A B-cell receptor-related gene signature predicts survival in mantle cell lymphoma: results from the Fondazione Italiana Linfomi MCL-0208 trial.

Mantle cell lymphoma patients have variable clinical courses, ranging from indolent cases that do not require immediate treatment to aggressive, rapidly progressing diseases. Thus, diagnostic tools capable of stratifying patients according to their risk of relapse and death are needed. This study included 83 samples from the Fondazione Italiana Linfomi MCL-0208 clinical trial. Through gene expression profiling and quantitative real-time PCR we analyzed 46 peripheral blood and 43 formalin-fixed paraffin-embedded lymph node samples. A prediction model to classify patients was developed. By analyzing the transcriptome of 27 peripheral blood samples, two subgroups characterized by a differential expression of genes from the B-cell receptor pathway (B-cell receptorlow and B-cell receptorhigh) were identified. The prediction model based on the quantitative real-time PCR values of six representative genes (AKT3, BCL2, BTK, CD79B, PIK3CD, and SYK), was used to classify the 83 cases (43 B-cell receptorlow and 40 B-cell receptorhigh). The B-cell receptorhigh signature associated with shorter progression-free survival (P=0.0074), selected the mantle cell lymphoma subgroup with the shortest progression-free survival and overall survival (P=0.0014 and P=0.029, respectively) in combination with high (>30%) Ki-67 staining, and was an independent predictor of short progression- free survival along with the Mantle Cell Lymphoma International Prognostic Index-combined score. Moreover, the clinical impact of the 6- gene signature related to the B-cell receptor pathway identified a mantle cell lymphoma subset with shorter progression-free survival intervals also in an external independent mantle cell lymphoma cohort homogenously treated with different schedules. In conclusion, this 6-gene signature associates with a poor clinical response in the context of the MCL- 0208 clinical trial. (clinicaltrials.gov identifier: 02354313).

Targeted chitosan nanobubbles as a strategy to down-regulate microRNA-17 into B-cell lymphoma models.

Introduction: MicroRNAs represent interesting targets for new therapies because their altered expression influences tumor development and progression. miR-17 is a prototype of onco-miRNA, known to be overexpressed in B-cell non-Hodgkin lymphoma (B-NHL) with peculiar clinic-biological features. AntagomiR molecules have been largely studied to repress the regulatory functions of up-regulated onco-miRNAs, but their clinical use is mainly limited by their rapid degradation, kidney elimination and poor cellular uptake when injected as naked oligonucleotides. Methods: To overcome these problems, we exploited CD20 targeted chitosan nanobubbles (NBs) for a preferential and safe delivery of antagomiR17 to B-NHL cells. Results: Positively charged 400 nm-sized nanobubbles (NBs) represent a stable and effective nanoplatform for antagomiR encapsulation and specific release into B-NHL cells. NBs rapidly accumulated in tumor microenvironment, but only those conjugated with a targeting system (antiCD20 antibodies) were internalized into B-NHL cells, releasing antagomiR17 in the cytoplasm, both in vitro and in vivo. The result is the down-regulation of miR-17 level and the reduction in tumor burden in a human-mouse B-NHL model, without any documented side effects. Discussion: Anti-CD20 targeted NBs investigated in this study showed physico-chemical and stability properties suitable for antagomiR17 delivery in vivo and represent a useful nanoplatform to address B-cell malignancies or other cancers through the modification of their surface with specific targeting antibodies.

TP53 Mutations with Low Variant Allele Frequency Predict Short Survival in Chronic Lymphocytic Leukemia.

PURPOSE: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. EXPERIMENTAL DESIGN: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. RESULTS: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. CONCLUSIONS: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

The SIRT1/TP53 axis is activated upon B-cell receptor triggering via miR-132 up-regulation in chronic lymphocytic leukemia cells.

The B-cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). By global microRNA profiling of CLL cells stimulated or not stimulated by anti-IgM, significant up-regulation of microRNAs from the miR-132~212 cluster was observed both in IGHV gene unmutated (UM) and mutated (M) CLL cells. Parallel gene expression profiling identified SIRT1, a deacetylase targeting several proteins including TP53, among the top-ranked miR-132 target genes down-regulated upon anti-IgM exposure. The direct regulation of SIRT1 expression by miR-132 was demonstrated using luciferase assays. The reduction of SIRT1 mRNA and protein (P = 0.001) upon anti-IgM stimulation was associated with an increase in TP53 acetylation (P = 0.007), and the parallel up-regulation of the TP53 target gene CDKN1A. Consistently, miR-132 transfections of CLL-like cells resulted in down-regulation of SIRT1 and an induction of a TP53-dependent apoptosis. Finally, in a series of 134 CLL samples, miR-132, when expressed above the median value, associated with prolonged time-to-first-treatment in patients with M CLL (HR = 0.41; P = 0.02). Collectively, the miR-132/SIRT1/TP53 axis was identified as a novel pathway triggered by BCR engagement that further increases the complexity of the interactions between tumor microenvironments and CLL cells.

Potential therapeutic role of antagomiR17 for the treatment of chronic lymphocytic leukemia.

Recently it was reported that microRNA from the miR-17 ~ 92 family may have a key role in chronic lymphocytic leukemia (CLL). Here, we designed specific oligonucleotides to target endogenous miR-17 (antagomiR17). In-vitro administration of antagomiR17 effectively reduced miR-17 expression and the proliferation of CLL-like MEC-1 cells. When injected in-vivo in tumor generated by the MEC-1 cells in SCID mice, antagomiR17 dramatically reduced tumor growth and significantly increase survival. Altogether, our results provide the rationale for the use of antagomiR17 as a novel potential therapeutic tool in CLL and in other lymphoproliferative disorders where miR-17 has a driver role in tumor progression.