High prevalence of EGFR R479K (rs2227983) polymorphism in Indian Head and Neck Cancer Patients: Association with Unfavourable Clinical Outcome

High prevalence of EGFR R479K (rs2227983) polymorphism in Indian Head and Neck Cancer Patients: Association with Unfavourable Clinical Outcome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High prevalence of EGFR R479K (rs2227983) polymorphism in Indian Head and Neck Cancer Patients: Association with Unfavourable Clinical Outcome Arjita Ghosh, Anbalagan Moorthy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5924397/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Epidermal growth factor receptor (EGFR) gene, is one of the most altered genes reported across various cancers. Polymorphism exists in the EGFR gene, and R497K (rs2227983) is a common single nucleotide polymorphism (SNP) of the gene that affects the protein's function. The current study was done to screen for EGFR R497K polymorphism in Indian patients with Head and Neck squamous cell carcinoma (HNSCC) and study the effect of this polymorphism on disease prognosis. In HNSCC patients (n = 50), genomic DNA was extracted, and polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) technique was applied for screening. In the cohort, 84% of patients carried the EGFR R497K variant in either homozygous (6%) or heterozygous (78%) condition and only 16% were wild type. We re-confirmed our PCR-RFLP data with Sanger sequencing. Kaplan-Meier statistical analysis was used to correlate the SNPs in patients to survival factors. Analysis revealed a decreased overall survival (OS) and progression-free survival (PFS) in the patients carrying the R497K polymorphism compared to the wild-type patients. This study concludes that the prevalence of R497K of the EGFR gene is high in Indian HNSCC patients and it is associated with poor prognosis. EGFR gene Polymerase Chain Reaction coupled with Restriction Fragment Length Polymorphism (PCR-RFLP) Single Nucleotide Polymorphism (SNP) R497K Head and Neck Squamous Cell Carcinoma (HNSCC) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Head and Neck Squamous cell carcinoma is one of the most prevalent cancers in the World. It stands sixth in the list of most commonly occurring cancers. Each year about 600,000 cases get reported, out of which, about 355,000 are reported to die mostly due to the development of resistance to therapeutic agents [ 1 ]. The sites for HNSCC occurrence are the larynx, pharynx, oral cavity, paranasal sinuses, head and neck, etc. In India, HNSCC accounts for one-third of all reported cancer cases [ 2 ]. Mostly men are affected by this cancer compared to females due to tobacco usage both by chewing and smoking. Alcohol consumption, betel nut chewing, Epstein Barr virus, etc are the other minor causes of HNSCC [ 3 ]. The epidermal growth factor receptor (EGFR) belongs to the family of receptor tyrosine kinases which is activated by its ligand, epidermal-like growth factor (EGF). Ligand–receptor interaction leads to the activation of several signalling pathways associated with cell proliferation and survival such as PI3K/Akt/mTOR, JAK/STAT, and MAPK [ 4 ] Deregulation of EGFR pathway utilizing overexpression, mutation, and receptor variants all three are implicated in various types of cancer; including HNSCC [ 5 ]. The role of EGFR in cancer is evident by the fact that expression levels and mutations in the receptor are considered major prognostic marker in cancer patients [ 6 ] In HNSCC patients, 80–90% of cases show either overexpression of the receptor or some kind of mutation in their cohort, which is reported to impact the overall survival and progression-free survival of the patients usually towards poor prognosis [ 7 ]. Across populations, polymorphisms occur in both coding and non-coding sequences of the genome which influences the cause and response to treatment in cancer patients. Some of the major forms of polymorphisms reported in EGFR are − 191C/A, -216G/T, and R497K. The non-coding polymorphisms − 191 C/A and − 216 G/T are located in the promoter region of the EGFR gene and contribute to the EGFR protein levels by regulating the transcription of the gene. R497K (rs2227983) is a variant form of the gene where arginine is substituted with lysine amino acid at codon 497 of the gene [ 8 , 9 ]. This variant form of EGFR is reported to affect the cause, progression, and response to treatment of various cancers. [ 10 ]. Cancer patients with EGFR R497K variant are reported to have poor prognostic factors. Studies have shown R497K functions in growth stimulation, ligand binding; and also, activation of tyrosine kinase leading to activation of proto-oncogenes like C-Fos, C-Myc, and C-Jun [ 11 ]. In the absence of any information on the distribution of EGFR R497K variant in the Indian HNSCC population, this work was carried out to study the distribution of the gene variant in the Indian HNSCC cohort and compare their survival functions by calculating overall survival and progression-free survival. Materials and Methods Patients and Specimens: Tumour samples were obtained from 50 HNSCC patients for this study. Patient recruitment was conducted at Apollo Hospital, Chennai where due to logistical challenges, and time constraints, only 50 patient samples were allowed to be collected. The tumour samples after resection were snap-frozen and transported. Informed consent forms were obtained from all the patients and the institute’s human ethical committee approved this study (IEC/IRB No: IECH/2013/Dec18‑006). Patients were staged following the standard TNM staging system. Specimens were majorly from the oral cavity and oropharynx with major sub-sites, tongue, and buccal mucosa. Patients with HNSCC had records with/without tobacco usage for duration 10 years. As mentioned extensively in Table 1 . The samples were obtained from Indian patients from different parts of the country like West Bengal, Manipur, Andhra Pradesh, Tamil Nadu, and Pondicherry. This study was done in accordance with the ethical standards laid down by the 1964 Declaration of Helsinki and its later amendments. A smaller sample size can cause statistical limitations to this study, however rigorous inclusion, exclusion criteria, and data analysis were employed to ensure the validity of our study. Exclusion and Inclusion Criteria: The criteria for inclusion observed for this study were as follows: 1) patients with HNC of non-nasopharyngeal origin such as cancers of the laryngeal, oropharyngeal and oral cavity; 2) patient’s biopsy with squamous cell cancer; 3) patients who are undertaking surgery or radical radiation therapy (accompanying chemoradiation therapy); 4) patients from stages I-IV A with potentially curable disease; and 5) Patient who consented to this study and also its follow-up. The conditions for exclusion for this study were observed as follows: 1) patients diagnosed with nasopharyngeal or thyroid cancer; 2) patients carrying metastatic diseases in any visceral organs like liver, lungs or bones; 3) patients having a prior history of disease or have previously undergone radiation or chemotherapy; 4) Patients with expected survival less than six months and having poor performance status (KPS < 70); and iv) Patients carrying Li Fraumeni syndrome. Also, there was no age cut-off. Table 1 Patient Demography All Patients Criteria Total n = 50 R497K Heterozygous Variant (Arg/Lys) n = 39 R497K Homozygous Variant (Lys/Lys) n = 3 Wild Type n = 8 45 years 28 (56%) 23 (59%) 3 (100%) 2 (25%) Gender Male 40 (80%) 33 (84.7%) 3 (100%) 4 (50%) Female 10 (20%) 6 (15.3%) 0 4 (50%) Diagnosis Oral cavity 35 (70%) 29 (74.3%) 2 (66.7%) 4 (50%) Oropharynx 5 (10%) 3 (7.7%) 0 2 (25%) Hypopharynx 4 (8%) 4 (10.25%) 0 0 Larynx 2 (4%) 0 1 (33.3%) 1 (12.5%) Maxilla 1 (2%) 1 (2.57%) 0 0 Others 3 (6%) 2 (5.18%) 0 1 (12.5%) Sub-site Tongue 12 (24%) 10 (25.6%) 2 (66.7%) 0 Buccal mucosa 16 (32%) 13 (33%) 0 3 (37.5%) Gingivo-buccal sulcus 6 (12%) 5 (12.8%) 1 (33.3%) 0 Hard palate 2 (4%) 2 (5.1%) 0 0 Base of tongue 3 (6%) 2 (5.1%) 0 1 (12.5%) Pyriform fossa 3 (6%) 3 (8.3%) 0 0 Tonsil 1 (2%) 0 0 1 (12.5%) Vocal cord 1 (2%) 1 (2.5%) 0 0 Maxilla 2 (4%) 1 (2.5%) 0 1 (12.5%) Others 4 (8%) 2 (5.1%) 0 2 (25%) Stage Stage I 13 (26%) 10 (25.6%) 0 3 (37.5%) Stage II 4 (8%) 2 (5.1%) 0 2 (25%) Stage III 10 (20%) 10 (25.6%) 0 0 Stage IV a 16 (32%) 12 (30.3%) 1 (33.3%) 3 (37.5%) Stage IV b 5 (10%) 3(8.3%) 2 (66.7%) 0 Stage IV c 2 (4%) 2 (5.1%) 0 0 Grade Grade I 16 (32%) 10 (25.6%) 0 6 (75%) Grade II 22 (44%) 19 (48.8%) 1 (33.3%) 2 (25%) Grade III 12 (24%) 10 (25.6%) 2 (66.7%) 0 Symptoms Ulcer 37 (74%) 31 (79.1%) 3 (100%) 3 (37.5%) Bleeding 4 (8%) 2 (5.1%) 0 2 (25%) Cheek swelling 3 (6%) 1 (2.5%) 0 2 (25%) Swallowing difficulty 3 (6%) 3 (8.3%) 0 0 Voice change 2 (4%) 1 (2.5%) 0 1 (12.5%) Foreign body sensation 1 (2%) 1 (2.5%) 0 0 Symptom duration 6 months 11 (22%) 7 (18%) 3 (100%) 1 (12.5%) Tobacco Usage Yes 33 (66%) 28 (71.8%) 2 (66.7%) 3 (37.5%) No 17 (34%) 11 (28.2%) 1 (33.3%) 5 (62.5%) Tobacco usage duration No tobacco usage 17 (34%) 11 (28.2%) 1 (33.3%) 5 (62.5%) 10 yrs 6 (12%) 5 (12.8%) 0 1 (12.5%) Treatment Radical Surgery only 10 (20%) 8 (20%) 0 2 (25%) Surgery + Post-OP RT 38 (76%) 30 (77.5%) 3 (100%) 5 (62.5%) Radical RT 2 (4%) 1 (2.5%) 0 1 (12.5%) DNA Extraction: High salt method was applied to extract DNA from tumour samples. The tumour samples were ground with liquid nitrogen in a mortar and pestle. The tissues were then digested in 1ml buffer (0.6% SDS, 100mM EDTA, 6M NaCl, 10mM Tris, and proteinase K 20mg/ml) overnight at 45⁰C and incubated overnight. After the overnight treatment 227uL of 6M NaCl was mixed, and the solution was centrifuged for 10 minutes at 12,000 rpm. The supernatant was transferred and to it, equal amounts of 100% ethanol were added. This solution is again centrifuged for 10 minutes at 12,000 rpm. The supernatant was then removed and the DNA pellet remained. This pellet was washed and then air-dried. Then in 20-100ul of sterile water, the DNA was suspended. The DNA was quantified using a Nano-drop instrument (Thermo-Fisher Scientific). To check the quality of DNA it was also run through agarose gel electrophoresis. The DNA was then visualized and documented in Axygen documentation system. PCR-RFLP: The genomic DNA of 50 patient samples was used as a template to PCR amplify a 155bp DNA region spanning the variant sequence using forward primer of sequence -TGCTGTGACCCACTCTGTCT and reverse primer of sequence -CCAGAAGGTTGCACTTGTCC. The primers were designed specifically to amplify the region encompassing EGFR R497K (rs2227983). This specific sequence of EGFR was retrieved from the NCBI GenBank database (AH006650.2). The tool NCBI- --Primer BLAST was used to design primers for this study. The 50µl PCR mixture consisted of 5µl of 10X buffer, 2µl of dNTP (2.5mM each dNTP), 2µl each of primers (100µM concentration each), 0.35µl of Taq polymerase (3 units/ µl) (Genei, India), 0.5µl of DMSO, 1µl of gDNA (50ng/ µl), and 37.15µl of sterile water. The PCR conditions were 94⁰C of initial denaturation for 5 minutes, 40 cycles of denaturation − 94⁰C for 40 seconds, primer annealing for 60.1⁰C for 1 minute, primer extension of 72⁰C for 1 minute, and then followed by final extension for 72⁰C for 5 minutes. An aliquot of PCR products was separated on a 10% polyacrylamide gel (PAGE) to observe 155 bp DNA bands. The PCR products were then digested by restriction enzyme BstNI (ThermoFisher 10U/ µl) at 37⁰C for two hours. The restriction-digested PCR products were then separated in 12% PAGE gel. The banding pattern of the samples was then recorded and documented in the gel documentation system (Axygen). Sanger Sequence Analysis: To collaborate the PCR-RFLP data Sanger sequencing was performed on random samples. The PCR product of homozygous, heterozygous, and wild-type samples was gel purified via a gel purification kit (Qiagen) and then sent for analysis. Statistical Analysis: Analysis of data was done using SPSS 20 statistical software. Overall survival and progression-free survival rates of patients were checked by using Kaplan- Meier survival analysis. Results Screening of EGFR R497K polymorphism by PCR-RFLP: In the codon 497 of exon 13 of the EGFR gene, due to a polymorphic variation of a single nucleotide, G is turned to A, which causes the amino acid arginine (Arg) to be substituted to lysine (Lys) [ 4 ]. The 155bp PCR product of a specific sequence carrying the site of polymorphism, when digested with BstNI restriction enzyme, cuts the PCR product in two sites, creating three fragments of length- 67, 50, and 38 base pairs. If there is R497K substitution, one of the restriction site gets abolished, and the PCR product produces only two fragments of length- 117, and 38 base pairs. This is a homozygous variant (Lys/Lys). If the variant is of a heterozygous nature (Arg/Lys), 4 DNA fragments are obtained of the length- 117, 67, 50, and 38 base pairs. As seen in Fig. 1 . Of the 50 patient samples that were screened, 42 patients showed the presence of R497K substitution; of which 39 were heterozygous (Arg/Lys) and 3 were homozygous (Lys/Lys). The remaining 8 patients were wild type (Arg/Arg). Sequencing Analysis of Samples: Sanger sequencing results collaborated with our PCR-RFLP data. Forward primer was used to analyse the PCR products. The wild-type sequence was A G G, whereas the whereas the SNP was represented by A A G. The samples given below represent homozygous (Fig. 2 a), heterozygous (Fig. 2 b), and wild-type samples (Fig. 2 c and 2 d). Statistical Analysis: When analysing the Overall survival (OS) by using the Kaplan Meir curve, the following observations were made- the patients who carried a homozygous variant of this gene survived for 30.8 months (SE ± 3.302) with a 95% Confidence Interval (CI) - (24.39–37.36), the heterozygous variant survived for 34.7 months (SE ± 2.152) (95% CI -30.51- 38.94). Patients with wild-type genotypes survived for 41.4 months (SE ± 0.511) (95% CI -39.958–42.374) (p-value = 0.409). The Kaplan-Meier curve for OS is depicted in Fig. 3 . The progression-free survival (PFS) analysis also showed a similar trend to the overall survival. Where patients with the homozygous gene variant survived for 30 months (SE ± 3.952) (95% CI -13.075–47.093), patients with the heterozygous variant survived for 33.5 months (SE ± 2.404) (95% CI = 28.660–38.390). In contrast, patients without the variant survived for 40.4 months (SE ± 1.162) (95% CI 39.49–41.435) (p-value = 0.553). The p-value is not statistically significant as number of incidences is lower. The Kaplan-Meier curve for PFS is depicted in Fig. 4 . Due, to the small sample size and low incidence rate the p-value may not be statistically significant. However, the trend observed shows that the polymorphism has an adverse effect on the clinical outcome of the patients. Discussion Single nucleotide polymorphisms are associated with cause, response to treatment, and progression of various diseases including cancer. In the current study, we screened for R497K (rs2227983) polymorphism of the EGFR gene in the Indian population of HNSCC patients to determine the relationship between the susceptibility to HNSCC and response to treatment of the patients with the variant gene. Out of a total of 50 patients included in the study, 84% of them (42 out of 50) carried the R497K variant, of which 3 (6%) were homozygous (Lys/Lys), and 39 (78%) were heterozygous (Lys/Arg) and rest 8 (16%) were found to be wild type (Arg/Arg). The prevalence of this gene variant in HNSCC patients differs across the geographical location. In Spain, it was found to be 12% homozygous, 35% heterozygous and 53% were wild type [ 11 ] In the Iranian population of oral cancer patients, 62.50% carried heterozygous polymorphism, and the rest (47.5%) were wild type [ 12 ]. In the German population of HNSCC patients out of 45, 17 (37.7%) patients carried the variant either in homozygous or heterozygous condition [ 8 ]. Thus, by comparing these studies, our cohort consisted of the highest percentage (84%) of HNSCC patients carrying the R497K variant. Hence, we can conclude that people with the EGFR R497K variant gene are susceptible to HNSCC, which is supported by a study done by Nagalakshmi et. al. [ 13 ]. To strengthen the hypothesis further, we compared the percentage of the non-cancerous Indian population carrying the variant gene. As per the data available in the database (Indigen public database), this gene variant was present in only 31.6% of the population either in homozygous or heterozygous form. A similar study carried out in patients with oral squamous cell carcinoma (OSCC) also concluded that individuals with the EGFR R497K variant gene are common in the cancer patients compared to that of control individuals, suggesting individuals with EGFR R497K variant gene are related to OSCC susceptibility [ 12 ]. Disease progression and response to treatment are reflected by employing analysing overall survival (OS) and progression-free survival (PFS), hence OS and PFS were calculated for HNSCC patients carrying Arg/Arg, Arg/Lys, and Lys/Lys variants of EGFR R497K gene. Compared to the Arg/Arg variant, the presence of the Lys variant either in homozygous condition or heterozygous condition decreased both OS and PFS. OS for Arg/Arg, Arg/Lys, and Lys/Lys was 41.1, 34.7, and 30.8 months respectively; PFS for Arg/Arg, Arg/Lys, and Lys/Lys were 40.4, 33.5 and 30.0 months respectively. In other cohorts with HNSCC patients also it is reported that even the heterozygous form of the gene variant had significantly less prognosis in patients treated with EGFR inhibitor cetuximab [ 8 ] [ 14 ]A poorer survival rate was also observed in colorectal cancer patients with EGFR R497K variant. [ 15 ], supporting our observation that HNSCC patients with EGFR R497K gene variant have a poor prognosis. Even in recent years the prevalence of this SNP remains poorly investigated, hence this study was conducted to further learn the prevalence of EGFR R497K in the HNSCC population. Limitations: The sample size is small, and low incidence rates have caused challenges in achieving statistically significant results. Therefore, these findings should be considered exploratory and a study of a larger population cohort is needed. The method we use in our study is PCR-RFLP, which provides for rapid and easy way to screen for gene mutations but this technique focuses only on the restriction enzyme recognition sequences. The use of next-generation DNA sequencing technique would have provided global mutations in the whole genome of the patients. Also, the status of mutation in heavily pre-treated or progressive HNSCC couldn’t be checked, since the sampling of this study is completed and more than 70% of patients in the cohort are no more. Hence it was not possible to check and report further molecular changes that have occurred in the tumor during the disease's progression or response to treatment protocol. Conclusion Our study concludes that the SNP R497K (rs2227983) of the EGFR gene has a higher prevalence in the Indian cohort compared to studies done in other cohort populations. When we correlated the variant status to the survival rates of the patients it was found, that homozygous and heterozygous variants have declining rates of OS and PFS compared to wild-type patients. Our findings conclude that the R497K (rs2227983) SNP of the EGFR gene has a negative effect on HNSCC patients. Hence, we suggest that this polymorphism can act as a biomarker for clinical treatment outcomes and disease-related mortality in patients. Declarations Competing Conflict of Interest: The authors report there is no competing conflict of interest to declare. Funding Details: Author Contribution A.M contributed to conceptualization , reviewing, and correcting the article.A.G contributed to writing , performing the experiment and data analysis of the article.All authors contributed equally to the preparation of this article. Acknowledgement The authors acknowledge Dr. Debnarayan Dutta, Apollo Hospital, Chennai for his valuable support for this study. Data Availability The data used in this work can be made available by requesting the authors of this manuscript. References Sathishkumar K, Chaturvedi M, Das P, Stephen S, Mathur P (2022) Cancer incidence estimates for 2022 & projection for 2025: Result from National Cancer Registry Programme, India. Indian J Med Res 156:598–607 Badola A, Mehta P, Mehra S, Sood S (2023) Epidemiology and survival analysis of head and neck cancer: Results from comprehensive care center in North India. Oral Oncol Rep 6:100022 Barsouk A, Aluru JS, Rawla P, Saginala K, Barsouk A, Epidemiology (2023) Risk Factors, and Prevention of Head and Neck Squamous Cell Carcinoma. Med Sci 11:42 Wang WS, Chen PM, Chiou TJ, Liu JH, Lin JK, Lin TC et al (2007) Epidermal growth factor receptor R497K polymorphism is a favorable prognostic factor for patients with colorectal carcinoma. Clin Cancer Res 13:3597–3604 KROHN V, WIEGAND S, WERNER JA (2011) EGFR Codon 497 Polymorphism – Implications for Receptor Sensitivity to Inhibitors in HNSCC Cell Lines. Anticancer Res 31:59–66 Wang Y, Zha L, Liao D, Li X (2014) A meta-analysis on the relations between EGFR R521K polymorphism and risk of cancer. Int J Genomics. ;2014 Nair S, Bonner JA, Bredel M (2022) EGFR Mutations in Head and Neck Squamous Cell Carcinoma. Int J Mol Sci MDPI JAN S-W, LUISE O GERHARDE (2012) Polymorphisms of the Epidermal Growth Factor Receptor (EGFR) and Survival in Patients with Advanced Cancer of the Head and Neck (HNSCC). Anticancer Res 32:421–426 Sharafinski ME, Ferris RL, Ferrone S, Grandis JR (2010) Epidermal growth factor receptor targeted therapy of squamous cell carcinoma of the head and neck. Head Neck. p. 1412–1421 Guo H, Xing Y, Mu A, Li X, Li T, Bian X et al (2016) Correlations between EGFR gene polymorphisms and pleural metastasis of lung adenocarcinoma. Onco Targets Ther 9:5257–5270 Bandrés E, Barricarte R, Cantero C, Honorato B, Malumbres R, Zárate R et al (2007) Epidermal growth factor receptor (EGFR) polymorphisms and survival in head and neck cancer patients. Oral Oncol 43:713–719 Saravani S, Parsamanesh N, Miri-Moghaddam E (2020) Role of EGFR gene polymorphisms in oral squamous cell carcinoma patients of Southeast Iran: A case-control study. Casp J Intern Med 11:391–397 Nagalakshmi K, Jamil K, Pingali U, Reddy MV, Attili SSV (2014) Epidermal growth factor receptor (EGFR) mutations as biomarker for head and neck squamous cell carcinomas (HNSCC). Biomarkers 19:198–206 Carcereny E, Castellvi-Bel S, Alonso V, Garcia-Albeniz X, Muñoz J, Gallego R et al (2008) EGFR polymorphisms as predictors of clinical outcome in patients with advanced colorectal cancer (ACRC) treated with cetuximab and panitumumab. Journal of Clinical Oncology [Internet]. [cited 2025 Jan 19];26:4124–4124. Available from: https://ascopubs.org/doi/10.1200/jco.2008.26.15_suppl.4124 Martinelli M, Ugolini G, Scapoli L, Rivetti S, Lauriola M, Mattei G et al (2010) The EGFR R521K polymorphism influences the risk to develop colorectal cancer. Cancer Biomarkers 8:61–65 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5924397","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":409503244,"identity":"191bc484-ebe7-4049-a28f-93b78c499804","order_by":0,"name":"Arjita Ghosh","email":"","orcid":"","institution":"Vellore Institute of Technology (VIT)","correspondingAuthor":false,"prefix":"","firstName":"Arjita","middleName":"","lastName":"Ghosh","suffix":""},{"id":409503246,"identity":"3365a511-8ef2-4dd7-8bfa-a21775165c98","order_by":1,"name":"Anbalagan Moorthy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3RMQrCMBSA4VcEF1NcM7VXSBHURbxKitCpis5q6aSTzoKXqAiCWyXQLsU5ooNHCAjiIGJLdU11E8w/vSEfLyEAKtVPhgDO+aSdgeRTWEhoPpUIEPIdKWN4r5FVXc52go7GZgMqzhD171CdhhrrSwg+7TuYRrG19fXNEaUXwwkFtpCt4S7BtBxpQahvDovsLRyAIYkwuVu70UfUzsggI2YRIdytY3sysjMCIiWkiFi85zTtedgJmL7GgtSQldi+lBi8y7i4eq0gnq0EvRuGETN2kZFXLP3IfEoPa34xAPA+OaRSqVT/2hPrVk8lC+wsmAAAAABJRU5ErkJggg==","orcid":"","institution":"Vellore Institute of Technology (VIT)","correspondingAuthor":true,"prefix":"","firstName":"Anbalagan","middleName":"","lastName":"Moorthy","suffix":""}],"badges":[],"createdAt":"2025-01-29 13:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5924397/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5924397/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75408505,"identity":"04fd78c5-d023-43b2-9b4d-78745b4e5518","added_by":"auto","created_at":"2025-02-04 08:59:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282140,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of 50 patient samples using PCR-RFLP method. A 50 bp marker was used as a reference, and the bands were separated in 12% PAGE gel. The Homozygous variant is represented by two bands (117 and 38 bp), as seen in samples -10, 12, and 25. The heterozygous variant is represented by 4 bands ( 117, 67, 50, 38 bp) as seen in samples – 1, 2, 3, 4, 5, 6, 9, 11, 13, 14, 18, 19, 21, 23, 24, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. The wild type is represented by 3 bands (67, 50, 38 bp), as seen in samples 7, 8, 15, 16, 17, 20, 22, 37.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5924397/v1/aa3fadce0c1773669b71386f.jpeg"},{"id":75408508,"identity":"22f1df0a-5271-47ba-85eb-abb1b685eaef","added_by":"auto","created_at":"2025-02-04 08:59:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":151424,"visible":true,"origin":"","legend":"\u003cp\u003eSanger sequencing of the patient samples. (a) The homozygous variant represented by sequence AAG, the conversion of G to A, as seen in the figure. The nucleotides are represented by the following colours C- blue, T- red, A- green, and G- black. (b) Heterozygous variant. The overlapping peak of A/G is seen (overlapping of green and black peaks). (c and, d) Wild type variant (AGG).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5924397/v1/dda1a532c86774f28538be8e.jpeg"},{"id":75408509,"identity":"b7877e38-d175-42f2-8ec1-b2302c932b76","added_by":"auto","created_at":"2025-02-04 08:59:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16466,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier curve for overall survival analysis of 50 HNSCC patients.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5924397/v1/895869e6265cc64a5e66fb70.png"},{"id":75410274,"identity":"0406301b-5790-4561-816d-56c541f9a89c","added_by":"auto","created_at":"2025-02-04 09:07:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15396,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier curve for progression-free survival analysis of 50 HNSCC patients.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5924397/v1/51553d7150bc88e5fc184a40.png"},{"id":75643360,"identity":"ad921550-10f3-4329-97c3-bcca9aa4f0b6","added_by":"auto","created_at":"2025-02-06 16:01:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1288196,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5924397/v1/968a16fc-1721-441f-b285-c7a62e85f689.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High prevalence of EGFR R479K (rs2227983) polymorphism in Indian Head and Neck Cancer Patients: Association with Unfavourable Clinical Outcome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and Neck Squamous cell carcinoma is one of the most prevalent cancers in the World. It stands sixth in the list of most commonly occurring cancers. Each year about 600,000 cases get reported, out of which, about 355,000 are reported to die mostly due to the development of resistance to therapeutic agents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The sites for HNSCC occurrence are the larynx, pharynx, oral cavity, paranasal sinuses, head and neck, etc. In India, HNSCC accounts for one-third of all reported cancer cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mostly men are affected by this cancer compared to females due to tobacco usage both by chewing and smoking. Alcohol consumption, betel nut chewing, Epstein Barr virus, etc are the other minor causes of HNSCC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe epidermal growth factor receptor (EGFR) belongs to the family of receptor tyrosine kinases which is activated by its ligand, epidermal-like growth factor (EGF). Ligand\u0026ndash;receptor interaction leads to the activation of several signalling pathways associated with cell proliferation and survival such as PI3K/Akt/mTOR, JAK/STAT, and MAPK [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Deregulation of EGFR pathway utilizing overexpression, mutation, and receptor variants all three are implicated in various types of cancer; including HNSCC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The role of EGFR in cancer is evident by the fact that expression levels and mutations in the receptor are considered major prognostic marker in cancer patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] In HNSCC patients, 80\u0026ndash;90% of cases show either overexpression of the receptor or some kind of mutation in their cohort, which is reported to impact the overall survival and progression-free survival of the patients usually towards poor prognosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcross populations, polymorphisms occur in both coding and non-coding sequences of the genome which influences the cause and response to treatment in cancer patients. Some of the major forms of polymorphisms reported in EGFR are \u0026minus;\u0026thinsp;191C/A, -216G/T, and R497K. The non-coding polymorphisms \u0026minus;\u0026thinsp;191 C/A and \u0026minus;\u0026thinsp;216 G/T are located in the promoter region of the EGFR gene and contribute to the EGFR protein levels by regulating the transcription of the gene. R497K (rs2227983) is a variant form of the gene where arginine is substituted with lysine amino acid at codon 497 of the gene [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This variant form of EGFR is reported to affect the cause, progression, and response to treatment of various cancers. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cancer patients with EGFR R497K variant are reported to have poor prognostic factors. Studies have shown R497K functions in growth stimulation, ligand binding; and also, activation of tyrosine kinase leading to activation of proto-oncogenes like C-Fos, C-Myc, and C-Jun [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the absence of any information on the distribution of EGFR R497K variant in the Indian HNSCC population, this work was carried out to study the distribution of the gene variant in the Indian HNSCC cohort and compare their survival functions by calculating overall survival and progression-free survival.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Specimens:\u003c/h2\u003e \u003cp\u003eTumour samples were obtained from 50 HNSCC patients for this study. Patient recruitment was conducted at Apollo Hospital, Chennai where due to logistical challenges, and time constraints, only 50 patient samples were allowed to be collected. The tumour samples after resection were snap-frozen and transported. Informed consent forms were obtained from all the patients and the institute\u0026rsquo;s human ethical committee approved this study (IEC/IRB No: IECH/2013/Dec18‑006). Patients were staged following the standard TNM staging system. Specimens were majorly from the oral cavity and oropharynx with major sub-sites, tongue, and buccal mucosa. Patients with HNSCC had records with/without tobacco usage for duration\u0026thinsp;\u0026lt;\u0026thinsp;5 to \u0026gt;\u0026thinsp;10 years. As mentioned extensively in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The samples were obtained from Indian patients from different parts of the country like West Bengal, Manipur, Andhra Pradesh, Tamil Nadu, and Pondicherry. This study was done in accordance with the ethical standards laid down by the 1964 Declaration of Helsinki and its later amendments. A smaller sample size can cause statistical limitations to this study, however rigorous inclusion, exclusion criteria, and data analysis were employed to ensure the validity of our study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExclusion and Inclusion Criteria:\u003c/h3\u003e\n\u003cp\u003eThe criteria for inclusion observed for this study were as follows: 1) patients with HNC of non-nasopharyngeal origin such as cancers of the laryngeal, oropharyngeal and oral cavity; 2) patient\u0026rsquo;s biopsy with squamous cell cancer; 3) patients who are undertaking surgery or radical radiation therapy (accompanying chemoradiation therapy); 4) patients from stages I-IV A with potentially curable disease; and 5) Patient who consented to this study and also its follow-up.\u003c/p\u003e \u003cp\u003eThe conditions for exclusion for this study were observed as follows: 1) patients diagnosed with nasopharyngeal or thyroid cancer; 2) patients carrying metastatic diseases in any visceral organs like liver, lungs or bones; 3) patients having a prior history of disease or have previously undergone radiation or chemotherapy; 4) Patients with expected survival less than six months and having poor performance status (KPS\u0026thinsp;\u0026lt;\u0026thinsp;70); and iv) Patients carrying Li Fraumeni syndrome. Also, there was no age cut-off.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient Demography\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAll Patients Criteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal n\u0026thinsp;=\u0026thinsp;50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR497K Heterozygous Variant (Arg/Lys)\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;39\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR497K Homozygous Variant (Lys/Lys)\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWild Type\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;8\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;45 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (44%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (75%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;45 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 (56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23 (59%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 (80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (84.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (15.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDiagnosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOral cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 (70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29 (74.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOropharynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (7.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypopharynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (10.25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaxilla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.57%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSub-site\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTongue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBuccal mucosa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGingivo-buccal sulcus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHard palate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBase of tongue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyriform fossa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (8.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTonsil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVocal cord\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaxilla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage IV a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (30.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage IV b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3(8.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage IV c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrade\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (75%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (44%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19 (48.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSymptoms\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUlcer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37 (74%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31 (79.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBleeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCheek swelling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSwallowing difficulty\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (8.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVoice change\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForeign body sensation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSymptom duration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (30.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u0026ndash;6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (46%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (51.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTobacco Usage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33 (66%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28 (71.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (28.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTobacco usage duration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo tobacco usage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (28.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026ndash;10 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (39%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadical Surgery only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurgery\u0026thinsp;+\u0026thinsp;Post-OP RT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38 (76%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (77.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadical RT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eDNA Extraction:\u003c/h3\u003e\n\u003cp\u003eHigh salt method was applied to extract DNA from tumour samples. The tumour samples were ground with liquid nitrogen in a mortar and pestle. The tissues were then digested in 1ml buffer (0.6% SDS, 100mM EDTA, 6M NaCl, 10mM Tris, and proteinase K 20mg/ml) overnight at 45⁰C and incubated overnight. After the overnight treatment 227uL of 6M NaCl was mixed, and the solution was centrifuged for 10 minutes at 12,000 rpm. The supernatant was transferred and to it, equal amounts of 100% ethanol were added. This solution is again centrifuged for 10 minutes at 12,000 rpm. The supernatant was then removed and the DNA pellet remained. This pellet was washed and then air-dried. Then in 20-100ul of sterile water, the DNA was suspended. The DNA was quantified using a Nano-drop instrument (Thermo-Fisher Scientific). To check the quality of DNA it was also run through agarose gel electrophoresis. The DNA was then visualized and documented in Axygen documentation system.\u003c/p\u003e\n\u003ch3\u003ePCR-RFLP:\u003c/h3\u003e\n\u003cp\u003eThe genomic DNA of 50 patient samples was used as a template to PCR amplify a 155bp DNA region spanning the variant sequence using forward primer of sequence -TGCTGTGACCCACTCTGTCT and reverse primer of sequence -CCAGAAGGTTGCACTTGTCC. The primers were designed specifically to amplify the region encompassing EGFR R497K (rs2227983). This specific sequence of EGFR was retrieved from the NCBI GenBank database (AH006650.2). The tool NCBI- --Primer BLAST was used to design primers for this study. The 50\u0026micro;l PCR mixture consisted of 5\u0026micro;l of 10X buffer, 2\u0026micro;l of dNTP (2.5mM each dNTP), 2\u0026micro;l each of primers (100\u0026micro;M concentration each), 0.35\u0026micro;l of Taq polymerase (3 units/ \u0026micro;l) (Genei, India), 0.5\u0026micro;l of DMSO, 1\u0026micro;l of gDNA (50ng/ \u0026micro;l), and 37.15\u0026micro;l of sterile water. The PCR conditions were 94⁰C of initial denaturation for 5 minutes, 40 cycles of denaturation \u0026minus;\u0026thinsp;94⁰C for 40 seconds, primer annealing for 60.1⁰C for 1 minute, primer extension of 72⁰C for 1 minute, and then followed by final extension for 72⁰C for 5 minutes. An aliquot of PCR products was separated on a 10% polyacrylamide gel (PAGE) to observe 155 bp DNA bands. The PCR products were then digested by restriction enzyme BstNI (ThermoFisher 10U/ \u0026micro;l) at 37⁰C for two hours. The restriction-digested PCR products were then separated in 12% PAGE gel. The banding pattern of the samples was then recorded and documented in the gel documentation system (Axygen).\u003c/p\u003e\n\u003ch3\u003eSanger Sequence Analysis:\u003c/h3\u003e\n\u003cp\u003eTo collaborate the PCR-RFLP data Sanger sequencing was performed on random samples. The PCR product of homozygous, heterozygous, and wild-type samples was gel purified via a gel purification kit (Qiagen) and then sent for analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eAnalysis of data was done using SPSS 20 statistical software. Overall survival and progression-free survival rates of patients were checked by using Kaplan- Meier survival analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eScreening of EGFR R497K polymorphism by PCR-RFLP:\u003c/h2\u003e \u003cp\u003eIn the codon 497 of exon 13 of the EGFR gene, due to a polymorphic variation of a single nucleotide, G is turned to A, which causes the amino acid arginine (Arg) to be substituted to lysine (Lys) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The 155bp PCR product of a specific sequence carrying the site of polymorphism, when digested with BstNI restriction enzyme, cuts the PCR product in two sites, creating three fragments of length- 67, 50, and 38 base pairs. If there is R497K substitution, one of the restriction site gets abolished, and the PCR product produces only two fragments of length- 117, and 38 base pairs. This is a homozygous variant (Lys/Lys). If the variant is of a heterozygous nature (Arg/Lys), 4 DNA fragments are obtained of the length- 117, 67, 50, and 38 base pairs. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eOf the 50 patient samples that were screened, 42 patients showed the presence of R497K substitution; of which 39 were heterozygous (Arg/Lys) and 3 were homozygous (Lys/Lys). The remaining 8 patients were wild type (Arg/Arg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSequencing Analysis of Samples:\u003c/h2\u003e \u003cp\u003eSanger sequencing results collaborated with our PCR-RFLP data. Forward primer was used to analyse the PCR products. The wild-type sequence was A\u003cb\u003eG\u003c/b\u003eG, whereas the whereas the SNP was represented by A\u003cb\u003eA\u003c/b\u003eG. The samples given below represent homozygous (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), heterozygous (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and wild-type samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eWhen analysing the Overall survival (OS) by using the Kaplan Meir curve, the following observations were made- the patients who carried a homozygous variant of this gene survived for 30.8 months (SE\u0026thinsp;\u0026plusmn;\u0026thinsp;3.302) with a 95% Confidence Interval (CI) - (24.39\u0026ndash;37.36), the heterozygous variant survived for 34.7 months (SE\u0026thinsp;\u0026plusmn;\u0026thinsp;2.152) (95% CI -30.51- 38.94). Patients with wild-type genotypes survived for 41.4 months (SE\u0026thinsp;\u0026plusmn;\u0026thinsp;0.511) (95% CI -39.958\u0026ndash;42.374) (p-value\u0026thinsp;=\u0026thinsp;0.409). The Kaplan-Meier curve for OS is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe progression-free survival (PFS) analysis also showed a similar trend to the overall survival. Where patients with the homozygous gene variant survived for 30 months (SE\u0026thinsp;\u0026plusmn;\u0026thinsp;3.952) (95% CI -13.075\u0026ndash;47.093), patients with the heterozygous variant survived for 33.5 months (SE\u0026thinsp;\u0026plusmn;\u0026thinsp;2.404) (95% CI\u0026thinsp;=\u0026thinsp;28.660\u0026ndash;38.390). In contrast, patients without the variant survived for 40.4 months (SE\u0026thinsp;\u0026plusmn;\u0026thinsp;1.162) (95% CI 39.49\u0026ndash;41.435) (p-value\u0026thinsp;=\u0026thinsp;0.553). The p-value is not statistically significant as number of incidences is lower. The Kaplan-Meier curve for PFS is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eDue, to the small sample size and low incidence rate the p-value may not be statistically significant. However, the trend observed shows that the polymorphism has an adverse effect on the clinical outcome of the patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSingle nucleotide polymorphisms are associated with cause, response to treatment, and progression of various diseases including cancer. In the current study, we screened for R497K (rs2227983) polymorphism of the EGFR gene in the Indian population of HNSCC patients to determine the relationship between the susceptibility to HNSCC and response to treatment of the patients with the variant gene. Out of a total of 50 patients included in the study, 84% of them (42 out of 50) carried the R497K variant, of which 3 (6%) were homozygous (Lys/Lys), and 39 (78%) were heterozygous (Lys/Arg) and rest 8 (16%) were found to be wild type (Arg/Arg). The prevalence of this gene variant in HNSCC patients differs across the geographical location. In Spain, it was found to be 12% homozygous, 35% heterozygous and 53% were wild type [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] In the Iranian population of oral cancer patients, 62.50% carried heterozygous polymorphism, and the rest (47.5%) were wild type [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the German population of HNSCC patients out of 45, 17 (37.7%) patients carried the variant either in homozygous or heterozygous condition [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, by comparing these studies, our cohort consisted of the highest percentage (84%) of HNSCC patients carrying the R497K variant. Hence, we can conclude that people with the EGFR R497K variant gene are susceptible to HNSCC, which is supported by a study done by Nagalakshmi et. al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To strengthen the hypothesis further, we compared the percentage of the non-cancerous Indian population carrying the variant gene. As per the data available in the database (Indigen public database), this gene variant was present in only 31.6% of the population either in homozygous or heterozygous form. A similar study carried out in patients with oral squamous cell carcinoma (OSCC) also concluded that individuals with the EGFR R497K variant gene are common in the cancer patients compared to that of control individuals, suggesting individuals with EGFR R497K variant gene are related to OSCC susceptibility [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDisease progression and response to treatment are reflected by employing analysing overall survival (OS) and progression-free survival (PFS), hence OS and PFS were calculated for HNSCC patients carrying Arg/Arg, Arg/Lys, and Lys/Lys variants of EGFR R497K gene. Compared to the Arg/Arg variant, the presence of the Lys variant either in homozygous condition or heterozygous condition decreased both OS and PFS. OS for Arg/Arg, Arg/Lys, and Lys/Lys was 41.1, 34.7, and 30.8 months respectively; PFS for Arg/Arg, Arg/Lys, and Lys/Lys were 40.4, 33.5 and 30.0 months respectively. In other cohorts with HNSCC patients also it is reported that even the heterozygous form of the gene variant had significantly less prognosis in patients treated with EGFR inhibitor cetuximab [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]A poorer survival rate was also observed in colorectal cancer patients with EGFR R497K variant. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], supporting our observation that HNSCC patients with EGFR R497K gene variant have a poor prognosis.\u003c/p\u003e \u003cp\u003eEven in recent years the prevalence of this SNP remains poorly investigated, hence this study was conducted to further learn the prevalence of EGFR R497K in the HNSCC population.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLimitations:\u003c/h2\u003e \u003cp\u003eThe sample size is small, and low incidence rates have caused challenges in achieving statistically significant results. Therefore, these findings should be considered exploratory and a study of a larger population cohort is needed. The method we use in our study is PCR-RFLP, which provides for rapid and easy way to screen for gene mutations but this technique focuses only on the restriction enzyme recognition sequences. The use of next-generation DNA sequencing technique would have provided global mutations in the whole genome of the patients. Also, the status of mutation in heavily pre-treated or progressive HNSCC couldn\u0026rsquo;t be checked, since the sampling of this study is completed and more than 70% of patients in the cohort are no more. Hence it was not possible to check and report further molecular changes that have occurred in the tumor during the disease's progression or response to treatment protocol.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study concludes that the SNP R497K (rs2227983) of the EGFR gene has a higher prevalence in the Indian cohort compared to studies done in other cohort populations. When we correlated the variant status to the survival rates of the patients it was found, that homozygous and heterozygous variants have declining rates of OS and PFS compared to wild-type patients. Our findings conclude that the R497K (rs2227983) SNP of the EGFR gene has a negative effect on HNSCC patients. Hence, we suggest that this polymorphism can act as a biomarker for clinical treatment outcomes and disease-related mortality in patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Conflict of Interest:\u003c/h2\u003e \u003cp\u003eThe authors report there is no competing conflict of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eDetails:\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.M contributed to conceptualization , reviewing, and correcting the article.A.G contributed to writing , performing the experiment and data analysis of the article.All authors contributed equally to the preparation of this article.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge Dr. Debnarayan Dutta, Apollo Hospital, Chennai for his valuable support for this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data used in this work can be made available by requesting the authors of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSathishkumar K, Chaturvedi M, Das P, Stephen S, Mathur P (2022) Cancer incidence estimates for 2022 \u0026amp; projection for 2025: Result from National Cancer Registry Programme, India. Indian J Med Res 156:598\u0026ndash;607\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadola A, Mehta P, Mehra S, Sood S (2023) Epidemiology and survival analysis of head and neck cancer: Results from comprehensive care center in North India. Oral Oncol Rep 6:100022\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarsouk A, Aluru JS, Rawla P, Saginala K, Barsouk A, Epidemiology (2023) Risk Factors, and Prevention of Head and Neck Squamous Cell Carcinoma. Med Sci 11:42\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang WS, Chen PM, Chiou TJ, Liu JH, Lin JK, Lin TC et al (2007) Epidermal growth factor receptor R497K polymorphism is a favorable prognostic factor for patients with colorectal carcinoma. Clin Cancer Res 13:3597\u0026ndash;3604\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKROHN V, WIEGAND S, WERNER JA (2011) EGFR Codon 497 Polymorphism \u0026ndash; Implications for Receptor Sensitivity to Inhibitors in HNSCC Cell Lines. Anticancer Res 31:59\u0026ndash;66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zha L, Liao D, Li X (2014) A meta-analysis on the relations between EGFR R521K polymorphism and risk of cancer. Int J Genomics. ;2014\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair S, Bonner JA, Bredel M (2022) EGFR Mutations in Head and Neck Squamous Cell Carcinoma. Int J Mol Sci MDPI\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJAN S-W, LUISE O GERHARDE (2012) Polymorphisms of the Epidermal Growth Factor Receptor (EGFR) and Survival in Patients with Advanced Cancer of the Head and Neck (HNSCC). Anticancer Res 32:421\u0026ndash;426\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharafinski ME, Ferris RL, Ferrone S, Grandis JR (2010) Epidermal growth factor receptor targeted therapy of squamous cell carcinoma of the head and neck. Head Neck. p. 1412\u0026ndash;1421\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Xing Y, Mu A, Li X, Li T, Bian X et al (2016) Correlations between EGFR gene polymorphisms and pleural metastasis of lung adenocarcinoma. Onco Targets Ther 9:5257\u0026ndash;5270\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBandr\u0026eacute;s E, Barricarte R, Cantero C, Honorato B, Malumbres R, Z\u0026aacute;rate R et al (2007) Epidermal growth factor receptor (EGFR) polymorphisms and survival in head and neck cancer patients. Oral Oncol 43:713\u0026ndash;719\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaravani S, Parsamanesh N, Miri-Moghaddam E (2020) Role of EGFR gene polymorphisms in oral squamous cell carcinoma patients of Southeast Iran: A case-control study. Casp J Intern Med 11:391\u0026ndash;397\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagalakshmi K, Jamil K, Pingali U, Reddy MV, Attili SSV (2014) Epidermal growth factor receptor (EGFR) mutations as biomarker for head and neck squamous cell carcinomas (HNSCC). Biomarkers 19:198\u0026ndash;206\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarcereny E, Castellvi-Bel S, Alonso V, Garcia-Albeniz X, Mu\u0026ntilde;oz J, Gallego R et al (2008) EGFR polymorphisms as predictors of clinical outcome in patients with advanced colorectal cancer (ACRC) treated with cetuximab and panitumumab. Journal of Clinical Oncology [Internet]. [cited 2025 Jan 19];26:4124\u0026ndash;4124. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ascopubs.org/doi/10.1200/jco.2008.26.15_suppl.4124\u003c/span\u003e\u003cspan address=\"https://ascopubs.doi/10.1200/jco.2008.26.15_suppl.4124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinelli M, Ugolini G, Scapoli L, Rivetti S, Lauriola M, Mattei G et al (2010) The EGFR R521K polymorphism influences the risk to develop colorectal cancer. Cancer Biomarkers 8:61\u0026ndash;65\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"EGFR gene, Polymerase Chain Reaction coupled with Restriction Fragment Length Polymorphism (PCR-RFLP), Single Nucleotide Polymorphism (SNP), R497K, Head and Neck Squamous Cell Carcinoma (HNSCC)","lastPublishedDoi":"10.21203/rs.3.rs-5924397/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5924397/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Epidermal growth factor receptor (EGFR) gene, is one of the most altered genes reported across various cancers. Polymorphism exists in the EGFR gene, and R497K (rs2227983) is a common single nucleotide polymorphism (SNP) of the gene that affects the protein's function. The current study was done to screen for EGFR R497K polymorphism in Indian patients with Head and Neck squamous cell carcinoma (HNSCC) and study the effect of this polymorphism on disease prognosis. In HNSCC patients (n\u0026thinsp;=\u0026thinsp;50), genomic DNA was extracted, and polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) technique was applied for screening. In the cohort, 84% of patients carried the EGFR R497K variant in either homozygous (6%) or heterozygous (78%) condition and only 16% were wild type. We re-confirmed our PCR-RFLP data with Sanger sequencing. Kaplan-Meier statistical analysis was used to correlate the SNPs in patients to survival factors. Analysis revealed a decreased overall survival (OS) and progression-free survival (PFS) in the patients carrying the R497K polymorphism compared to the wild-type patients. This study concludes that the prevalence of R497K of the EGFR gene is high in Indian HNSCC patients and it is associated with poor prognosis.\u003c/p\u003e","manuscriptTitle":"High prevalence of EGFR R479K (rs2227983) polymorphism in Indian Head and Neck Cancer Patients: Association with Unfavourable Clinical Outcome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 08:59:42","doi":"10.21203/rs.3.rs-5924397/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e7f9d9b-fe8c-4edf-84f8-6109f443977a","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-06T15:53:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-04 08:59:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5924397","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5924397","identity":"rs-5924397","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}