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Background: Mounting evidence suggests that sex plays a critical role in various aspects of cancer such as immune responses. However, a male bias exists in human and non-human studies including immunotherapy trials. The role of sex on immune responses in pancreatic ductal adenocarcinoma (PDA) is unclear. Methods: Here, tumor tissues (tumor and stroma separately) and corresponding blood samples from patients with PDA (n = 52) were systematically analyzed by immunohistochemistry and multiplex cytokine measurements and compared by sex. Results: Females showed a stronger systemic immune response with higher levels of CXCL9, IL1B, IL6, IL10 and IL13. Additionally, more peripheral white blood cells were detected in females. In the microenvironment, males showed higher tumoral levels of CXCL12. No differences were detected in the stroma. Females showed a tendency towards an anti-tumoral immune cell profile. CXCL12 blockade indicated a differential microenvironmental effect by sex in an independent immunotherapy trial cohort of patients with PDA (one female, five males). The overall survival did not differ by sex in our cohort. Conclusion: Systemic and local immune responses differ between sexes in PDA. Accordingly, sex-dependent differences need to be considered in human studies and for specific immunological interventions before clinical translation.

Simple Summary Gallbladder cancer (GBC) is an aggressive malignancy with poor prognosis. Currently, therapeutic options are mostly limited to palliative chemotherapy. Considering the advances of immunotherapy, we assessed the expression of the programmed cell death ligand-1 (PD-L1) as the most widely used predictive marker for immunotherapy response in a large Western-world GBC cohort. Additionally, we quantified the expression of the T-cell immunoreceptor with Ig and ITIM domains TIGIT/CD155 axis as an emerging immune checkpoint. Our results indicate that PD-L1 is heterogeneously expressed in Western-world GBC and associated with distinct histomorphological tumor subtypes and increased immune cell densities. We show that a high tumoral PD-L1 expression is a significant negative prognosticator. In a subset of patients, we identified expression of TIGIT in scattered immune cells, which correlated with tumoral expression of its ligand CD155. Our results suggest a subset of GBC patients to be candidates for immunotherapy via (combined) PD-L1 and TIGIT/CD155 inhibition. Abstract Inhibition of the programmed cell death protein-1/ligand-1 (PD-1/PD-L1) axis has opened a new era in the treatment of solid cancers. However, there is no data on the expression and relevance of PD-L1 in Western gallbladder cancer (GBC). We assessed PD-L1 immunohistochemically in 131 GBC patients as Tumor Proportion Score (TPS), Immune Cell Score (IC) and Combined Positivity Score (CPS). Tumor cells expressed PD-L1 in a subset of 14.7% GBC patients at a TPS cut-off of 1%. Higher PD-L1 levels above 10% and 25% TPS were reached in 4.7% and 3.1% of GBC cases, respectively. At a 10% cut-off, TPS was associated with distinct histomorphological subtypes and correlated with poor tumor differentiation. Survival analysis revealed a TPS above 10% to be a highly significant and independent negative prognosticator in GBC. PD-L1 expression was associated with increased CD4+, CD8+ and PD-1+ immune cell densities. In 14.8% of the cases, scattered immune cells expressed T-cell immunoreceptor with Ig and ITIM domains (TIGIT), which was correlated to tumoral expression of its ligand CD155. We here show that a high PD-L1 expression confers a negative prognostic value in Western-world GBC and highlight the TIGIT/CD155 immune checkpoint as a potential new target for GBC immunotherapy.

Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

Oncogenic gene fusions are important drivers in many cancer types, including carcinomas, with diagnostic and therapeutic implications. Hence, sensitive and rapid methods for parallel profiling in formalin-fixed and paraffin-embedded (FFPE) specimens are needed. In this study we analyzed gene fusions in a cohort of 517 cases where standard treatment options were exhausted. To this end the Archer&reg, DX Solid tumor panel (AMP, 285 cases) and the Oncomine Comprehensive Assay v3 (OCA, 232 cases) were employed. Findings were validated by Sanger sequencing, fluorescence in situ hybridization (FISH) or immunohistochemistry. Both assays demonstrated minimal dropout rates (AMP: 2.4%, n = 7/292, OCA: 2.1%, n = 5/237) with turnaround times of 6&ndash, 9 working days (median, OCA and AMP, respectively). Hands-on-time for library preparation was 6 h (AMP) and 2 h (OCA). We detected n = 40 fusion-positive cases (7.7%) with TMPRSS2::ERG in prostate cancer being most prevalent (n = 9/40, 22.5%), followed by other gene fusions identified in cancers of unknown primary (n = 6/40, 15.0%), adenoid cystic carcinoma (n = 7/40, 17.5%), and pancreatic cancer (n = 7/40, 17.5%). Our results demonstrate that targeted RNA-sequencing of FFPE samples is feasible, and a well-suited approach for the detection of gene fusions in a routine clinical setting.