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// Fabian Dominik Mairinger 1 , Claudia Vollbrecht 2, 3, 4 , Elena Flom 1 , Daniel Christian Christoph 5 , Kurt-Werner Schmid 1 , Jens Kollmeier 6 , Helmut Hans Popper 7 , Thomas Mairinger 8 and Robert Fred Henry Walter 1, 9 1 Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 2 Institute of Pathology, Division of Molecular Pathology, Charite, Berlin, Germany 3 German Cancer Consortium (DKTK), Germany 4 German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Department of Medical Oncology, West German Cancer Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 6 Department of Pneumology, Helios Klinikum Emil von Behring, Berlin, Germany 7 Department of Pathology, Division of Molecular Lung- and Pleurapathology, Medical University of Graz, Graz, Austria 8 Department of Pathology, Helios Klinikum Emil von Behring, Berlin, Germany 9 Ruhrlandklinik, West German Lung Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany Correspondence to: Fabian Dominik Mairinger, email: fabian.mairinger@uk-essen.de Keywords: pleural mesothelioma, pemetrexed, thymidylate synthethase, folylpolyglutamate synthase, personalized therapy Received: October 12, 2016 Accepted: March 01, 2017 Published: March 21, 2017 ABSTRACT Background: Malignant pleural mesothelioma (MPM) is a rare tumor linked to a dismal prognosis. Even the most effective chemotherapeutical regime of pemetrexed combined with cisplatin leads to a remission-rate of only about 40%. The reasons for the rather poor efficacy remain largely unknown. Results: Phenotypes were significantly associated with progression (p=0.0279) and remission (p=0.0262). Cox-regression revealed significant associations between SLC19A1 / TYMS -ratio (p=0.0076) as well as FPGS / TYMS -ratio (p=0.0026) and OS. For differentiation by risk-groups, COXPH identified a strong correlation (p=0.0008). Methods: 56 MPM specimens from patients treated with pemetrexed were used for qPCR analysis. Phenotypes and risk groups were defined by their expression levels of members of the folic acid metabolism and correlated to survival and objective response. Conclusion: Our results indicate that the balance between folic acid uptake, activation and metabolism plays a crucial role in response to pemetrexed-based chemotherapy and the prognosis of MPM patients. Implementing this marker profile in MPM stratification may help to individualize MPM-therapy more efficiently.

Objectives Pulmonary adenocarcinomas (ADC) can be sub-grouped based on dominant oncogenic drivers. EGFR mutations define an entity of metastatic ADC with favorable prognosis and high susceptibility to EGFR tyrosine kinase inhibition. In contrast, the clinical impact of additional ERBB family members in ADC is less defined. To this end we prospectively studied HER2 expression, gene amplification, and mutation in relation to outcome of patients with advanced or metastatic ADC. Materials and methods Diagnostic tumor biopsies from 193 sequential patients with stage III/IV ADC were prospectively studied for HER2 expression by immunohistochemistry (IHC). Cases with IHC scores 2+ or 3+ were analyzed by HER2 chromogenic in situ hybridization (CISH), and sequencing of HER2 exons 20 and 23. Additional prospectively determined biomarkers included PTEN, cMET, pAKT, and pERK expression, KRAS , EGFR , BRAF and PIK3CA mutations, and ALK fluorescence ISH (FISH) . Results and conclusion HER2–IHC was feasible in 176 (91.2%) cases. Of 53 (30%) cases with IHC scores 2+/3+, 45 (85%) could be studied by CISH and 34 (64%) by sequencing. The lower number of HER2 -mutational analyses resulted from exhaustion of tumor tissue and DNA following mutational analysis of KRAS , EGFR , BRAF and PIK3CA . HER2 amplification was detected in 4 cases (2.3%), while no mutation was found. HER2 expression correlated with expression of pAKT and cMET. Expression of HER2 and pAKT was associated with favorable overall survival in stage IV disease. HER2-expressing ADC more frequently harbored KRAS mutations, while HER2 expression was absent in all 4 cases with BRAF mutation. HER2–IHC was not predictive of HER2 gene amplification or mutation, which both were rare events in prospectively studied patients with advanced or metastatic ADC. Expression of HER2 and pAKT define a population of patients with stage IV ADC with a distinct disease course, who could benefit from specifically tailored pharmacotherapies.

Introduction Monoclonal antibodies and tyrosine kinase inhibitors (TKI) targeting the EGFR are approved or under investigation for the systemic treatment of patients with HNSCC. However, primary and acquired resistance against EGFR-targeted therapies are frequently observed. Mutational activation of the EGFR signaling cascade as well as molecular changes caused by HPV infection are the most abundant oncogenic alterations in HNSCC. Against this background we have used preclinical cancer models to study the impact of activated PI3K/AKT and RAS/RAF/MAPK pathways or HPV16 oncoprotein expression on the response to anti-EGFR therapies. Materia and methods Cetuximab- and erlotinib-sensitive cancer cells were retrovirally transduced to express HPV16 oncoproteins E6 or E7, conditionally active AKT and RAF-1 or a RASG12V mutant. HPV16-positive cancer cells and cells harboring endogenously mutated PIK3CA or RAS were used as alternative models. Cell proliferation, induction of apoptosis, clonogenic survival and tumor growth in immune-compromised mice were analyzed in relation to treatment with anti-EGFR antibodies. In support, tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16INK4a expression, an established biomarker of HPV infection and retrospectively analyzed concerning the response rates (RR) and progression free survival (PFS) in relation to HPV status. Results Activation of the PI3K/AKT and the RAS/RAF/MAPK pathways strongly protected HNSCC cells against anti-EGFR therapies. Mechanistically, this resistance was mediated by changes in the expression of anti-apoptotic proteins and could be overcome by co-treatment with specific inhibitors of EGFR downstream signaling. In contrast, the endogenous HPV status of HNSCC cells or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. In addition, response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16INK4A-positive and p16INK4A-negative tumors. Conclusion Activation of the PI3K/AKT and the RAS/RAF/MAPK pathways functionally interfere with the activity of anti-EGFR therapies. In contrast, HPV status of HNSCC did not influence the response to cetuximab in our retrospective analyses and in our preclinical models. In conclusion, cetuximab treatment should be administered independently of HPV status.