Evidence of mutations in tumour suppressor genes among oral cancer in Naswar, smokeless tobacco users
DOI:
https://doi.org/10.2340/aos.v84.43778Keywords:
Oral squamous cell carcinoma, tumour suppressor genes, whole exome sequencing, mutations, targeted therapyAbstract
Objective: Smokeless tobacco has been linked to the genetic modification of oral squamous cell carcinoma (OSCC). Our study aims to further investigate the disease among Naswar users at the genomic level to understand genetic diversity and discover new targeted therapy.
Methods: A multi-centre descriptive cross sectional research was designed comprising a total of 80 cases of OSCC who were habitual users of Naswar. Out of the 80 cases, whole exome sequencing (WES) was applied to 7 formalin fixed paraffin embedded (FFPE) tissues of OSCC. We further investigated immunohistochemical expression of mutant TP53 and CDKN2A protein in tissues of 80 OSCC samples. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 19.
Results: Among the total 2,216 somatic variants identified in tumour suppressor genes (TSGs), we compared the high frequency mutation genes reported in OSCC in Catalogue of Somatic Mutations in Cancer (COSMIC) database with research samples, and found that TP53 (85.7%), NOTCH1 (85.7%), and FAT1 (85.7%) showed higher rate of mutation. Among single nucleotide variants, higher prevalence of C/T and G/A base change was noted. Interestingly, a distinct panel of 12 genes was detected to be mutated in 100% samples which was not previously reported compared to Single Nucleotide Polymorphism Database (dbSNP). PTPRT mutation (rs2867655) was present in seven samples and IGF2R (rs629849) was seen in two samples. A statistically significant relation was observed between mutant TP53 protein expression and duration of Naswar use and clinical stages while difference in CDKN2A protein expression was found to be statistically significant with respect to stage only.
Conclusions: Our study presented preliminary data of genetic aberrations in patients exposed to known risk factor (Naswar). These findings can enhance the understanding of genetic aetiology and serve as basis for innovative targets of therapy.
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References
Ferlay J, Ervik M, Lam F, Laversanne M, Colombet M, Mery L, et al. Global cancer observatory: Cancer Today [Internet]. Lyon: International Agency for Research on Cancer; 2024 [cited 24 Jun 2024]. Available from: https://gco.iarc.who.int/today
Ong T, Murphy C, Smith A, Kanatas A, Mitchell D. Survival after surgery for oral cancer: a 30-year experience. Br J Oral Maxillofac Surg. 2017;55(9):911–6. https://doi.org/10.1016/j.bjoms.2017.08.362 DOI: https://doi.org/10.1016/j.bjoms.2017.08.362
Alsahafi E, Begg K, Amelio I, Raulf N, Lucarelli P, Sauter T, et al. Clinical update on head and neck cancer: molecular biology and ongoing challenges. Cell death Dis. 2019;10(8):540. https://doi.org/10.1038/s41419-019-1769-9 DOI: https://doi.org/10.1038/s41419-019-1769-9
Siddiqi K, Scammell K, Huque R, Khan A, Baral S, Ali S, et al. Smokeless tobacco supply chain in South Asia: a comparative analysis using the who framework convention on tobacco control. Nicotine Tob Res. 2015;18(4):424–30. https://doi.org/10.1093/ntr/ntv067 DOI: https://doi.org/10.1093/ntr/ntv067
Sinha DN, Naveen Agarwal NA, Gupta P. Prevalence of smokeless tobacco use and number of users in 121 countries. Br J Med Med Res. 2015;9(6):1–20. https://doi.org/10.9734/BJMMR/2015/16285 DOI: https://doi.org/10.9734/BJMMR/2015/16285
Ali J, Sabiha B, Jan HU, Haider SA, Khan AA, Ali SS. Genetic etiology of oral cancer. Oral Oncol. 2017;70:23–8. https://doi.org/10.1016/j.oraloncology.2017.05.004 DOI: https://doi.org/10.1016/j.oraloncology.2017.05.004
Khan Z. Naswar (smokeless tobacco product), oral cancer and tobacco control in Khyber Pakhtunkhwa, Pakistan. KMUJ [Internet]. 2016;8(3):113. [cited 28 Jun 2024] Available from: https://www.kmuj.kmu.edu.pk/article/view/16886
Saeed M, Asad M, Shahzad M, Akram M, Khan Z. Biochemical composition of a smokeless tobacco product (NASWAR) used in Pakistan. J King Saud Univ Sci. 2024;36(5):103168. https://doi.org/10.1016/j.jksus.2024.103168 DOI: https://doi.org/10.1016/j.jksus.2024.103168
Khan Z, Dreger S, Shah SMH, Pohlabeln H, Khan S, Ullah Z, et al. Oral cancer via the bargain bin: the risk of oral cancer associated with a smokeless tobacco product (Naswar). PLoS One. 2017;12(7):e0180445. https://doi.org/10.1371/journal.pone.0180445 DOI: https://doi.org/10.1371/journal.pone.0180445
Batta N, Pandey M. Mutational spectrum of tobacco associated oral squamous carcinoma and its therapeutic significance. World J Surg Oncol. 2019;17:1–12. https://doi.org/10.1186/s12957-019-1741-2 DOI: https://doi.org/10.1186/s12957-019-1741-2
Ghias K, Rehmani SS, Razzak SA, Madhani S, Azim MK, Ahmed R, et al. Mutational landscape of head and neck squamous cell carcinomas in a South Asian population. Genet Mol Biol. 2019;42:526–42. https://doi.org/10.1590/1678-4685-GMB-2018-0005 DOI: https://doi.org/10.1590/1678-4685-gmb-2018-0005
Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333(6046):1157–60. https://doi.org/10.1126/science.1208130 DOI: https://doi.org/10.1126/science.1208130
Al‐hebshi NN, Li S, Nasher AT, El‐Setouhy M, Alsanosi R, Blancato J, et al. Exome sequencing of oral squamous cell carcinoma in users of A rabian snuff reveals novel candidates for driver genes. Int J Cancer. 2016;139(2):363–72. https://doi.org/10.1002/ijc.30068 DOI: https://doi.org/10.1002/ijc.30068
Alshahrani SA, Al-Qahtani WS, Almufareh NA, Domiaty DM, Albasher GI, Safhi FA, et al. Oral cancer among Khat users: finding evidence from DNA analysis of nine cancer-related gene mutations. BMC Oral Health. 2021;21:1–9. https://doi.org/10.1186/s12903-021-01981-7 DOI: https://doi.org/10.1186/s12903-021-01981-7
Zhang W, Wang M, Wu Q, Zhu Q, Jiao Y, Zhu Y, et al. Mutational signatures and the genomic landscape of betel quid chewing‐associated tongue carcinoma. Cancer Med. 2019;8(2):701–11. https://doi.org/10.1002/cam4.1888 DOI: https://doi.org/10.1002/cam4.1888
Yang W, Qin N, Song X, Jiang C, Li T, Ji P, et al. Genomic signature of mismatch repair deficiency in areca nut–related oral cancer. J Dent Res. 2020;99(11):1252–61. https://doi.org/10.1177/0022034520930641 DOI: https://doi.org/10.1177/0022034520930641
El-Naggar AK, Chan JKC, Grandis JR, Takata T, Slootweg PPJ, editors. WHO classification of head and neck tumours. 4th ed. Lyon: IARC; 2017.
Chen Y, Chen Y, Shi C, Huang Z, Zhang Y, Li S, et al. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience. 2018;7(1):gix120. https://doi.org/10.1093/gigascience/gix120 DOI: https://doi.org/10.1093/gigascience/gix120
Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754–60. https://doi.org/10.1093/bioinformatics/btp324 DOI: https://doi.org/10.1093/bioinformatics/btp324
Ebbert MT, Wadsworth ME, Staley LA, Hoyt KL, Pickett B, Miller J, et al. Evaluating the necessity of PCR duplicate removal from next-generation sequencing data and a comparison of approaches. BMC Bioinform. 2016;17:491–500. https://doi.org/10.1186/s12859-016-1097-3 DOI: https://doi.org/10.1186/s12859-016-1097-3
Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38(16):e164-e. https://doi.org/10.1093/nar/gkq603 DOI: https://doi.org/10.1093/nar/gkq603
School of Biomedical Informatics UTHealth. Tumour Supressor Gene Database (TSGene) [Internet]. 2016 [cited 2024 May 8]. Available from: https://bioinfo.uth.edu/TSGene/
Sondka Z, Dhir NB, Carvalho-Silva D, Jupe S, Madhumita, McLaren K, et al. COSMIC: a curated database of somatic variants and clinical data for cancer. Nucleic Acids Res. 2024;52(D1):D1210–D7. https://doi.org/10.1093/nar/gkad986 DOI: https://doi.org/10.1093/nar/gkad986
Li Y, Zhang J. Expression of mutant p53 in oral squamous cell carcinoma is correlated with the effectiveness of intra-arterial chemotherapy. Oncol Lett. 2015;10(5):2883–7. https://doi.org/10.3892/ol.2015.3651 DOI: https://doi.org/10.3892/ol.2015.3651
Tokuzen N, Nakashiro K-I, Tojo S, Goda H, Kuribayashi N, Uchida D. Human papillomavirus‑16 infection and p16 expression in oral squamous cell carcinoma. Oncol Lett. 2021;22(1):1–6. https://doi.org/10.3892/ol.2021.12789 DOI: https://doi.org/10.3892/ol.2021.12789
Nakagaki T, Tamura M, Kobashi K, Koyama R, Fukushima H, Ohashi T, et al. Profiling cancer-related gene mutations in oral squamous cell carcinoma from Japanese patients by targeted amplicon sequencing. Oncotarget. 2017;8(35):59113. https://doi.org/10.18632/oncotarget.19262 DOI: https://doi.org/10.18632/oncotarget.19262
Lin L-H, Chou C-H, Cheng H-W, Chang K-W, Liu C-J. Precise identification of recurrent somatic mutations in oral cancer through whole-exome sequencing using multiple mutation calling pipelines. Front Oncol. 2021;11:741626. https://doi.org/10.3389/fonc.2021.741626 DOI: https://doi.org/10.3389/fonc.2021.741626
Campbell BR, Chen Z, Faden DL, Agrawal N, Li RJ, Hanna GJ, et al. The mutational landscape of early‐and typical‐onset oral tongue squamous cell carcinoma. Cancer. 2021;127(4):544–53. https://doi.org/10.1002/cncr.33309 DOI: https://doi.org/10.1002/cncr.33309
Chen X, Zhao W, Chen S, Yu D. Mutation profiles of oral squamous cell carcinoma cells. Adv Oral Maxillofac Surg. 2021;2:100026. https://doi.org/10.1016/j.adoms.2021.100026 DOI: https://doi.org/10.1016/j.adoms.2021.100026
Nakagaki T, Tamura M, Kobashi K, Omori A, Koyama R, Idogawa M, et al. Targeted next-generation sequencing of 50 cancer-related genes in Japanese patients with oral squamous cell carcinoma. Tumor Biol. 2018;40(9):1010428318800180. https://doi.org/10.1177/1010428318800180 DOI: https://doi.org/10.1177/1010428318800180
Osawa Y, Aoyama K-I, Hosomichi K, Uchibori M, Tajima A, Kimura M, et al. Somatic mutations in oral squamous cell carcinomas in 98 Japanese patients and their clinical implications. Cancer Treat Res Commun. 2021;29:100456. https://doi.org/10.1016/j.ctarc.2021.100456 DOI: https://doi.org/10.1016/j.ctarc.2021.100456
Patel K, Bhat FA, Patil S, Routray S, Mohanty N, Nair B, et al. Whole-exome sequencing analysis of oral squamous cell carcinoma delineated by tobacco usage habits. Front Oncol. 2021;11:660696. https://doi.org/10.3389/fonc.2021.660696 DOI: https://doi.org/10.3389/fonc.2021.660696
Sharma V, Kumar D, Kumar S, Singh H, Sharma N, Gupta S. Impact of tobacco smoking on oral cancer genetics—a next‐generation sequencing perspective. Imeta. 2022;1(3):e44. https://doi.org/10.1002/imt2.44 DOI: https://doi.org/10.1002/imt2.44
Zanaruddin SNS, Yee PS, Hor SY, Kong YH, Ghani WMNWA, Mustafa WMW, et al. Common oncogenic mutations are infrequent in oral squamous cell carcinoma of Asian origin. PLoS One. 2013;8(11):e80229. https://doi.org/10.1371/journal.pone.0080229 DOI: https://doi.org/10.1371/journal.pone.0080229
Liu G-M, Lu T-C, Liu Y, Luo Y-G. Gene expression analysis of primary gingival cancer by whole exome sequencing in thirteen Chinese patients. Int J Clin Exp Pathol. 2020;13(7):1909.
Pansare K, Gardi N, Kamat S, Dange P, Previn R, Gera P, et al. Establishment and genomic characterization of gingivobuccal carcinoma cell lines with smokeless tobacco associated genetic alterations and oncogenic PIK3CA mutation. Sci Rep. 2019;9(1):8272. https://doi.org/10.1038/s41598-019-44143-0 DOI: https://doi.org/10.1038/s41598-019-44143-0
Cao C-H, Liu R, Lin X-R, Luo J-Q, Cao L-J, Zhang Q-J, et al. LRP1B mutation is associated with tumor HPV status and promotes poor disease outcomes with a higher mutation count in HPV-related cervical carcinoma and head & neck squamous cell carcinoma. Int J Biol Sci. 2021;17(7):1744. https://doi.org/10.7150/ijbs.56970 DOI: https://doi.org/10.7150/ijbs.56970
Chen L-C, Lo Y-S, Ho H-Y, Lin C-C, Chuang Y-C, Chang W-C, et al. LDL Receptor-related protein 1B polymorphisms associated with increased risk of lymph node metastasis in oral cancer group with diabetes mellitus. Int J Mol Sci. 2024;25(7):3963. https://doi.org/10.3390/ijms25073963 DOI: https://doi.org/10.3390/ijms25073963
Shaikh MH, Dawson A, Prokopec SD, Barrett JW, Zeng PY, Khan MI, et al. Loss of LRP1B expression drives acquired chemo and radio-resistance in HPV-positive head and neck cancer. Oral Oncol. 2023;146:106580. https://doi.org/10.1016/j.oraloncology.2023.106580 DOI: https://doi.org/10.1016/j.oraloncology.2023.106580
He J, Xu T, Zhao F, Guo J, Hu Q. SETD2-H3K36ME3: an important bridge between the environment and tumors. Front Genet. 2023;14:1204463. https://doi.org/10.3389/fgene.2023.1204463 DOI: https://doi.org/10.3389/fgene.2023.1204463
Liu T-Y, Lee C-C, Chen Y-C, Chang Y-S, Huang H-Y, Lee Y-T, et al. Mutation analysis of second primary tumors in oral cancer in Taiwanese patients through next-generation sequencing. Diagnostics. 2022;12(4):951. https://doi.org/10.3390/diagnostics12040951 DOI: https://doi.org/10.3390/diagnostics12040951
Wang H-C, Moi S-H, Chan L-P, Wu C-C, Du J-S, Liu P-L, et al. The role of the genomic mutation signature and tumor mutation burden on relapse risk prediction in head and neck squamous cell carcinoma after concurrent chemoradiotherapy. Exp Mol Med. 2023;55(5):926–38. https://doi.org/10.1038/s12276-023-00984-4 DOI: https://doi.org/10.1038/s12276-023-00984-4
India Project Team of the International Cancer Genome Consortium. Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups. Nat Commun. 2013;4:2873. https://doi.org/10.1038/ncomms3873 DOI: https://doi.org/10.1038/ncomms3873
Das LP, Pitty RH, Asokan K, Krithika C, Anandi M, Ramanathan A. Analysis of ARID2 gene mutation in oral squamous cell carcinoma. Asian Pac J Cancer Prev. 2017;18(10):2679. https://doi.org/10.22034/apjcp.2017.18.10.2679
Vettore AL, Ramnarayanan K, Poore G, Lim K, Ong CK, Huang KK, et al. Mutational landscapes of tongue carcinoma reveal recurrent mutations in genes of therapeutic and prognostic relevance. Genome Med. 2015;7:1–15. https://doi.org/10.1186/s13073-015-0219-2 DOI: https://doi.org/10.1186/s13073-015-0219-2
Haft S, Ren S, Xu G, Mark A, Fisch K, Guo TW, et al. Mutation of chromatin regulators and focal hotspot alterations characterize human papillomavirus–positive oropharyngeal squamous cell carcinoma. Cancer. 2019;125(14):2423–34. https://doi.org/10.1002/cncr.32068 DOI: https://doi.org/10.1002/cncr.32068
Payungwong T, Angkulkrerkkrai K, Chaiboonchoe A, Lausoontornsiri W, Jirawatnotai S, Chindavijak S. Comparison of mutation landscapes of pretreatment versus recurrent squamous cell carcinoma of the oral cavity: the possible mechanism of resistance to standard treatment. Cancer Rep. 2024;7(3):e2004. https://doi.org/10.1002/cnr2.2004 DOI: https://doi.org/10.1002/cnr2.2004
Daniels SR, Liyasova M, Kales SC, Nau MM, Ryan PE, Green JE, et al. Loss of function Cbl-c mutations in solid tumors. PLoS One. 2019;14(7):e0219143. https://doi.org/10.1371/journal.pone.0219143 DOI: https://doi.org/10.1371/journal.pone.0219143
Rajendran S, Muthupalani RS, Ramanathan A. Lack of RING finger domain (RFD) mutations of the c-Cbl gene in oral squamous cell carcinomas in Chennai, India. Asian Pac J Cancer Prev. 2013;14(2):1073–5. https://doi.org/10.7314/apjcp.2013.14.2.1073 DOI: https://doi.org/10.7314/APJCP.2013.14.2.1073
Lui VWY, Peyser ND, Ng PK-S, Hritz J, Zeng Y, Lu Y, et al. Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer. Proc Natl Acad Sci U S A. 2014;111(3):1114–9. https://doi.org/10.1073/pnas.1319551111 DOI: https://doi.org/10.1073/pnas.1319551111
Li X, Z Li, Gao Q, Peng Y, Yu Y, Hu T, Wang W. Correlation of DNA methylation of DNMT3A and TET2 with oral squamous cell carcinoma. Discov Oncol. 2024; 15(1):15. https://doi.org/10.1007/s12672-024-00866-9 DOI: https://doi.org/10.1007/s12672-024-00866-9
Brett JO, Spring LM, Bardia A, Wander SA. ESR1 mutation as an emerging clinical biomarker in metastatic hormone receptor-positive breast cancer. Breast Cancer Res. 2021;23:1–15. https://doi.org/10.1186/s13058-021-01462-3 DOI: https://doi.org/10.1186/s13058-021-01462-3
Bulk E, Yu J, Hascher A, Koschmieder S, Wiewrodt R, Krug U, et al. Mutations of the EPHB6 receptor tyrosine kinase induce a pro-metastatic phenotype in non-small cell lung cancer. PLoS One. 2012;7(12):e44591. https://doi.org/10.1371/journal.pone.0044591 DOI: https://doi.org/10.1371/journal.pone.0044591
Kolegova E, Schegoleva A, Vorobev R, Fedorova I, Kulbakin D, Polyakov A, et al. Genetic features of tongue cancer recurrence in young adults. Bull Exp Biol Med. 2024;177(3):359–62. https://doi.org/10.1007/s10517-024-06189-7 DOI: https://doi.org/10.1007/s10517-024-06189-7
Kumar S, Rangarajan A, Pal D. Somatic mutation analyses of stem-like cells in gingivobuccal oral squamous cell carcinoma reveals DNA damage response genes. Genomics. 2022;114(2):110308. https://doi.org/10.1016/j.ygeno.2022.110308 DOI: https://doi.org/10.1016/j.ygeno.2022.110308
Su S-C, Lin C-W, Liu Y-F, Fan W-L, Chen M-K, Yu C-P, et al. Exome sequencing of oral squamous cell carcinoma reveals molecular subgroups and novel therapeutic opportunities. Theranostics. 2017;7(5):1088. https://doi.org/10.7150/thno.18551 DOI: https://doi.org/10.7150/thno.18551
Zhou X, Cai X, Jing F, Li X, Zhang J, Zhang H, et al. Genomic alterations in oral multiple primary cancers. Int J Oral Sci. 2024;16(1):13. https://doi.org/10.1038/s41368-023-00265-w DOI: https://doi.org/10.1038/s41368-023-00265-w
Gawande M, Chaudhary M, Sharma P, Hande A, Patil S, Sonone A. Expression of p53 at invasive front of oral squamous cell carcinoma and negative histopathological surgical margins to establish correlation at 3-year survival. J Oral Maxillofac Pathol. 2020;24(3):582. https://doi.org/10.4103/jomfp.jomfp_106_20 DOI: https://doi.org/10.4103/jomfp.JOMFP_106_20
Giri R, Hota SK, Sahu SK, Senapati U. Expression of TP53 in oral squamous cell carcinoma and its correlation with adverse histopathological features. J Cancer Res Ther. 2023;19(2):278–82. https://doi.org/10.4103/jcrt.jcrt_1963_21 DOI: https://doi.org/10.4103/jcrt.jcrt_1963_21
Hyodo T, Kuribayashi N, Fukumoto C, Komiyama Y, Shiraishi R, Kamimura R, et al. The mutational spectrum in whole exon of p53 in oral squamous cell carcinoma and its clinical implications. Sci Rep. 2022;12(1):21695. https://doi.org/10.1038/s41598-022-25744-8 DOI: https://doi.org/10.1038/s41598-022-25744-8
Shyamsundar V, Thangaraj SV, Krishnamurthy A, Vimal S, Kesavan P, Babu A, et al. Exome sequencing with validations and expression of p16/cdkn2a shows no association with HPV in oral cancers. Asian Pac J Cancer Prev. 2022;23(1):191. https://doi.org/10.31557/apjcp.2022.23.1.191 DOI: https://doi.org/10.31557/APJCP.2022.23.1.191
Agarwal A, Kamboj M, Shreedhar B. Expression of p16 in oral leukoplakia and oral squamous cell carcinoma and correlation of its expression with individual atypical features. J Oral Biol Craniofac Res. 2019;9(2):156–60. https://doi.org/10.1016/j.jobcr.2019.03.002 DOI: https://doi.org/10.1016/j.jobcr.2019.03.002
Lim AM, Do H, Young RJ, Wong SQ, Angel C, Collins M, et al. Differential mechanisms of CDKN2A (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome. Int J Cancer. 2014;135(4):887–95. https://doi.org/10.1002/ijc.28727 DOI: https://doi.org/10.1002/ijc.28727
Anwar N, Chundriger Q, Awan S, Moatter T, Ali TS, Abdul Rasheed M, et al. Prevalence of high-risk human papillomavirus in oral squamous cell carcinoma with or without chewing habits. PLoS One. 2024;19(5):e0300354. https://doi.org/10.1371/journal.pone.0300354 DOI: https://doi.org/10.1371/journal.pone.0300354
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