Despite great enthusiasm towards immunotherapy, reliable biomarkers are still lacking. The importance of subsets based on human papillomavirus (HPV) status is supported by a growing body of evidence. However, role of other possible subgroups such as anatomic localization of primary tumor remains controversial. Our objective was to investigate immune cell infiltrate and checkpoint inhibitor proteins in above-mentioned head and neck cancer subsets. Archival tumor samples of 106 laryngeal, oropharyngeal, and hypopharyngeal cancer patients were stained with PD-L1, PD-L2, PD-1, and CTLA-4 antibodies. Proportion of tumor-infiltrating lymphocytes was assessed as well. In HPV-negative tumors, PD-L1 immune cell positivity was associated with better disease-specific survival. PD-L1 expression on immune cells correlated with improved disease-specific survival in laryngeal tumors. Furthermore, PD-L1 immune cell positivity correlated with CTLA-4 expression on immune cells and it was accompanied by high tumor-infiltrating lymphocyte rate. PD-L1 expression on tumor cells and PD-1 status showed strong correlation in all patients and in oropharyngeal and laryngeal localization, but not in hypopharynx. HPV-negative oropharyngeal cancers showed negative PD-L1 status on tumor cells. CTLA-4 positivity was observed in 49.5% and 20.6% on immune cells and on tumor cells, respectively. We concluded that PD-L1 expression on immune cells indicates better prognosis in laryngeal squamous cell carcinoma and in HPV-negative head and neck squamous cell carcinoma. We have not found any essential differences between anatomic subgroups. A possibly distinct role of hypopharyngeal localization regarding immune activity requires further clarification.

Pancreatic cancer is an increasing cause of cancer related death worldwide. KRAS is the dominant oncogene in this cancer type and molecular rationale would indicate, that inhibitors of the downstream target MEK could be appropriate targeted agents, but clinical trials have failed so far to achieve statistically significant benefit in unselected patients. We aimed to identify predictive molecular biomarkers that can help to define subgroups where MEK inhibitors might be beneficial alone or in combination. Next-generation sequencing data of 50 genes in three pancreatic cancer cell lines (MiaPaCa2, BxPC3 and Panc1) were analyzed and compared to the molecular profile of 138 clinical pancreatic cancer samples to identify the molecular subtypes of pancreatic cancer these cell lines represent. Luminescent cell viability assay was used to determine the sensitivity of cell lines to kinase inhibitors. Western blot was used to analyze the pathway activity of the examined cell lines. According to our cell viability and pathway activity data on these model cell lines only cells harboring the rare G12C KRAS mutation and low EGFR expression are sensitive to single MEK inhibitor (trametinib) treatment. The common G12D KRAS mutation leads to elevated baseline Akt activity, thus treatment with single MEK inhibitors fails. However, combination of MEK and Akt inhibitors are synergistic in this case. In case of wild-type KRAS and high EGFR expression MEK inhibitor induced Akt phosphorylation leads to trametinib resistance which necessitates for MEK and EGFR or Akt inhibitor combination treatment. In all we provide strong preclinical rational and possible molecular mechanism to revisit MEK inhibitor therapy in pancreatic cancer in both monotherapy and combination, based on molecular profile analysis of pancreatic cancer samples and cell lines. According to our most remarkable finding, a small subgroup of patients with G12C KRAS mutation may still benefit from MEK inhibitor monotherapy.

The poor prognosis of head and neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable predictive markers. Connexin 43 (Cx43) protein and its cell-communication channels have been assigned tumor suppressor functions while the anti-apoptotic Bcl-2 (B-cell lymphoma-2) protein has been associated with negative prognostic significance in cancer. This study aimed to test the role of Cx43 protein on Bcl-2 expression, tumor progression and response to taxane-based treatment in HNSCC. Human papillomavirus (HPV) negative HNSCC cell lines were tested for paclitaxel sensitivity through measuring apoptosis induction, cell viability and changes in Cx43 and Bcl-2 levels using flow cytometry, cell viability assay, immunocytochemistry and western blot. Inhibition of Cx43 expression using siRNA increased Bcl-2 protein levels in SCC25 (tongue squamous cell carcinoma) cells, while forced Cx43 expression reduced Bcl-2 levels and supported paclitaxel cytotoxicity in FaDu (hypopharynx squamous cell carcinoma) cells. In vitro results were in line with protein expression and clinicopathological features tested in tissue microarray samples of HNSCC patients. Our data demonstrate that elevated Cx43 and reduced Bcl-2 levels may indicate HNSCC sensitivity to taxane-based treatments. On the contrary, silencing of the Cx43 gene GJA1 (gap junction protein alpha-1) can result in increased Bcl-2 expression and reduced paclitaxel efficiency. Clinical tumor-based analysis also confirmed the inverse correlation between Cx43 and Bcl-2 expression.

The poor prognosis of head-and-neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable prognostic and predictive markers. The Ras/Raf/MEK/ERK signaling pathway is often activated by overexpressed epidermal growth factor receptor (EGFR) and stimulates the progression of HNSCCs. Our research was performed on three human papillomavirus (HPV)-negative HNSCC-cell lines: Detroit 562, FaDu and SCC25. Changes in cell viability upon EGFR and/or MEK inhibitors were measured by the MTT method. The protein-expression and phosphorylation profiles of the EGFR-initiated signaling pathways were assessed using Western-blot analysis. The EGFR expression and pY1068-EGFR levels were also studied in the patient-derived HNSCC samples. We found significant differences between the sensitivity of the tumor-cell lines used. The SCC25 line was found to be the most sensitive to the MEK inhibitors, possibly due to the lack of feedback Akt activation through EGFR. By contrast, this feedback activation had an important role in the FaDu cells. The observed insensitivity of the Detroit 562 cells to the MEK inhibitors might have been caused by their PIK3CA mutation. Among HNSCC cell lines, EGFR-initiated signaling pathways are particularly versatile. An ERK/EGFR feedback loop can lead to Akt-pathway activation upon MEK inhibition, and it is related not only to increased amounts of EGFR but also to the elevation of pY1068-EGFR levels. The presence of this mechanism may justify the combined application of EGFR and MEK inhibitors.

Head and neck squamous cell carcinomas (HNSCC) have a high mortality rate, although several potential therapeutic targets have already been identified. Gonadotropin-releasing hormone receptor (GnRH-R) expression is less studied in head and neck cancers, hence, we investigated the therapeutic relevance of GnRH-R targeting in HNSCC patients. Our results indicate that half of the patient-derived samples showed high GnRH-R expression, which was associated with worse prognosis, making this receptor a promising target for GnRH-based drug delivery. Photodynamic therapy is a clinically approved treatment for HNSCC, and the efficacy and selectivity may be enhanced by the covalent conjugation of the photosensitizer to a GnRH-R targeting peptide. Several native ligands, gonadotropin-releasing hormone (GnRH) isoforms, are known to target GnRH-R effectively. Therefore, different ⁴Lys(Bu) modified GnRH analogs were designed and conjugated to protoporphyrin IX. The receptor binding potency of the novel conjugates was measured on human pituitary and human prostate cancer cells, indicating only slightly lower GnRH-R affinity than the peptides. The in vitro cell viability inhibition was tested on Detroit-562 human pharyngeal carcinoma cells that express GnRH-R in high levels, and the results showed that all conjugates were more effective than the free protoporphyrin IX.