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    -  Mehra S
    -  Tiwari AK
    -  Mehta SP
    -  Sachdev R
    -  Rajvanshi C
    -  Chauhan R
    -  Saini A
    -  Vaid A

 
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ORIGINAL ARTICLE
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The utility of the Ion Torrent PGM next generation sequencing for analysis of the most commonly mutated genes among patients with colorectal cancer in India


1 Molecular and Transplant Immunology Laboratory, Department of Transfusion Medicine, Gurgaon, Haryana, India
2 Department of Histopathology, Medanta-The Medicity, Gurgaon, Haryana, India
3 Department of Medical and Haemato Oncology, Medanta-The Medicity, Gurgaon, Haryana, India

Date of Submission10-Aug-2019
Date of Decision15-Jun-2020
Date of Acceptance02-Jul-2020
Date of Web Publication21-Mar-2021

Correspondence Address:
Aseem Kumar Tiwari,
Molecular and Transplant Immunology Laboratory, Department of Transfusion Medicine, Gurgaon, Haryana
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijc.IJC_723_19

PMID: 33753628

  Abstract 


Background: The requirement for the mutation analysis for Kirsten rat sarcoma viral oncogene (KRAS) in colorectal cancer (CRC) is rapidly increasing as it is a predictive biomarker and also, its absence signifies response to anti-epidermal growth factor receptor (anti-EGFR) antibody treatment. The aim of our study was to investigate the pathological diagnosis and distribution of KRAS mutations in colorectal cancer with the use of next generation sequencing platform (Ion Torrent).
Methods: A total of 56 CRC samples were tested to identify the genetic mutations, especially KRAS using the primers which included ~2800 COSMIC mutations of 50 oncogenes. Ion Torrent personal genome machine (semiconductor-based sequencing) was used for the sequencing and analysis. Along with KRAS, other 49 genes were also studied for COSMIC mutations.
Results: KRAS mutation 25 (44.6%) had the highest frequency, followed by TP53 10 (17.9%) and PIK3CA mutation 4 (7.1%). Of all the KRAS mutations identified, mutations in codon 12 were most frequent followed by mutations in codon 13 and 61. The most frequent substitution was glycine to aspartate mutation in codon 12 (p.Gly12Asp) followed by glycine to valine (p.Gly12Val). Combinations of mutations were also studied. Our study revealed that seven cases (12.5%) had both KRAS and TP53 mutations (highest of all the combinations).
Conclusion: The analysis of KRAS mutation frequency and its mutational subtype analysis in human CRCs by using semiconductor-based platform in routine clinical practices have been performed in Indian population. The findings were similar to earlier published reports from the Western literature.


Keywords: Colorectal cancer, DNA sequencing, K-Ras protein
Key Message Detection of KRAS single-point mutations on next-generation sequencing (NGS) platform in colorectal cancer can help provide appropriate treatment and evaluate prognosis. Detection of KRAS, NRAS, BRAF, and other related mutations by NGS saves cost, time, and offers overall practicality of individual genome sequencing.



How to cite this URL:
Mehra S, Tiwari AK, Mehta SP, Sachdev R, Rajvanshi C, Chauhan R, Saini A, Vaid A. The utility of the Ion Torrent PGM next generation sequencing for analysis of the most commonly mutated genes among patients with colorectal cancer in India. Indian J Cancer [Epub ahead of print] [cited 2021 Oct 23]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=311635





  Introduction Top


Colorectal cancer (CRC) is the second most common malignancy in women, third most common malignancy in men, and second most common cause of cancer death.[1] Combination of environmental and genetic interactions have been involved in the CRC development. Genetic factors have been responsible for only 5%–10% of CRC. CRC tumors with Kirsten rat sarcoma viral oncogene (KRAS) mutations generally do not respond to commonly used drugs targeting epidermal growth factor receptor (EGFR).[2] Specific monoclonal antibodies such as cetuximab and panitumumab are being used to treat the CRCs in patients having wild type KRAS mutations.

KRAS single-point mutations occurrence and association with other genes in CRC can help understand the disease process and also provide better treatment approach for CRC-affected patients by combining the “conventional” and “monoclonal antibodies” therapies. The same can be accomplished by profiling an individual cancer genome by using next generation sequencing (NGS) which saves cost, time, and overall practicality of individual genome sequencing.

The recent availability of NGS, which differs from other sequencing methods, makes individual genome sequencing possible as it does not depend on modified nucleotides. It has now become essential to do extended-RAS mutational testing along with KRAS. Other mutations such as V-raf murine sarcoma viral oncogene homolog B (BRAF), neuroblastoma RAS viral oncogene homolog (NRAS), phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), and phosphatase and tensin homolog (PTEN) should also be tested.[3] There are studies on commonly mutated genes among patients of CRC in the Caucasian population, but none in the Indian population.

In this study, we have used NGS to analyze 56 clinical CRC samples to identify the genetic mutations, with focus on KRAS mutation, using the primers which include ~2800 COSMIC mutations of 50 oncogenes.


  Methods Top


Patient and samples

A total of 56 consecutive patients with biopsy diagnosed CRC were studied in a tertiary care center, Medanta-The Medicity, India from January 2015 to June 2016. Demographic and clinical characteristics, including age, gender, and anatomical location of tumor and histology of the tumor were obtained from patient records. The percentage of tumor cellularity required for the sample was minimum 10%. Out of 56 patients, 40 (71.4%) were men and 16 (28.5%) were women. Mean age of men was 59.5 years (range: 26–84 years) and for women, it was 58 years (range: 39–78 years). Tumor metastasis was involved in 27 (48%) of cases.

NGS analysis

Mutation analysis was carried out using NGS platform-Ion Torrent personal genome machine (PGM). DNA was extracted using nucleospin column-based method from the formalin-fixed paraffin-embedded (FFPE) tissue following the manufacturer recommendations and the kit used was ReliaPrep™ FFPE gDNA Miniprep System (Promega Corporation, WI, USA). The extracted DNA was stored at −20°C. The DNA concentration was quantified using Qubit® 2.0 Fluorometer and adjusted to 10 ng/μL for one reaction. An Ion Torrent adapter-ligated library was constructed with the Ion AmpliSeq Library Kit 2.0 following the manufacturer's protocol. The Ion AmpliSeq™ cancer hotspot panel v2 primer pool was used to detect the mutations. This panel contains the primers against 50 oncogenes and tumor suppressor genes. Prepared library was then templated and amplified on ion sphere particles (ISPs) and the amplified ISPs were enriched using Ion One Touch™ 2 System. The amplified product was loaded in Ion 316™ Chip v2. Sequencing of the amplified product was performed in Ion PGM™ System as per the manufacturer protocol.

The data obtained were processed in Ion Torrent platform-specific pipeline software-Torrent Suite V.4.0 to generate sequence reads, trim adapter sequences, filter, and remove poor signal-profile reads. Variant calling from the obtained filter data was done with a plug-in “Variant caller.” Further, ion reporter software was used to find out the clinically relevant mutations and visualization was done using integrated genome viewer (IGV). A minimum coverage of 1000× and a frequency of 5% was considered significant and confirmed by the IGV visual inspection.

The Ion AmpliSeq™ cancer hotspot panel v2 include ABL1, EGFR, GNAS, KRAS, PTPN11, AKT1, ERBB2, GNAQ, MET, RB1, ALK, ERBB4, HNF1A, MLH1, RET, APC, EZH2, HRAS, MPL, SMAD4, ATM, FBXW7, IDH1, NOTCH1, SMARCB1, BRAF, FGFR1, JAK2, NPM1, SMO, CDH1, FGFR2, JAK3, NRAS, SRC, CDKN2A, FGFR3, IDH2, PDGFRA, STK11, CSF1R, FLT3, KDR, PIK3CA, TP53, CTNNB1, GNA11, KIT, PTEN, and VHL.

Statistical analysis

Ordinal data obtained were expressed in terms of mean and standard deviation. All the calculations were done using SPSS Software.

Institutional Review

The study has been approved by the Institutional Review Board (IRB) and the approval letter number is 1212/2020.


  Results Top


One or more mutations were detected in 36/56 (64%) tumors in 14 of the 50 genes included in the panel [Table 1].
Table 1: 50 multiple gene mutations analysis by the Ion AmpliSeq™ Cancer Hotspot Panel v2 in routine samples of CRCs

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[Figure 1]a and [Figure 1]b shows the proportions of KRAS mutations found according to gender and KRAS point mutation frequency in codon 12, 13, and 61 in the tumors. As shown in [Figure 1]a, out of 56 CRCs, 25 (44.6%) colonic tumors were having KRAS mutation. None of the cases showed both KRAS and EGFR. None of these cases had EGFR mutation. Mutations in codon 12 were found to be 22 (88%) which is much more common than mutations in 61 and 13. Among codon 12 mutations, p.Gly12Asp mutation type had the highest frequency 15 (60%) followed by p.Gly12Val 4 (16%). Combination of point mutations was also studied in all 56 CRCs samples [Figure 1]b. Fourteen cases had more than one mutation, 18 cases had only one mutation, and 24 cases showed no mutation. As shown in [Figure 1]b, seven (12.5%) cases out of all CRCs showed KRAS and TP53 mutation in combination. Frequency of all other mutations found is also reported [Table 2]. KRAS mutations 25 (44.6%) had the highest frequency, followed by TP53 10 (17.9%) and PIK3CA mutation 4 (7.1%). Other mutations were also found (CTNNB1, SMAD4, ERBB2, etc.) but at lesser frequencies.
Figure 1: (a) Distribution of point mutations in KRAS among males and females. (b) Frequency of single-gene mutations and with the combination of mutations

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Table 2: Number and percentage of mutated cases of each gene

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  Discussion Top


KRAS mutations have been reported in up to 35.7%–50% of all human CRC.[4],[5],[6] Similarly, in our study, 44.6% of all CRCs samples showed the KRAS mutation which is consistent with the frequency reported in World Health Organization's histological classification. None of the CRC has shown the EGFR mutation which is consistent with the other studies.[7] In the majority of cases, KRAS mutations are missense mutations which introduce an amino acid substitution at position 12, 13, or 61. Our study revealed that mutations in codon 12 were more common than mutations in codon 61 and 13, which is consistent with Jauhri et al.'s study.[6] A cohort study of Kodaz et al.[7] showed that in CRCs, p.Gly12Asp mutations were the most common mutations and had the frequency 36%, followed by the p.Gly12Val mutation having frequency of 21.8%. Similarly, our study revealed the similar distribution of point mutations; p.Gly12Asp 15 (60%) were more common than other mutations followed by p.Gly12 Val 4 (16%).

Tumor protein p53 (TP53) gene abnormality is the most common event in CRCs and plays an important role in tumorigenesis of colorectal epithelial cells. TP53 mutation is a relatively late event in the generation of CRCs from an adenoma to a malignant tumor as it is followed by adenomatous polyposis coli (APC) and KRAS mutation.[5] TP53 mutation was found in multiple exons in our study. A total of 10 cases (17.9%) had TP53 mutation. Out of which, 2 (20%) of TP53 mutations were found in exon 4 (p.Pro72Arg), 3 (30%) in exon 5 (p.Arg175His), 2 (20%) in exon 7 (p.Gly245Asp and p.Arg248Gln), and 3 (30%) in exon 8 (p.Arg273His and p.Arg273Cys), which is consistent with the study by Jauhri M et al.[6]

Phosphatidylinositol-4, 5-biphosphate-3-kinase, catalytic subunit α (PIK3CA) is a lipid kinase which is capable of phosphorylating the phosphoinositides. Mutation in PIK3CA leads to an increase in lipid kinase activity and activation of downstream Akt signaling. Of all the CRCs, an estimated 10%–30% has PIK3CA mutations.[8] Similarly, 4 of our 56 CRCs samples (7.1%) harbored PIK3CA mutations; three were found in exon 10 and one was found in exon 21.

Several studies have evaluated the combination frequency of KRAS with other genes and found the most common mutation combinations are TP53 and APC, whereas TP53 and KRAS mutation combinations were rare;[9] our study revealed that six cases (10.7%) had both KRAS and TP53 mutations (highest of all the combinations). However, other studies from India have shown the significant association of KRAS with APC followed by KRAS and TP53 and KRAS and PIK3CA.[6]

In our study, CTNNB1 mutation was detected in three (5.4% of the total cases), in which, two (66.6% of total CTNNB1 mutation) cases was associated with at least one or other concurrent mutation, especially KRAS. This finding was also concordant with the study by Fearon.[10]


  Conclusion Top


In summary, it is a retrospective study which has been performed for the first time on Indian population to provide KRAS mutation frequencies data in Indian population using NGS platform (Ion Torrent). Our study results are consistent with the previously described mutation rates for KRAS and other mutations in worldwide data. Also, our data confirm that CRCs consist of a group of heterogeneous disorders with a large number of genetic changes set in oncogenes and tumor suppressor genes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Maxwell Parkin D. Author's reply to: Lung cancer mortality in sub-Saharan Africa. Int J Cancer 2011;129:1539.  Back to cited text no. 1
    
2.
Silvestri A, Pin E, Huijbers A, Pellicani R, Parasido EM, Pierobon M, et al. Individualized therapy for metastatic colorectal cancer. J Intern Med 2013;274:1-24.  Back to cited text no. 2
    
3.
Sepulveda AR, Hamilton SR, Allegra CJ, Grody W, Cushman-Vokoun AM, Funkhouser WK, et al. Molecular biomarkers for the evaluation of colorectal cancer: Guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. Am J Clin Pathol 2017;147:221-60.  Back to cited text no. 3
    
4.
Zhang K, Xu J, Yan L, Liu X, Xu F, Liu Y. Detection of KRAS, NRAS and BRAF gene mutations in colorectal carcinoma. Chinese journal of pathology 2015;44:254-7.  Back to cited text no. 4
    
5.
Barber TD, Vogelstein B, Kinzler KW, Velculescu VE. Somatic mutations of EGFR in colorectal cancers and glioblastomas. N Engl J Med 2004;351:2883.  Back to cited text no. 5
    
6.
Jauhri M, Bhatnagar A, Gupta S, Bp M, Minhas S, Shokeen Y, et al. Prevalence and coexistence of KRAS, BRAF, PIK3CA, NRAS, TP53, and APC mutations in Indian colorectal cancer patients: Next-generation sequencing–based cohort study. Tumour Biol. 2017;39:1-11.  Back to cited text no. 6
    
7.
Kodaz H, Hacibekiroglu I, Erdogan B, Turkmen E, Tozkir H, Albayrak D, et al. Association between specific KRAS mutations and the clinicopathological characteristics of colorectal tumors. Mol Clin Oncol 2015;3:179-84.  Back to cited text no. 7
    
8.
Stintzing S, Lenz HJ. A small cog in a big wheel: PIK3CA mutations in colorectal cancer. J Natl Cancer Inst 2013;105:1775-6.  Back to cited text no. 8
    
9.
Smith G, Carey FA, Beattie J, Wilkie MJ, Lightfoot TJ, Coxhead J, et al. Mutations in APC, Kirsten-ras, and p53—alternative genetic pathways to colorectal cancer. Proc Natl Acad Sci U S A 2002;99:9433-8.  Back to cited text no. 9
    
10.
Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol 2011;6:479-507.  Back to cited text no. 10
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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