FREQUENCIES of altered genes RECURRENT variants & mutated genes Characterisation of Somatic Mutations in Proximal and Distal Colorectal Cancer Patients R. I. Mohd Yunos1*, N. S. Ab Mutalib1, J. Khor2, S. Saidin1, N. Md Nadzir1, Z. Abd Razak1, I. Md. Rose3, N. Mohd Mokhtar4, I. Sagap5, A. R. Jamal1   1 UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur 2 Life Science Group, Thermo Fisher Scientific Inc. Singapore 3 Department of Pathology, 4Department of Physiology, 5Department of Surgery, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, Malaysia.  Colorectal cancer (CRC) is the commonest cancer for men in Malaysia and majority of patients presented with advanced disease at diagnosis in particular proximal CRC. The anatomical location of the cancer may correlate with the clinical outcome of the patient. However, the molecular events that lead to the different outcomes between proximal and distal CRCs are still poorly understood. This study has successfully identified novel and druggable mutations in proximal and distal CRC. The comparison of the genomic mutation between both sides of colorectal cancer may provide us a new insight into the molecular events in the early step of colorectal carcinogenesis as well as leading to the discovery of novel, superior treatment options for patients. INTRODUCTION OBJECTIVES To characterise molecular alterations and related pathways unique to proximal versus distal colorectal cancers. To identify actionable mutations in proximal and distal colorectal cancers. 1 2 FREQUENCIES of altered genes APC is the most frequently mutated gene in CRC patients. TP53 is the second most frequently mutated gene. ATM and KRAS were exclusively altered in proximal sided CRC with frequency of 40 % and 60 % respectively METHODOLOGY DNA EXTRACTION DNA was extracted using QIAamp DNA Mini Kit (Qiagen), quantified using Qubit & Gel electrophoresis SAMPLE COLLECTION 10 Samples of CRCs (5 proximal & 5 distal matched tumor-normal tissues) H&E STAINING > 80% of tumor cells DATA ANALYSIS Torrent Suite 4.4.2 Annovar IGV RECURRENT variants & mutated genes LIBRARY PREPARATION Ion Plus Fragment Library kit Ion TargetSeq Exome Kit 56 tumor variants in 49 genes 11 true somatic variants in 11 genes (confirmed by IGV) 8 novel somatic variants in 8 genes SEQUENCING Ion Proton Sequencer Proximal 37 tumor variants in 32 genes 1 true somatic variants in 1 gene (confirmed by IGV) 1 novel somatic variants in 1 genes VALIDATION Sanger Sequencing Distal RESULTS Sample ID Exonic Function Start End Ref Alt Gene Mutation Type R1T,R5T Frameshift deletion 39932907 T - BCOR Novel R2T,R5T 132377900 1.32E+08 C C9orf50 R1T,R4T Nonsynonymous SNV 110301081 1.1E+08 G A GPR6 Known R1T,R2T 25398284 KRAS Frameshift substitution 149426304 1.49E+08 AG GC KRBA1 R3T,R5T 152419157 1.52E+08 NEB 21623290 OR5AU1 55886294 OR6C68 4852106 PLIN3 R3T,R4T Stopgain SNV 25657029 ZNF337 148963763 ZNF783 L3T,L5T Nonframeshift substitution 1887018 1887019 TA TG CFAP74 CONCLUSIONS APC remains as the most frequently altered gene in both proximal & distal CRCs KRAS and ATM were uniquely altered in proximal CRC. TP53 mutations frequency were equal in both sides of the colon. TGF-beta signaling and PI3K signaling pathway were exclusively altered in patients with proximal CRCs which could potentially explain the poor prognosis 90% of the patients in the present study harbored at least one druggable alterations Eight and one recurrent variant and mutated genes were found in proximal and distal CRCs, respectively Wnt signaling pathway was altered in 90% of all CRC patients (100% in proximal; 80% in distal). TGF-beta signaling and PI3K signaling pathway were predominantly altered in proximal. RTK-RAS and p53 signaling pathway were altered in both proximal and distal CRC.