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// Verena Wieser 1 , Inge Gaugg 1 , Martina Fleischer 1 , Giridhar Shivalingaiah 2, 5 , Soeren Wenzel 2 , Susanne Sprung 3 , Sigurd F. Lax 4 , Alain G. Zeimet 1 , Heidelinde Fiegl 1 and Christian Marth 1 1 Department of Obstetrics and Gynecology, Medical University of Innsbruck, Innsbruck 6020, Austria 2 Division of Human Genetics, Medical University of Innsbruck, Innsbruck 6020, Austria 3 Institute of Pathology, Medical University of Innsbruck, Innsbruck 6020, Austria 4 Department of Pathology, Hospital Graz Sud-West, Academic Teaching Hospital of the Medical University Graz, Graz 8020, Austria 5 Present address: Division Biological Chemistry, Biocenter, Innsbruck, Medical University of Innsbruck, Innsbruck 6020, Austria Correspondence to: Heidelinde Fiegl, email: Heidelinde.Fiegl@i-med.ac.at Keywords: ovarian cancer; PD-1; PD-L1; IFNγ; BRCA1/2 Received: July 31, 2017 Accepted: February 27, 2018 Published: April 03, 2018 ABSTRACT Checkpoint molecules such as programmed cell death protein-1 (PD-1) and its ligand PD-L1 are critically required for tumor immune escape. The objective of this study was to investigate tumoral PD-1 and PD-L1 mRNA-expression in a cohort of ovarian cancer (OC) patients in relation to tumor mutations. We analyzed mRNA expression of PD-1 , PD-L1 and IFNG by quantitative real-time PCR in tissue of 170 patients with low grade-serous (LGSOC), high-grade serous (HGSOC), endometrioid and clear cell OC compared to 28 non-diseased tissues (ovaries and fallopian tubes) in relation to tumor protein 53 ( TP53 ) and breast cancer gene 1/2 ( BRCA1/2 ) mutation status. TP53 -mutated OC strongly expressed PD-L1 compared to TP53 wild-type OC ( p = 0.028) and BRCA1/2 -mutated OC increasingly expressed PD-1 ( p = 0.024) and PD-L1 ( p = 0.012) compared to BRCA1/2 wild-type OC. For the first time in human, we noted a strong correlation between tumoral IFNG and PD-1 or PD-L1 mRNA-expression, respectively ( p < 0.001). OC tissue increasingly expressed PD-1 compared to healthy controls (vs. ovaries: p < 0.001; vs. tubes: p = 0.018). PD-1 and PD-L1 mRNA-expression increased with higher tumor grade ( p = 0.008 and p = 0.027, respectively) and younger age (< median age, p = 0.001). Finally, in the major subgroup of our cohort, FIGO stage III/IV HGSOC, high PD-1 and PD-L1 mRNA-expression was associated with reduced progression-free ( p = 0.024) and overall survival ( p = 0.049) but only in the univariate analysis. Our study suggests that in OC PD-1 / PD-L1 mRNA-expression is controlled by IFNγ and affected by TP53 and BRCA1/2 mutations. We suggest that these mutations might serve as potential predictive factors that guide anti- PD1 / PD-L1 immunotherapy.

Free circulating DNA is increased in the serum/plasma of cancer patients, and methylation of certain genes has been found to be characteristic for malignancy. Therefore, we investigated the prognostic value of two promising genes, PITX2 and RASSF1A, in peripheral blood-plasma (PB-P) and bone marrow plasma (BM-P) of breast cancer patients. Peripheral blood and bone marrow samples from patients with primary breast cancer were prospectively collected during primary surgery at the Department of Obstetrics and Gynecology in Innsbruck (n = 428) from June 2000 to December 2006. The study has been approved by the ethical committee of the Medical University of Innsbruck. Methylation analysis was performed using MethyLight, a methylation-specific quantitative PCR-method. In univariate survival analysis, methylated PITX2 in PB-P was found to be a significant indicator for poor overall survival (OAS) and distant disease-free survival (DDFS) (P = 0.001 and P = 0.023). Methylated RASSF1A in PB-P was also an indicator for poor OAS and DDFS (P = 0.001 and P = 0.004). RASSF1A had also significant prognostic potential when determined in BM-P (P = 0.016). In multivariate survival analysis methylated PITX2 and RASSF1A in PB-P remained as therapy-independent prognostic factors for OAS (P = 0.021, P < 0.001). For DDFS only RASSF1A in PB-P showed prognostic significance (P = 0.002). Methylated RASSF1A and PITX2 in PB-P appear to have promising potential as prognostic markers in clinical use.

Interferons are known to modulate several cellular functions by the induction of different proteins. In our study interferon-gamma and -alpha augmented presentation of one specific ovarian cancer antigen recognized by the antibody OV632 on the cell surface on one of four ovarian carcinoma cell lines in vitro (HTB-77). The interferon-gamma effect reached a maximum after 24 of treatment. Both HLA-DR and OV632 antigen expression were increased by interferon-gamma on HTB-77 cells whereas the two cell lines OVCAR-3 and 2780, which respond to an interferon-gamma treatment with a reduced proliferation and induction of HLA-DR, and also the interferon-resistant CRL-1572 were found to be resistant regarding OV632 epitope modulation. The demonstration of OV632 expression provides an important insight into the potential regulatory mechanism governing this tumor marker.