[Study of genetic variants in 169 non-small cell lung cancer patients]. / Estudio de variantes genéticas en 169 pacientes de cáncer de pulmón no microcítico.

INTRODUCTION: Lung cancer is the leading cause of cancer death in our country. Non-small cell lung cancer (NSCLC) represents the paradigm of personalized medicine. The main objective of this study is analysing the distribution of the most frequently described clinically significant variants in NSCLC, in our environment. MATERIAL AND METHODS: We studied the immunohistochemical expression of TTF1, p40 and PD-L1 and the genetic variants frequency using Next-Generation Sequencing (NGS) with a panel of 52 genes, in 174 NSCLC paraffin-embedded samples in 169 patients (111 men and 52 women) from the province of Cádiz. RESULTS: The immunohistochemical expression of TTF1, p40 and PD-L1 was positive in 87%, 0% and 46% in adenocarcinoma, and 0%, 100% and 41% in squamous cell carcinoma. In NGS, the most common single nucleotide variants (SNVs) were KRAS (36%), EGFR (14%), BRAF (10%), PIK3CA (8%), and MET (3%). The most frequent copy number variants (CNVs) were amplifications in NF1 (30%), EGFR (18%), CCND1 (9%), MYC (9%) and KRAS (7%). In women, SNV in EGFR are more frequent than in men (P<.0001). Adenocarcinoma is the most frequent histological type with SNV in KRAS (P=.007361) or in EGFR (P<.0001). Gene fusions were detected in 16 patients (9.47%), in 9 cases in the MET gene. CONCLUSIONS: We detected associations, not described so far, between immunohistochemical expression and specific gene variants, which could have an impact on the treatment of NSCLC patients.

Identification of prognostic factors in classic Hodgkin lymphoma by integrating whole slide imaging and next generation sequencing.

Digital pathology and genomics are increasingly used to improve our understanding of lymphoid neoplasms. Algorithms for quantifying cell populations in the lymph node and genetics can be integrated to identify new biomarkers with prognostic impact in classic Hodgkin lymphoma (cHL). In 16 cHL patients, we have performed whole slide imaging (WSI) analysis and quantification of CD30+, CD20+, CD3+ and MUM1+ cells in whole tissue slides, and Next Generation Sequencing (NGS) in formalin fixed paraffin-embedded (FFPE) tissue, using a widely used NSG panel (Oncomine® Focus Assay) to define genetic variants underlying tumor development. The different cell populations could be successfully identified in scanned slides of cHL, supporting the inclusion of WSI in the histopathological evaluation of cHL as an adequate method for the quantification of different cell populations. We also performed genetic profiling in FFPE samples of cHL leading to the identification of copy number variations in the Neurofibromin 1 gene (17q11.2) and the Androgen Receptor gene (Xq12) accompanied by chromosomal gains and losses in CDK4, KRAS and FGFR2 genes. Progression-free survival (PFS) was statistically significantly higher in cHL patients with amplification in the NF1 gene combined with CD3+ cells above 28.6% (p = 0.006) and MUM1+ cells above 21.8% (p < 0.001). Moreover, patients with MUM1+ cells above 21.8% showed a statistically significantly higher PFS when combined with amplification of the AR gene (p < 0.001) and wild-type KRAS (p < 0.001). The integration of WSI analysis and DNA sequencing could be useful to improve our understanding of the biology of cHL and define risk subgroups.

The Need for Standardization in Next-Generation Sequencing Studies for Classic Hodgkin Lymphoma: A Systematic Review.

Classic Hodgkin lymphoma (cHL) constitutes a B cell-derived neoplasm defined by a scarce tumoral population, termed Hodgkin and Reed-Sternberg (HRS) cells, submerged into a histologically heterogeneous microenvironment. The paucity of HRS cells has historically hampered genetic studies, rendering the identification of the recurrent genetic lesions and molecular pathways deregulated in this lymphoma difficult. The advent of high-throughput sequencing methods such as next-generation sequencing (NGS) could sensibly optimize the identification of the mutational landscape of cHL. However, there is no current consensus either in the design of panels for targeted NGS or in its most relevant clinical applications. In this work, we systematically review the current state of NGS studies of cHL, stressing the need for standardization both in the candidate genes to be analyzed and the bioinformatic pipelines. As different institutions have developed and implemented their own customized NGS-based protocols, to compare and systematically review the major findings of this ongoing research area could be of added value for centers that routinely perform diagnostic, monitoring and genotyping strategies in cHL samples. The results of this systematic review should contribute to the interdepartmental harmonization and achievement of a consensus in the current clinical applications of NGS studies of cHL.

Prognostic Role of the Expression of Latent-Membrane Protein 1 of Epstein-Barr Virus in Classical Hodgkin Lymphoma.

The prognostic impact of the presence of Epstein-Barr virus (EBV) in classical Hodgkin lymphoma (cHL) is controversial. Previous studies reported heterogeneous results, rendering difficult the clinical validation of EBV as a prognostic biomarker in this lymphoma. The objective of this study was to evaluate the survival impact of the expression of EBV Latent-Membrane Protein 1 (EBV-LMP1) in tumoral Hodgkin-Reed-Sternberg (HRS) cells of primary diagnostic samples of cHL. Formalin-Fixed Paraffin-Embedded (FFPE) lymph node samples from 88 patients with cHL were analyzed. Patients were treated with the standard first-line chemotherapy (CT) with Adriamycin, Bleomycin, Vinblastine and Dacarbazine (ABVD) followed by radiotherapy. The Kaplan-Meier method and the Cox proportional hazards model were used for carrying out the survival analysis. In order to investigate whether the influence of EBV was age-dependent, analyses were performed both for patients of all ages and for age-stratified subgroups. In bivariate analysis, the expression of EBV was associated with older age (p = 0.011), mixed cellularity subtype cHL (p < 0.001) and high risk International Prognostic Score (IPS) (p = 0.023). Overall survival (OS) and progression-free survival (PFS) were associated with the presence of bulky disease (p = 0.009) and advanced disease at diagnosis (p = 0.016). EBV-positive cases did not present a significantly lower OS and PFS in comparison with EBV-negative cases, for all ages and when stratifying for age. When adjusted for covariates, absence of bulky disease at diagnosis (HR: 0.102, 95% CI: 0.02-0.48, p = 0.004) and limited disease stages (I-II) (HR: 0.074, 95% CI: 0.01-0.47, p = 0.006) were associated with a significant better OS. For PFS, limited-disease stages also retained prognostic impact in the multivariate Cox regression (HR: 0.145, 95% CI: 0.04-0.57, p = 0.006). These results are of importance as the early identification of prognostic biomarkers in cHL is critical for guiding and personalizing therapeutic decisions. The prognostic role of EBV in cHL could be modulated by the type of CT protocol employed and interact with the rest of presenting features.

Potential Diagnostic Value of the Differential Expression of Histone H3 Variants between Low- and High-Grade Gliomas.

Glioblastoma (GB) is the most aggressive form of glioma and is characterized by poor prognosis and high recurrence despite intensive clinical interventions. To retrieve the key factors underlying the high malignancy of GB with potential diagnosis utility, we combined the analysis of The Cancer Gene Atlas and the REMBRANDT datasets plus a molecular examination of our own collection of surgical tumor resections. We determined a net reduction in the levels of the non-canonical histone H3 variant H3.3 in GB compared to lower-grade astrocytomas and oligodendrogliomas with a concomitant increase in the levels of the canonical histone H3 variants H3.1/H3.2. This increase can be potentially useful in the clinical diagnosis of high-grade gliomas, as evidenced by an immunohistochemistry screening of our cohort and can be at least partially explained by the induction of multiple histone genes encoding these canonical forms. Moreover, GBs showing low bulk levels of the H3.1/H3.2 proteins were more transcriptionally similar to low-grade gliomas than GBs showing high levels of H3.1/H3.2. In conclusion, this study identifies an imbalanced ratio between the H3 variants associated with glioma malignancy and molecular patterns relevant to the biology of gliomas, and proposes the examination of the H3.3 and H3.1/H3.2 levels to further refine diagnosis of low- and high-grade gliomas in future studies.

Differential epigenetic regulation between the alternative promoters, PRDM1α and PRDM1ß, of the tumour suppressor gene PRDM1 in human multiple myeloma cells.

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1ß isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1ß isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1ß promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1ß promoter in non-expressing cell lines compared to PRDM1ß-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1ß promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1ß promoters as components of novel therapeutic approaches.