: Out of 277 candidate records, a total of 20 studies qualified for inclusion in this review. In all, 11 studies reported a total of 48 miRNAs with expression patterns associated with specific molecular DLBCL subtypes, and 14 studies reported a total of 30 miRNAs associated with patient outcome. However, only few miRNAs showed significant results in more than one study. CONCLUSION: MiRNAs qualify as potential diagnostic and prognostic biomarkers in DLBCL. However, more clinical validation including prospective and cross-centre studies are required before specific miRNAs can be integrated into the daily practice as biomarkers in DLBCL, which would contribute to an era of more personalised medicine.

MicroRNAs in B-cells: from normal differentiation to treatment of malignancies.

MicroRNAs (miRNAs) are small non-coding RNAs that play important post-transcriptional regulatory roles in a wide range of biological processes. They are fundamental to the normal development of cells, and evidence suggests that the deregulation of specific miRNAs is involved in malignant transformation due to their function as oncogenes or tumor suppressors. We know that miRNAs are involved in the development of normal B-cells and that different B-cell subsets express specific miRNA profiles according to their degree of differentiation. B-cell-derived malignancies contain transcription signatures reminiscent of their cell of origin. Therefore, we believe that normal and malignant B-cells share features of regulatory networks controlling differentiation and the ability to respond to treatment. The involvement of miRNAs in these processes makes them good biomarker candidates. B-cell malignancies are highly prevalent, and the poor overall survival of patients with these malignancies demands an improvement in stratification according to prognosis and therapy response, wherein we believe miRNAs may be of great importance. We have critically reviewed the literature, and here we sum up the findings of miRNA studies in hematological cancers, from the development and progression of the disease to the response to treatment, with a particular emphasis on B-cell malignancies.

The clinical application of UGT1A1 pharmacogenetic testing: gene-environment interactions.

Over the past decade, the number of pharmacogenetic tests has increased considerably, allowing for the development of our knowledge of their clinical application. The uridine diphosphate glucuronosyltransferase 1A1 gene ( UGT1A1 ) assay is an example of a pharmacogenetic test. Numerous variants have been found in UGT1A1 , the main conjugating enzyme of bilirubin and drugs such as the anticancer drug irinotecan. Recently, the US Food and Drug Administration (FDA) recommended testing for the presence of UGT1A1*28 , an allele correlated with decreased transcriptional activity, to predict patients at risk of irinotecan toxicity. The administration of other drugs - such as inhibitors of the UGT1A1 enzyme - can clinically mimic the *28 phenotype, whereas inducers of UGT1A1 can increase the glucuronidation rate of the enzyme. The *28 polymorphism is not present in all ethnicities at a similar frequency, which suggests that it is important to study different populations to determine the clinical relevance of testing for UGT1A1*28 and to identify other clinically relevant UGT1A1 variants. Environmental factors such as lifestyle can also affect UGT1A1 activity. This review is a critical analysis of studies on drugs that can be affected by the presence of UGT1A1*28 , the distribution of this polymorphism around the globe, distinct variants that may be clinically significant in African and Asian populations and how lifestyle can affect treatment outcomes that depend on UGT1A1 activity.