Eliana Bignotti

Oxford Classic–Defined EMT Risk Stratification of High-Grade Serous Ovarian Cancer for Guiding Treatment Decisions

Abstract Purpose: The association between epithelial-to-mesenchymal transition (EMT) in high-grade serous ovarian cancer (HGSOC) and poor prognosis is known. However, molecularly defining a subset of tumors that reproducibly associates with poor prognosis has been an elusive goal in this disease. A molecular signature that can robustly identify patients with poor prognosis and guide treatment decisions, including surgical strategy and targeted therapies, can improve survival rates. Experimental Design: We carried out RNA sequencing of 139 tumor samples (Brescia cohort); an external validation on 362 and 126 patients from the Scottish and Garsed cohorts, respectively; and a meta-analysis of 1,023 tumors to develop clinically useful risk groups. Identification of therapeutic targets was carried out by transcriptomic analyses of fluorescence-activated cell sorted (FACS) tumor epithelial cells and multiplex immunofluorescence assessment of tissue sections. Results: In this study, we have validated the prognostic strength of the Oxford Classic–defined EMT in three independent patient cohorts: Brescia [HR = 3.6; 95% confidence interval (CI) of 1.59–7.97; P = 1.99e−03], Scottish (HR = 1.71; 95% CI of 1.08–2.70; P = 2.23e−02), and Garsed (Kruskal–Wallis P = 0.00071). OxC-based risk stratification of HGSOC could robustly identify poor-risk patients with a 5-year median survival for OxC high-risk and OxC low-risk groups of 13% and 50%, respectively (95% CI of 7.1%–23.5% vs. 36.1%–69.3%) in the Brescia cohort. Further analysis of the risk groups suggests that an alternative surgical strategy and a combination therapy involving EMT targeting drugs and immunomodulators could elicit improved clinical response in poor-risk patients. Conclusions: This study provides a clinically useful risk stratification strategy for HGSOC, as well as targeted treatment options for high-risk patients. See related commentary by Venegas et al., p. 10

Integrated mutational landscape analysis of endometrial stromal sarcoma

Endometrial stromal sarcoma (ESS) is a rare uterine malignancy with limited treatment options. We performed integrated whole-genome, whole-exome, and transcriptome sequencing on 80 ESS tumors, comprising 32 low-grade (LG) and 48 high-grade (HG) tumors, to characterize their genetic landscape. The overall mutation burden was modest, with no significant difference between grades; however, we identified six hypermutated cases (7.5%) harboring POLE or mismatch repair mutations, genomic features predictive of immunotherapy response. We identified focal RAD54B amplifications in 15 tumors (18.8%), leading to elevated RAD54B expression and significantly shorter survival. This establishes RAD54B as an oncogenic driver in ESS. Known tumor suppressors (PTEN, TP53) were frequently mutated in HG-ESS but rare in LG-ESS, highlighting distinct grade-specific drivers of malignancy. HG-ESS exhibited widespread chromosomal gains, frequent loss of cell-cycle regulators (RB1, CDKN2A), and numerous private gene fusions arising from complex DNA rearrangements. In contrast, LG-ESS were defined by canonical fusions (e.g., JAZF1–SUZ12 ) and co-occurring deletions in metabolic regulator genes (TSC2, STK11). Finally, in an activating NRAS-mutant (p.Q61R) HG-ESS xenograft, the combination of MEK and FAK inhibition dramatically suppressed tumor growth and prolonged survival, highlighting a promising targeted treatment strategy. Overall, our comprehensive analysis defines the molecular basis of ESS and provides a strong preclinical rationale for precision therapies in this aggressive cancer.

L1CAM expression as a predictor of platinum response in high‐risk endometrial carcinoma

AbstractFor high‐risk endometrial cancer (EC) patients, adjuvant chemotherapy is recommended to improve outcome. Yet, predictive biomarkers for response to platinum‐based chemotherapy (Pt‐aCT) are currently lacking. We tested expression of L1 cell‐adhesion molecule (L1CAM), a well‐recognised marker of poor prognosis in EC, in tumour samples from high‐risk EC patients, to explore its role as a predictive marker of Pt‐aCT response. L1CAM expression was determined using RT‐qPCR and immunohistochemistry in a cohort of high‐risk EC patients treated with Pt‐aCT and validated in a multicentric independent cohort. The association between L1CAM and clinicopathologic features and L1CAM additive value in predicting platinum response were determined. The effect of L1CAM gene silencing on response to carboplatin was functionally tested on primary L1CAM‐expressing cells. Increased L1CAM expression at both genetic and protein level correlated with high‐grade, non‐endometrioid histology and poor response to platinum treatment. A predictive model adding L1CAM to prognostic clinical variables significantly improved platinum response prediction (C‐index 78.1%, P = .012). In multivariate survival analysis, L1CAM expression was significantly associated with poor outcome (HR: 2.03, P = .019), potentially through an indirect effect, mediated by its influence on response to chemotherapy. In vitro, inhibition of L1CAM significantly increased cell sensitivity to carboplatin, supporting a mechanistic link between L1CAM expression and response to platinum in EC cells. In conclusion, we have demonstrated the role of L1CAM in the prediction of response to Pt‐aCT in two independent cohorts of high‐risk EC patients. L1CAM is a promising candidate biomarker to optimise decision making in high‐risk patients who are eligible for Pt‐aCT.

TP53 mutations and survival in ovarian carcinoma patients receiving first‐line chemotherapy plus bevacizumab: Results of the MITO16A/MaNGO OV‐2 study

AbstractTo date, there are no biomarkers that define a patient subpopulation responsive to bevacizumab (BEV), an effective treatment option for advanced ovarian carcinoma (OC). In the context of the MITO16A/MaNGO OV‐2 trial, a Phase IV study of chemotherapy combined with BEV in first‐line treatment of advanced OC, we evaluated TP53 mutations by next‐generation sequencing and p53 expression by immunohistochemistry (IHC) on 202 and 311 cases, respectively. We further correlated TP53 mutations in terms of type, function, and site, and IHC data with patients' clinicopathological characteristics and survival. TP53 missense mutations of unknown function (named unclassified) represented the majority of variants in our population (44.4%) and were associated with a significantly improved overall survival (OS) both in univariable (hazard ratio [HR] = 0.43, 95% confidence interval [CI] = 0.20–0.92, p = .03) and multivariable analysis (HR = 0.39, 95% CI = 0.18–0.86, p = .02). Concordance between TP53 mutational analysis and IHC was 91%. We observed an HR of 0.70 for OS in patients with p53 IHC overexpression compared to p53 wild‐type, which however did not reach statistical significance (p = .31, 95% CI = 0.36–1.38). Our results indicate that the presence of unclassified TP53 mutations has favorable prognostic significance in patients with OC receiving upfront BEV plus chemotherapy. In particular, unclassified missense TP53 mutations characterize a subpopulation of patients with a significant survival advantage, independently of clinicopathological characteristics. Our findings warrant future investigations to confirm the prognostic impact of TP53 mutations in BEV‐treated OC patients and deserve to be assessed for their potential predictive role in future randomized clinical studies.

Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D / MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4 , a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib ( P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.