1. 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, 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, 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 T -
BCOR
Novel
R2T,R5T
1.32E+08 C
C9orf50
R1T,R4T
Nonsynonymous SNV
1.1E+08 G A
GPR6
Known
R1T,R2T
KRAS
Frameshift substitution
1.49E+08 AG GC
KRBA1
R3T,R5T
1.52E+08
NEB
OR5AU1
OR6C68
PLIN3
R3T,R4T
Stopgain SNV
ZNF337
ZNF783
L3T,L5T
Nonframeshift
substitution 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.