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HLA studies have been used to determine the admixture of different populations within the Iberian Peninsula including neighbouring regions with shared origins, such as Portugal and Castile and Leon. These studies certainly can be used to study human migration that could establish populations currently settled according to genetic distant analysis based on the HLA diversity and language variety.

[Introduction]: Molecular alterations leading progression of asymptomatic CLL-like high-count monoclonal B lymphocytosis (hiMBL) to chronic lymphocytic leukemia (CLL) remain poorly understood. Recently, genome-wide association studies have found 6p21.3, where the human leukocyte antigen (HLA) system is coded, to be a susceptibility risk region for CLL. Previous studies have produced discrepant results regarding the association between HLA and CLL development and outcome, but no studies have been performed on hiMBL. [Aims]: We evaluated the role of HLA class I (-A, -B and -C) and class II (-DRB1 and -DQB1) in hiMBL/CLL susceptibility, hiMBL progression to CLL, and treatment requirement in a large series of 263 patients diagnosed in our center with hiMBL (n = 156) or Binet A CLL (n = 107). [Results]: No consistent association between HLA specificities and hiMBL or CLL susceptibility was found. With a median follow-up of 7.7 years, 48/156 hiMBLs (33%) evolved to asymptomatic CLLs, while 16 hiMBLs (10%) and 44 CLLs (41%) required treatment. No HLA specificities were found to be significantly associated with hiMBL progression or treatment in the whole cohort. However, within antigen-experienced immunoglobulin heavy-chain (IGHV)-mutated hiMBLs, which represents the highest proportion of hiMBL cases (81%), the presence of HLA-DQB1∗03 showed a trend to a higher risk of progression to CLL (60% vs. 26%, P = 0.062). Moreover, HLA-DQB1∗02 specificity was associated with a lesser requirement for 15-year treatment (10% vs. 36%, P = 0.012). [Conclusion]: In conclusion, our results suggest a role for HLA in IGHV-mutated hiMBL prognosis, and are consistent with the growing evidence of the influence of 6p21 on predisposition to CLL. Larger non-biased series are required to enable definitive conclusions to be drawn.

[Purpose]: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma. [Experimental Design]: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR. [Results]: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. [Conclusions]: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.

Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20 % of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5 %) and 17p13.1 (2.5 %) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development.

[Context]: MiR-155 plays a critical role in the development of B-cell malignancies. Previous studies have shown a deregulation of miR-155 in specific cytogenetic subtypes of multiple myeloma (MM). However, the mechanisms that regulate miR-155 expression in MM are not fully understood. [Objective]: In the present study, we explored the regulation of miRNA-155 in MM by DNA methylation mechanisms and the impact of miR-155 expression in survival of MM patients. [Method]: Primary samples were obtained from 95 patients with newly diagnosed myeloma. Methylation was analyzed by Methylation Specific PCR, sequencing of bisulfite treated DNA and luciferase assay. [Results]: qRT-PCR analysis revealed that miR-155 was differentially expressed in MM and its upregulation was associated with longer survival. DNA methylation of CpG island present in the first exon of miR-155 host gene was associated with its low expression in MM cell lines and patient samples. Our results showed for the first time that in vitro methylation of part of the promoter and first exon abrogated the miR-155 expression. We further showed that miR-155 expression in MM cell lines was increased by demethylating 5-aza-dC treatment and decreased by RNA-directed DNA methylation. Additionally, we found that LPS >immunological challenge> was insufficient to induce miR-155 expression in MM cell lines with methylated DNA around transcription start site (TSS). [Conclusion]: This study provides evidence that DNA methylation contributes to miR-155 expression in myeloma cells. Interestingly, the survival data showed an association between miR-155 expression and outcome of MM.