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Early stages are under-represented in studies on the molecular and immune features of high-grade serous ovarian carcinoma (HGSOC), and specific studies focused on early-stage HGSOC are required for a better prognostic stratification and to personalize chemotherapy. The aim of this study was to determine the prognostic significance of CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs), tumoral cell PD-L1 expression, BRCA mutational status and tumor mutation burden (TMB) in early-stage HGSOC. A retrospective study was performed on stage I and II HGSOC from the Molecular Reclassification of Early Stages of Ovarian Cancer (RECLAMO) cohort from the Spanish Group of Ovarian Cancer Research (GEICO). Centralized histological typing was performed based on morphological and immunohistochemical features. Intraepithelial (i) and stromal (s) CD8+ and CD4+ T cells and PD-L1 were evaluated on tissue microarrays by immunohistochemistry. BRCA1 and BRCA2 mutation status and TMB were analyzed in tumor DNA using next-generation sequencing. The study included 124 tumors. High iCD8+ (>20 TILs/core), low/intermediate CD4+ (35/core) were associated with favorable outcomes. Tumor cell PD-L1 expression (TPS ≥ 1) was present in only 8% of tumors. In total, 11 (16%) and 6 (9%) out of 69 HGSOC tested carried pathogenic or likely pathogenic BRCA1 or BRCA2 mutations, respectively. Median TMB of 40 tumors analyzed was 5.04 mutations/Mb and only 6 tumors had 10 or more mutations/Mb. BRCA status and TMB were not associated with TILs or prognosis. When compared with studies on advanced HGSOC, our results suggested that prognostic variables differed according to stage and that more studies focused on early stages of HGSOC are needed to better stratify these tumors.

The relationship between the ductal and lobular components of invasive ductolobular carcinomas (IDLC) has not been fully elucidated. In this study, the molecular alterations of both components were analyzed in a series of 20 IDLC that were selected, not only by morphologic criteria, but also by the loss of E-cadherin expression in the lobular component. We found that 80% of tumors shared alterations of driver genes in both components, being PIK3CA the most common alteration. In addition, 45% of IDLC carried CDH1 mutations in their lobular component that were absent in the ductal component. Fluorescent in situ hybridization analysis of the CDH1 gene excluded homozygous CDH1 loss as a frequent cause of E-cadherin loss in tumors without CDH1 mutations. In addition, no pathogenic mutations of catenin genes were detected in this series of tumors. In 25% of tumors, actionable mutations in PIK3CA , AKT1 , and ERBB2 were found in only 1 component. Altogether, our results confirm that most IDLC derive from invasive carcinoma of no special type, in which a population of cells lose E-cadherin and acquire a lobular phenotype. The frequency of CDH1 mutations in IDLC appears to be lower than in conventional invasive lobular carcinomas, suggesting the implication of alternative mechanisms of E-cadherin loss. Moreover, molecular heterogeneity between ductal and lobular areas suggests the need for molecular characterization of both components to guide targeted therapies.

Uterine carcinosarcomas (UCS) account for approximately 2% of all malignancies of the uterine corpus. They are clinically aggressive, high-grade tumours that consist of an epithelial (carcinoma) and a mesenchymal (sarcoma) tumour component. Most UCSs are metaplastic carcinomas, as their malignant behaviour is primarily driven by the carcinomatous component. Recent studies centred on the molecular characterization of UCS have shed light on the genetic alterations shaping UCS pathogenesis. The most common mutational event in UCS is TP53 mutation, frequently accompanied by p16 overexpression. As a result of genomic instability secondary to TP53 mutation, UCS frequently carry a large number of gene copy number alterations, including amplification of important oncogenes, such as CCNE1 and C-MYC. The PI3K pathway is frequently affected in UCS by mutations of different genes or amplification of PIK3CA. While mutations in the frequently mutated genes of endometrial serous carcinomas, such as FBXW7 and PPP2R1A, are also frequent in UCS, genes typically mutated in endometrioid endometrial carcinomas, such as PTEN, ARID1A and CTNNB1, are infrequently mutated. Thus, most UCS belong to copy number high serous-like molecular subtype of endometrial carcinoma. However, a proportion of cases (20%) probably represent the progression of tumours with a copy number low endometrioid-like molecular subtype after the acquisition of TP53 mutations. Finally, only few UCS belong to the microsatellite-unstable hypermutated molecular type and the POLE-mutated ultramutated molecular type. This molecular heterogeneity among UCS may have treatment implications with emerging therapies.

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumours, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole exome sequencing of the endometrioid and undifferentiated components as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: a) hypermutated tumours with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); c) high copy number alterations (copy-number high) tumours group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%) ; and d) low copy number alterations (copy-number low) tumours with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumours. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumours, which may have prognostic value.

Heat shock proteins (HSPs), known to inhibit apoptosis and promote cellular survival, are overexpressed in many tumours. We analysed the expression of relevant HSPs and heat shock factor 1 (HSF1) in classical Hodgkin lymphoma (cHL) and their relationship with caspase signalling pathways and patient outcome.Using tissue microarrays (TMAs), most cases showed strong immunohistochemical expression of HSPs [10, 27, 40, 60, 70, 90, 110, HO1, cell division cycle 37 homolog (CDC37) and HSF1, which points to cHL as a potential candidate to stress-response inhibitors. Active caspases 3, 8 and 9 were detected in 55.1%, 55.4% and 96.2% of cases although cleaved poly (ADP-ribose) polymerase (PARP) was observed in only 16.1%, suggesting an improper functioning of apoptosis. Statistical analysis showed associations of HSP70 with active caspase 3 (P = 0.000); HSP40 with active caspase 9 (P = 0.031) and p53 (P = 0.003); HO1 with p53 (P = 0.006) and p21 (P = 0.005); and p53 with p21 (P = 0.015).Correlations between the expression of apoptotic markers and HSPs may suggest a role for the latter in modulating apoptosis in cHL, mainly through the HSP70-HSP40 system, and in the stabilization of p53. Survival analyses showed that absence of active caspase 8 and HO1 had a negative impact in patient outcome.

Epstein-Barr virus (EBV) seems to have an etiological role in the pathogenesis of classical Hodgkin's lymphoma (cHL). Studies of whole tissue DNA by polymerase-chain reaction (PCR) have shown a considerable number of cHL cases with co-infections by different EBV strains and variants, which apparently contradict the clonality of EBV in cHL previously demonstrated by Southern blot analysis. Due to the paucity of HRS cells in HL tissues, studies on single cell DNA are necessary to identify the specific cellular location (HRS cells and/or bystander B lymphocytes) of the EBV strains and variants present in tissue specimens. In the current study, the presence of EBV was determined by PCR of the 3' end of the LMP-1 gene and EBNA-3C gene in whole tissue and, consecutively, in isolated cells from 26 cases of cHL: 10 HIV-positive and 16 sporadic cHL cases. EBV EBERs were present in all but 2 sporadic cHL cases, which were used as negative controls. At isolated cell level, EBNA-3C gene PCR was more sensitive. Indeed, from the cHL cases in which dual-infection was present, it was observed that, in most of them, HRS cells were infected by type 1 virus, and B lymphocytes were co-infected by both types, which points towards EBV infection occurring early in cHL development. Moreover, the finding of 2 cases with dual-infection in HRS may suggest that, in a small percentage of cHL cases, HRS cells derive from different neoplastic clones, or that HRS cells are superinfected by other viral types after the establishment of the neoplastic clone.