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// Natalia Paez Arango 1 , Lauren Brusco 2 , Kenna R. Mills Shaw 2 , Ken Chen 3 , Agda Karina Eterovic 4 , Vijaykumar Holla 2 , Amber Johnson 2 , Beate Litzenburger 2 , Yekaterina B. Khotskaya 2 , Nora Sanchez 2 , Ann Bailey 2 , Xiaofeng Zheng 3 , Chacha Horombe 2 , Scott Kopetz 5 , Carol J. Farhangfar 6 , Mark Routbort 7 , Russell Broaddus 8 , Elmer V. Bernstam 9,10 , John Mendelsohn 2,11 , Gordon B. Mills 2,4 and Funda Meric-Bernstam 1,2,12 1 Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 6 Levine Cancer Institute, Carolinas Healthcare System, Charlotte, NC, USA 7 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 8 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 9 School of Biomedical Informatics, The University of Texas Health Science Center at Houston, TX, USA 10 Division of General Internal Medicine, Medical School, The University of Texas Health Science Center at Houston, TX, USA 11 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 12 Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Funda Meric-Bernstam, email: // Keywords : precision medicine, genomics, somatic mutation, CLIA, clinical trial Received : November 09, 2016 Accepted : February 27, 2017 Published : March 08, 2017 Abstract Purpose: Molecular profiling performed in the research setting usually does not benefit the patients that donate their tissues. Through a prospective protocol, we sought to determine the feasibility and utility of performing broad genomic testing in the research laboratory for discovery, and the utility of giving treating physicians access to research data, with the option of validating actionable alterations in the CLIA environment. Experimental design: 1200 patients with advanced cancer underwent characterization of their tumors with high depth hybrid capture sequencing of 201 genes in the research setting. Tumors were also tested in the CLIA laboratory, with a standardized hotspot mutation analysis on an 11, 46 or 50 gene platform. Results: 527 patients (44%) had at least one likely somatic mutation detected in an actionable gene using hotspot testing. With the 201 gene panel, 945 patients (79%) had at least one alteration in a potentially actionable gene that was undetected with the more limited CLIA panel testing. Sixty-four genomic alterations identified on the research panel were subsequently tested using an orthogonal CLIA assay. Of 16 mutations tested in the CLIA environment, 12 (75%) were confirmed. Twenty-five (52%) of 48 copy number alterations were confirmed. Nine (26.5%) of 34 patients with confirmed results received genotype-matched therapy. Seven of these patients were enrolled onto genotype-matched targeted therapy trials. Conclusion: Expanded cancer gene sequencing identifies more actionable genomic alterations. The option of CLIA validating research results can provide alternative targets for personalized cancer therapy.

// Genevieve M. Boland 1,2 , Sarina A. Piha-Paul 3 , Vivek Subbiah 3 , Mark Routbort 4 , Shelley M. Herbrich 5 , Keith Baggerly 6 , Keyur P. Patel 4 , Lauren Brusco 3 , Chacha Horombe 7 , Aung Naing 3 , Siqing Fu 3 , David S. Hong 3 , Filip Janku 3 , Amber Johnson 7 , Russell Broaddus 8 , Raja Luthra 4 , Kenna Shaw 7 , John Mendelsohn 7 , Gordon B. Mills 7,9 and Funda Meric-Bernstam 2,3,7 1 Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 2 Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 6 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 7 Department of Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 8 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 9 Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Funda Meric-Bernstam, email: // Keywords : genomic sequencing, actionable genes Received : November 10, 2014 Accepted : April 23, 2015 Published : May 08, 2015 Abstract Purpose: We determined the frequency of recurrent hotspot mutations in 46 cancer-related genes across tumor histologies in patients with advanced cancer. Methods: We reviewed data from 500 consecutive patients who underwent genomic profiling on an IRB-approved prospective clinical protocol in the Phase I program at the MD Anderson Cancer Center. Archival tumor DNA was tested for 740 hotspot mutations in 46 genes (Ampli-Seq Cancer Panel; Life Technologies, CA). Results: Of the 500 patients, 362 had at least one reported mutation/variant. The most common likely somatic mutations were within TP53 (36%), KRAS (11%), and PIK3CA (9%) genes. Sarcoma (20%) and kidney (30%) had the lowest proportion of likely somatic mutations detected, while pancreas (100%), colorectal (89%), melanoma (86%), and endometrial (75%) had the highest. There was high concordance in 62 patients with paired primary tumors and metastases analyzed. 151 (30%) patients had alterations in potentially actionable genes. 37 tumor types were enrolled; both rare actionable mutations in common tumor types and actionable mutations in rare tumor types were identified. Conclusion: Multiplex testing in the CLIA environment facilitates genomic characterization across multiple tumor lineages and identification of novel opportunities for genotype-driven trials .

Background: We developed and established a high depth targeted sequencing platform of 202 genes (all exons) to identify actionable DNA alterations in tumor samples. We aimed a minimum of 500x depth and defined actionable if the aberration had a direct link to an available clinical treatment or clinical trial. Our platform was optimized for FFPE specimens and low input DNA while the data analysis pipeline was designed to detect low frequency mutations and copy number alterations. Over 500 tumor samples and matched normal were analyzed using this platform. Methods: Libraries were made from a minimum of 170 ng of FFPE DNA (tumors) or blood DNA (matched normal). Capture of 202 genes (over 5,000 exons) was performed using Nimblegen probes to a minimum of 50x fold enrichment. Captured libraries were sequenced using Illumina HiSeq2000. Duplicate reads were removed from the raw data and the reads were mapped hg19 reference genome. We used VarScan2 for calling somatic, germline and loss of heterozygosity SNVs and short indels. Copy number variation was also reported when significant gain/loss on an exon was detected. Additionally, we used other tolls to annotate the functional consequence of the SNVs, such as CanDrA to predict if an SNV is driver or passenger event. Results: We sequenced over 500 tumor samples and their normal match (blood). The mean coverage for most of samples was over 1500x, which allowed us to detect low frequency mutations (5% or higher) with accuracy. The most common disease sites analyzed where we found genetic alterations were: breast, colon, brain, ovary, endometrial, skin, lung, prostate, head and neck, sarcoma, kidney and stomach. About 98% of all tumors analyzed had at least one somatic alteration (SNVs or copy number) and although the vast majority of the samples analyzed were derived from FFPE blocks, the number of samples that failed was less than 5%. Among the most common genes mutated are several “actionable genes” such as BRAF, EGFR and KRAS. The samples tested in this platform were also tested on ion torrent Ampliseq 46 gene panel (46 overlapping genes) in a CLIA certified laboratory at MD Anderson Cancer Center with a very high level of concordance. Conclusion: With a very low level of failure and high depth provided, our results indicate that this platform can be used for FFPE tumor samples with high level of accuracy and sensitivity, showing that this platform is reliable to be implemented in clinical settings. Citation Format: Karina Eterovic, Ken Chen, Hao Zhao, Funda Meric-Bernstam, Raja Luthra, Aldape Kenneth, Mark Routbort, Scott Kopetz, Michael Davies, John de Groot, Stacy Moulder, Yong Mao, Chacha Horombe, Lin-ya Tang, Song Ping, Zhang Qingxiu, Ezzeddine Nader, Lan Zhang, Kenna M. Shaw, John Mendelsohn, Gordon B. Mills. T200: a high depth targeted exome sequencing platform to identify actionable alterations in FFPE solid tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4276. doi:10.1158/1538-7445.AM2014-4276