Clonal tumor evolution under induction chemotherapy and concurrent radiochemotherapy (RCHT) in patients with resectable stage IIIA (N2) and selected IIIb non-small cell lung cancer (NSCLC): Molecular analysis of the ESPATUE  randomized phase III trial.

person Sayedmohammad Hasheminasab Clinical Cooperation Unit Translational Radiation Oncology, German Cancer Consortium (DKTK) Core Center Heidelberg, German Cancer Research Center (DKFZ), Germany, Heidelberg, Germany info_outline Sayedmohammad Hasheminasab, Maximilian Knoll, Christian Schwager, Ali Nowrouzi, Nicole Pfarr, Stefan Rieken, Christoph Pöttgen, Thomas Christoph Gauler, Wilfried Eberhardt, Martin H. Schuler, Dirk Theegarten, Clemens Aigner, Juergen Debus, Martin Stuschke, Amir Abdollahi, Wilko Weichert

Authors person Sayedmohammad Hasheminasab Clinical Cooperation Unit Translational Radiation Oncology, German Cancer Consortium (DKTK) Core Center Heidelberg, German Cancer Research Center (DKFZ), Germany, Heidelberg, Germany info_outline Sayedmohammad Hasheminasab, Maximilian Knoll, Christian Schwager, Ali Nowrouzi, Nicole Pfarr, Stefan Rieken, Christoph Pöttgen, Thomas Christoph Gauler, Wilfried Eberhardt, Martin H. Schuler, Dirk Theegarten, Clemens Aigner, Juergen Debus, Martin Stuschke, Amir Abdollahi, Wilko Weichert Organizations Clinical Cooperation Unit Translational Radiation Oncology, German Cancer Consortium (DKTK) Core Center Heidelberg, German Cancer Research Center (DKFZ), Germany, Heidelberg, Germany, Departments of Radiation Oncology, Neurology, Neurosurgery, Heidelberg University Hospital, National Center for Tumor Disease (NCT), UKHD and German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Core-Center Heidelberg, Heidelberg, Germany, German Cancer Research Center (DKFZ), Heidelberg and German Cancer Consortium (DKTK), Core Center Heidelberg, Heidelberg, Germany, DKTK Partner Site Munich and Pathology Department, TUM, Munich, Germany, Munich, Germany, Heidelberg Ion-Beam Therapy Center (HIT), Department of Radiation Oncology, Heidelberg University Hospital (UKHD), National Center for Tumor Diseases (NCT), UKHD and German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK) Core Center Heidelb, Heidelberg, Germany, Department of Radiation Oncology, West German Tumor Centre, Essen, Germany, Department of Medicine, West German Cancer Center, University Hospital Essen of the University Duisburg-Essen, Essen, Germany, Departments of Radiation Oncology, Pathology, Thoracic Surgery and Medical Oncology, West German Cancer Center (WTZ), University Medicine Essen; DKTK partner site University Hospital Essen, Essen, Germany., Essen, Germany, University Hospital Essen, Essen, Germany, Department of Pathology and Neuropathology, University Hospital Essen, University Duisburg-Essen, Essen, Germany, Department of Thoracic Surgery and Thoracic Endoscopy - Ruhrlandklinik, University Hospital Essen, University of Duisburg-Essen, Essen, Germany, German Cancer Consortium (DKTK) Core Center Heidelberg and DKTK Partner Site Essen, Essen, Germany, Technical University Munich, Institute of Pathology, Munich, Germany Abstract Disclosures Research Funding Other Background: A better understanding of molecular mechanisms governing clonal tumor evolution under RCHT is of utmost importance for development of novel biomarker and targeted therapies. We report here the first attempt to decipher RCHT induced cellular and molecular perturbation in NSCLC on an integrative multiscale level. Methods: Patients with stage III disease received induction chemotherapy with cisplatin and paclitaxel followed by concurrent RCHT with 45 Gy (1.5 Gy twice daily) and cisplatin/vinorelbine according to the ESPATUE protocol. Tumor tissue was sampled from tumor enriched areas marked by pathologists at diagnosis (biopsies, n= 23) and post RCHT during surgical resection (n=22, ESPATUE-Arm B) corresponding to 16 paired samples (PS). Transcriptome analysis (n=45, 16PS), methylome analysis (n=35, 12PS), deep whole exome sequencing (WES) including copy number variation (CNV) analysis by low-pass whole genome sequencing (WGS, n=34, 13PS) were performed. A confirmatory targeted ultra-deep NGS for 41 genes was conducted (n=20PS). Results: Similarity plots of delta transcriptome data identified three distinct clusters of tumor evolution under RCHT. Cluster 1 was highly enriched for STS (5 out of 7 Pat.) compared to cluster 3 enriched for LTS (4 out of 6), p<0.02. 146 transcripts were differentially expressed as the function of RCHT (FDR <0.05). Among them, 61 genes were upregulated and enriched for ECM and tissue remodeling (COL6A3/4, Col14A1, LAMA2, PAI1, MMP2), p53 signaling (p21, GADD45B) and stress response (FOSB, EGR1) pathways, p<0.01. 39 downregulated genes were enriched for genes attributed to cell cycle- and DDR signaling (FANCI, SLX1A) p<0.05. 4221 CpG were differentially methylated (FDR<0.05). Seven inversely regulated genes were found with SLIT3 and TBX5 being among upregulated and hypomethylated genes. WES analysis revealed patterns of tumor evolution with a range of clonal diversity. In 5/13 pairs the clonal composition remained unchanged after RCHT. Approximately 500 post RCHT exclusive mutations were found. Conclusions: Clonal, transcriptional and methylome dynamic of tumor evolution towards RCHT selection pressure is unrevealed in patients with locally advanced NSCLC. This multi-scale dynamic approach provides novel means for development of biomarker and therapeutic targets. Clinical trial information: ESPATUE.