Carlos de Andrea

The Proteomic Landscape of CTNNB1 Mutated Low-Grade Early-Stage Endometrial Carcinomas

Endometrial carcinoma is the most frequent gynecologic malignancy in western countries. In recent years, mutations in CTNNB1 have been associated with worse prognosis in low-risk carcinomas. However, there is a lack of understanding of the proteomic implications of CTNNB1 mutations in this type of tumor. In this study, we performed shotgun proteomics using Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of CTNNB1 mutated and wild-type low-risk endometrial carcinomas. A publicly available proteomic and transcriptomic database was used to validate results. Differential protein expression and Gene Set Enrichment Analysis revealed dysregulation of pathways associated with cell keratinization, immune response modulation, and intracellular calcium regulation. CTNNB1 mutated tumors showed immune dysregulation at multiple levels including cytokine secretion, cell adhesion, and lymphocyte activation. These results were supported by tissue multiplex immunofluorescence analysis, demonstrating reduced CD8 tumor-infiltrating lymphocytes and different immune spatial interaction patterns. Intracellular calcium dysfunction was associated with key transcript dysregulation. We found an increased expression of CAMK2A and ROR2, suggesting a potential role for non-canonical Wnt pathway activation in CTNNB1 mutated tumors.

The association between the tumor immune microenvironments and clinical outcome in low‐grade, early‐stage endometrial cancer patients

AbstractEndometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low‐grade, early‐stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD‐1, and PD‐L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune‐desert subtype, characterized by low immune cell densities. Tumor‐infiltrating lymphocyte (TIL)‐rich samples (4%) displayed high CD8+ T‐cell infiltration, as well as a high percentage of CD8/PD‐1+ cells. Immune‐exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD‐L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+/PD‐1+ and CK/PD‐L1+ cells. FOXP3 and macrophage‐rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T‐cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune‐exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single‐marker immunohistochemistry (IHC) performed on whole tissue sections. TIL‐rich tumors demonstrated increased CD8+ T‐cells by IHC, while immune‐exclusion tumors displayed a lack of CD8+ T‐cells and frequent expression of PD‐L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low‐grade, early‐stage endometrial carcinomas. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Analysis of Tumor Microenvironment Changes after Neoadjuvant Chemotherapy with or without Bevacizumab in Advanced Ovarian Cancer (GEICO-89T/MINOVA Study)

Abstract Purpose: The aim of our study was to elucidate the impact of bevacizumab added to neoadjuvant chemotherapy (NACT) on the tumor immune microenvironment and correlate the changes with the clinical outcome of the patients. Experimental Design: IHC and multiplex immunofluorescence for lymphoid and myeloid lineage markers were performed in matched tumor samples from 23 patients with ovarian cancer enrolled in GEICO 1205/NOVA clinical study before NACT and at the time of interval cytoreductive surgery. Results: Our results showed that the addition of bevacizumab to NACT plays a role mainly on lymphoid populations at the stromal compartment, detecting a significant decrease of CD4+ T cells, an increase of CD8+ T cells, and an upregulation in effector/regulatory cell ratio (CD8+/CD4+FOXP3+). None of the changes observed were detected in the intra-epithelial site in any arm (NACT or NACT-bevacizumab). No differences were found in myeloid lineage (macrophage-like). The percentage of Treg populations and effector/regulatory cell ratio in the stroma were the only two variables significantly associated with progression-free survival (PFS). Conclusions: The addition of bevacizumab to NACT did not have an impact on PFS in the GEICO 1205 study. However, at the cellular level, changes in CD4+, CD8+ lymphocyte populations, and CD8+/CD4+FOXP3 ratio have been detected only at the stromal site. On the basis of our results, we hypothesize about the existence of mechanisms of resistance that could prevent the trafficking of T-effector cells into the epithelial component of the tumor as a potential explanation for the lack of efficacy of ICI in the first-line treatment of advanced epithelial ovarian cancer. See related commentary by Soberanis Pina and Oza, p. 12