VISTA/CTLA4/PD1 coexpression on tumor cells confers a favorable immune microenvironment and better prognosis in high-grade serous ovarian carcinoma.

Introduction: Immunotherapy by blocking immune checkpoints programmed death/ligand (PD1/PDL1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) has emerged as new therapeutic targets in cancer. However, their efficacy has been limited due to resistance. A new- checkpoint V-domain Ig-containing suppressor of T cell activation (VISTA) has appeared, but the use of its inhibition effect in combination with antibodies targeting PDL1/PD1and CTLA4 has not been reported in ovarian cancer. Methods: In this study, we investigated the expressions of VISTA, CTLA4, and PDL1 using immunohistochemistry (IHC)on 135 Formalin-Fixed Paraffin-Embedded (FFPE)tissue samples of High-grade serous carcinoma (HGSOC). VISTA, CTLA4, PDL1, PD1, CD8, CD4, and FOXP3 mRNA extracted from 429 patients with ovarian cancer in the Cancer Genome Atlas (TCGA) database was included as a validation cohort. Correlations between these checkpoints, tumor-infiltrating- lymphocytes (TILs), and survival were analyzed. Results and discussion: CTLA4 was detectable in 87.3% of samples, VISTA in 64.7%, PD1 in 56.7%, and PDL1 in 48.1%. PDL1 was the only tested protein associated with an advanced stage (p=0.05). VISTA was associated with PDL1, PD1, and CTLA4 expressions (p=0.005, p=0.001, p=0.008, respectively), consistent with mRNA level analysis from the TCGA database. Univariate analyses showed only VISTA expression (p=0.04) correlated with overall survival (OS). Multivariate analyses showed that VISTA expression (p=0.01) and the coexpression of VISTA+/CTLA4+/PD1+ (p=0.05) were associated with better OS independently of the clinicopathological features. Kaplan-Meier analysis showed that the coexpression of the VISTA+/CTLA4+/PDL1+ and VISTA+/CTLA4+/PD1+ checkpoints on tumor cells (TCs)were associated with OS (p=0.02 and p<0.001; respectively). VISTA+/CTLA4+/PD1+ in TCs and CD4+/CD8+TILswere associated with better 2-yer OS. This correlation may refer to the role of VISTA as a receptor in the TCs and not in the immune cells. Thus, targeting combination therapy blocking VISTA, CTLA4, and PD1 could be a novel and attractive strategy for HGSOC treatment, considering the ambivalent role of VISTA in the HGSOC tumor cells.

VISTA+/CD8+ status correlates with favorable prognosis in Epithelial ovarian cancer.

Immunotherapy by blocking immune checkpoint regulators has emerged as a new targeted therapy for some cancers. Among them V-domain Ig suppressor of Tcell activation (VISTA) which is identified as a novel checkpoint regulator in ovarian cancer. This study aimed to investigate the VISTA role in Epithelial ovarian cancer (EOC), and its relationship with tumor-infiltrating lymphocytes (TILs) markers and its prognostic value. The expression of VISTA, CD3, CD8, CD4, FOXP3, and CD56 was assessed in 168 EOC tissue microarrays (TMA) by immunohistochemistry (IHC). In addition, associations between VISTA, TILs, clinicopathological variables, and overall survival (OS) were analyzed. VISTA expression in IGRov1 cells, as well as in PBMC of EOC patient, was evaluated by western blot. VISTA expression was detected in 64,28% of tissues, among which 42.3% were positive for tumor cells (TCs), and 47,9% were positive for immune cells (ICs). In univariate analysis, VISTA expression was significantly associated with a high density of TILs:CD3+ (p = 0,001), CD4+ (p = 0,002) and CD8+ (p≤0,001), in ICs but not in TCs. In terms of OS, multivariate analysis showed a significant association between the high density of CD8+ TILs and VISTA positive staining in ICs (p = 0,044), but not in TCs (p = 0,108). Kaplan-Meier curves demonstrated no correlation between VISTA expression and prolonged OS in both ICs (p = 0,841) and TCs (p = 0,090). Classification of EOC tumor microenvironment based on VISTA and CD8+TILs expression, demonstrated four immune subtypes: VISTA+/CD8+, VISTA+/CD8-, VISTA-/CD8+ and VISTA-/CD8-. The dual positive VISTA+/CD8+ subtype was significantly associated with prolonged OS in both TCs and ICs (p = 0,012 and p≤0,01, respectively), whereas patients with VISTA+/CD8- had the worst OS. Our results showed that VISTA is highly expressed in the IGRov1 cell line and LT-CD8 from a patient with EOC. Our results highlighted the association of VISTA expression and CD8+ TILs in EOC, with prolonged OS in patients with VISTA+/CD8+ and proposed VISTA as a potential immunotherapeutic target in EOC.

Association of ZEB1 and Vimentin with poor prognosis in metaplastic breast cancer.

Zinc finger E-box binding homeobox factor 1 (ZEB1) is a transcription factor involved in the epithelial to mesenchymal transition (EMT) process of metaplastic breast cancer (MBC). This study aimed to assess the expression of ZEB1 in MBC and explore its association with clinicopathological factors and prognosis. We analyzed the immunohistochemical expression of ZEB1 in 50 MBC tissue samples. ZEB1 was overexpressed in 36% (18/50) of cases. ZEB1 overexpression was significantly correlated to fibromatosis-like and spindle cell sarcoma subtypes (P < 0.001) and tended to be correlated to metastatic status (P = 0.069). Using the Kaplan-Meier method, ZEB1 expression was significantly associated with poor 5-years overall survival (OS) (P = 0.001) and relapse-free survival (RFS) (P = 0.0001). The multivariate Cox regression analysis showed that ZEB1 positive remained a significantly independent adverse prognostic factor for RFS and OS (HR = 4.9 [2.14-11.53]; P < 0.0001) and (HR = 4 [1.05-15.18]; P = 0.042), while Vimentin was an independent poor prognostic factor only for RFS (HR = 5.69 [1.79-18.11], P = 0.003). Our results indicated that ZEB1 and Vimentin overexpression might serve as adverse prognostic factors and potential therapeutic targets for MBC patients.