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Classic Hodgkin lymphoma (cHL) constitutes a B-cell neoplasm derived from germinal center lymphocytes. Despite high cure rates (80-90%) obtained with the current multiagent protocols, a significant proportion of cHL patients experience recurrences, characterized by a lower sensitivity to second-line treatments. The genomic background of chemorefractory cHL is still poorly understood, limiting personalized treatment strategies based on molecular features. In this study, using a targeted next-generation sequencing (NGS) panel specifically designed for cHL research, we compared chemosensitive and chemorefractory diagnostic tissue samples of cHL patients. Furthermore, we longitudinally examined paired diagnosis-relapsesamples of chemorefractory cHL in order to define patterns of dynamic evolution and clonal selection. Pathogenic variants in NOTCH1 and NOTCH2 genes frequently arise in cHL. Mutations in genes associated with epigenetic regulation (CREBBP and EP300) are particularly frequent in relapsed/refractory cHL. The appearance of novel clones characterized by mutations previously not identified at diagnosis is a common feature in cHL cases showing chemoresistance to frontline treatments. Our results expand current molecular and pathogenic knowledge of cHL and support the performance of molecular studies in cHL prior to the initiation of first-line therapies.

Aims: The prognostic impact of programmed death-ligand 1 (PD-L1) cells in classic Hodgkin lymphoma (cHL) tumour microenvironment remains undefined., Methods: Model development via Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis guidelines were followed. PD-L1+ and CD30+ tumoral Reed-Sternberg cells were quantified through whole slide imaging and digital image analysis in 155 digital histopathological slides of cHL. Univariate and multivariate survival analyses were performed. The analyses were reproduced for patients with advanced stages (IIB, III and IV) using the Advanced-stage cHL International Prognostic Index., Results: The PD-L1/CD30 ratio was statistically significantly associated with survival outcomes. Patients with a PD-L1/CD30 ratio above 47.1 presented a shorter overall survival (mean OS: 53.7 months; 95% CI: 28.7 to 78.7) in comparison with patients below this threshold (mean OS: 105.4 months; 95% CI: 89.6 to 121.3) (p=0.04). When adjusted for covariates, the PD-L1/CD30 ratio retained prognostic impact, both for the OS (HR: 1.005; 95% CI: 1.002 to 1.008; p=0.000) and the progression-free survival (HR: 3.442; 95% CI: 1.045 to 11.340; p=0.04) in a clinical and histopathological multivariate model including the male sex (HR: 3.551; 95% CI: 0.986 to 12.786; p=0.05), a percentage of tumoral cells ≥10.1% (HR: 1.044; 95% CI: 1.003 to 1.087; p=0.03) and high risk International Prognostic Score (≥3 points) (HR: 6.453; 95% CI: 1.970 to 21.134; p=0.002)., Conclusions: The PD-L1/CD30 ratio identifies a group of cHL patients with an increased risk of treatment failure. Its clinical application can be performed as it constitutes an easy to implement pathological information in the diagnostic work-up of patients with cHL., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (Copyright © 2023 The Authors. Publicado por Elsevier España, S.L.U. All rights reserved.)

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.

Classic Hodgkin lymphoma (cHL) constitutes the most frequent lymphoma in young adults. Its histopathology is unique as a scattered tumor population, termed Hodgkin Reed-Sternberg (HRS) cells is diluted in a prominent tumor microenvironment (TME) composed of T lymphocytes, B lymphocytes, macrophages, neutrophils, eosinophils and histiocytes. Traditionally, the identification of prognostic biomarkers in the cHL TME has required visual inspection and manual counting by pathologists. The advent of whole-slide imaging (WSI) and digital image analysis methods could significantly contribute to improve this essential objective in cHL research, as a 10-20% of patients are still refractory or relapsed after conventional chemotherapy. In this work, we have digitized a total of 255 diagnostic cHL slides and quantified the proportion of HRS cells (CD30), B cells (CD20) and T cells (CD3) by digital image analysis. Data obtained where then correlated with the overall survival (OS) and progression free survival (PFS) of cHL patients. Quantification of HRS cells, B cells and T cells reflects the biological heterogeneity of the different cHL histological subtypes analyzed. A percentage of 2.00% of HRS cells statistically significantly discriminated between patients achieving a complete metabolic response (CMR) and refractory or relapsed (R/R) patients both for the OS ( P =0.001) and PFS ( P =0.005). Furthermore, patients with a percentage of T cells below the 26.70% in the TME showed a statistically significantly shorter OS ( P =0.019) and PFS ( P=0.041 ) in comparison with patients above this threshold. A subgroup of patients with a low content of T cells and high content of HRS cells exhibited a special aggressive clinical course. Currently, there is the need to implement quantitative and easy scalable methods to enhance clinical translation, as the cHL TME plays a central role in the clinical course of the disease. The results of this study could contribute to the identification of prognostic biomarkers specifically looking at the cHL TME and their inclusion in future clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Santisteban-Espejo, Bernal-Florindo, Perez-Requena, Atienza-Cuevas, Maira-Gonzalez and Garcia-Rojo.)

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.

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.