Analysis of Micro-RNAs Associated to Treatment Failure in Diffuse Large B Cell Lymphoma

Introduction:

Micro-RNAs (miRNAs) are 19-24 nucleotide non-coding RNAs that regulate gene expression through the inactivation of their messenger RNA. Previous studies have demonstrated the important role of some miRNAs in the development of cancer. Specifically, in diffuse large B cell lymphoma (DLBCL), miRNAs involved in lymphomagenesis were identified but understanding their biological function continues to be a challenge. Nevertheless, the pathogenesis effects of some miRNAs such as miR-125a, miR-17-92 cluster or mi-R-155 are well characterized. Some studies suggest that miRNAs also possess a prognostic potential role in DLBCL. For this reason, our objective was to analyze the different miRNAs involved in the chemo-sensitivity or resistance to the first line treatment, their correlation with standard prognostic factors at diagnosis and the role of these miRNAs in survival in patients with DLBCL.

Material and methods:

Patients homogeneously treated with R-CHOP from 1999-2013 were reviewed from 3 Spanish centers. They were retrospectively obtained from Pathology Department registry to avoid selection bias. We included those patients with valid genomic material in formalin-fixed-paraffin-embedded tissue and with available clinical data. Samples were processed using the miRNA 4 Affymetrix microarrays kit in a discovery group that included 2 cohorts (patients with durable complete remission (CR) versus refractory or early relapsing patients). Those miRNAs with differential expression were validated in the whole series with quantitative RT-PCR. We also analyzed the role of these miRNAs as predictors of event-free survival (EFS) and overall survival (OS) and their relationship with standard prognostic factors in DLBCL.

Results:

We identified 156 patients homogeneously treated with R-CHOP. Finally, 96 samples were obtained with valid material for RNA extraction. To identify those miRNAs with prognostic implication, a discovery cohort of 12 patients was used in which all the cases had poor prognosis with high tumor load and advance disease (III-IV stage and unfavorable R-IPI). On this basis of poor prognosis, 2 groups were defined totally opposed from the point of view of the treatment response and evolution: chemo-resistance group (n=6) including refractory or relapsed (RR) patients (first 12 months) and chemo-sensitive group (n=6) including patients with at least 3 years of CR. A hierarchical clustering was performed in which 26 miRNAs differentially expressed were identified. A screening of miRNAs was carried out based on the fold-change and pathways involved and finally we obtained 10 miRNAs differentially expressed in RR group. The validation of these miRNAs was performed with quantitative RT-PCR in the whole series which finally included 68 samples with valid material. A univariate survival analysis including clinical prognostic factors and the selected 10 miRNAs was performed. We confirmed that 7 of them (miR-20b-5p, miR-1244, miR-6840-3p, miR-1231, miR-193b-5p, miR-6860-5p y miR-199a-5p) significantly influenced EFS and 6 of them (miR-1244, miR-1231, miR-193b-5p, miR-885-3p, miR-182-5p y miR-199a-5p) on OS. From these 10 miRNAs, only 3 had a significant prognosis role not only for EFS but also for OS and progression-free survival (PFS): miR-1244, miR-193b-5p and miR-1231. The overexpression of miR-1244 and miR-193-5p were associated with advance stage and worse clinical prognosis factors (p<0.05). A multivariate analysis was performed including clinical and biological variables and we obtained that the overexpression of miR-1231, high R-IPI and a reduction of relative dose intensity (RDI) >15% were independently associated with worse SG and EFS (Figure 1). In previously reported studies, these 3 miRNAs have been related with proliferative events such as the overexpression of myc or anti-apoptosis. Also, the miR-1231 may be related with new lymphomagenesis pathways associated to viral infections.

Conclusions:

Through a discovery group focused on progression/refractoriness, a group of new miRNAs differentially expressed on chemo-resistant patients with DLBCL was identified. The overexpression of miR-1244 and miR-193-5p was associated with more extensive disease and worse clinical prognostic factors. The high R-IPI, reduction of >15% RDI and the overexpression of miR-1231 were independently associated with worse EFS and OS.

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