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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 538-543
Impact of ERCC1 gene polymorphisms on response to cisplatin based therapy in oral squamous cell carcinoma (OSCC) patients


1 Department of Oral Medicine and Radiology, Kamineni Institute of Dental Sciences, Narketpally, Telangana, Ph.D Scholar (Oral Medicine and Radiology), Saveetha University, Chennai, Tamil Nadu, India
2 Department of Oral Medicine and Radiology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India
3 Department of Pathology and Microbiology, National Institute of Nutrition, Hyderabad, Telangana, India
4 Department of Radiation Oncology, MNJ Institute of Oncology and Regional Cancer Centre, Hyderabad, Telangana, India
5 Department of Research and Development, Saveetha University, Chennai, Tamil Nadu, India
6 Department of Prosthodontics, Kamineni Institute of Dental Sciences, Narketpally, Telangana, India

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Date of Submission07-Dec-2019
Date of Decision26-Jan-2020
Date of Acceptance26-Jan-2020
Date of Web Publication28-Oct-2020
 

   Abstract 


Background and Objectives: Cisplatin is one of the major drugs that used in the treatment of oral cancer.Excision repair cross-complementation group 1 (ERCC1) is a key DNA repair gene in the nucleotide excision repair pathway which is activated in the repair of intra- and interstrand DNA crosslink caused by platinum-based treatment. The aim of this study was to investigate the association between polymorphisms in ERCC1 (C118T & C8092A) genes and the response to cisplatin-based chemotherapy.
Methods: ERCC1polymorphisms (C118T & C8092A) were studied using PCR-RFLP method from 150 OSCC patients as cases as well as 150 normal tissues from the same patients were collected as controls for this study. Results: Frequencies of ERCC1 C118C, C118T and T118T genotypes were 60%, 28% and 12% in OSCC patients and 78%, 19% and 3% in the controls, respectively. The C118T & T118T genotype had a 1.69 and 4.97 -folds increased risk for OSCC. Frequencies of ERCC1 C8092C, C8092A and A8092A were 78%, 18% and 4% in the OSCC patients and 89%, 10%, amd 1% in the controls, respectively. The C8092A genotype showed a 1.97-fold increased risk for OSCC.
Interpretation & Conclusions: In conclusion, this study highlights the DNA repair gene polymorphisms that might play a role in mediating susceptibility to oral squamous cell carcinoma and cisplatin therapy. Our data suggest that the ERCC1 C118T, T118T and ERCC1 C8092A genotypes are genetic risk factors for Oral squamous cell carcinoma and ERCC1 118 C/T and C8092A polymorphisms have significant influence on clinical outcome.

Keywords: Cisplatin, ERCC, expression, oral squamous cell carcinoma, PCR-RFLP, polymorphism

How to cite this article:
Avinash Tejasvi M L, Maragathavalli G, Putcha UK, Ramakrishna M, Vijayaraghavan R, Anulekha Avinash C K. Impact of ERCC1 gene polymorphisms on response to cisplatin based therapy in oral squamous cell carcinoma (OSCC) patients. Indian J Pathol Microbiol 2020;63:538-43

How to cite this URL:
Avinash Tejasvi M L, Maragathavalli G, Putcha UK, Ramakrishna M, Vijayaraghavan R, Anulekha Avinash C K. Impact of ERCC1 gene polymorphisms on response to cisplatin based therapy in oral squamous cell carcinoma (OSCC) patients. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Dec 1];63:538-43. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/538/299338





   Introduction Top


Oral cancer ranks as the 11th most prevalent cancer worldwide, and among all oral malignancies, oral squamous cell carcinoma (OSCC) is the most predominant, constituting about 90% of all oral cancers.[1] The deoxyribonucleic acid (DNA) repair genes like Excision repair cross-complementation groups 1 (ERCC1) and 2 (ERCC2) are genes encoding two key enzymes in NER pathway.[2] Single nucleotide polymorphisms (SNPs) in the ERCC1 gene may affect the function of the encoding gene. The ERCC1 gene, on chromosome 19q13.2 - q13.3, encodes a protein of 297 amino acids. To date, there are 36 coding SNPs in ERCC1 have been reported. Two common polymorphisms of the ERCC1 gene, 118 C/T (rs11615) and C8092A (rs3212986), have been reported.[3] The single nucleotide polymorphism (SNP) at codon 118 causes a C > T change that codes for the same amino acid, asparagine, and it has been proposed to impair ERCC1 translation and reduce ERCC1 protein expression in cells, and thereby, effect the response to platinum based chemo-therapy.[3],[4] The C8092A polymorphism, located in position 8092 of the 3'-untranslated region of the gene consisting of a C > A change, may be involved in translational repression of ERCC1 mRNA,[5],[6] or affect ERCC1 mRNA stability resulting in impaired DNA repair capacity and has also been associated with the risk of cancer.[7]

Chemotherapy is the use of anticancer drugs designed to slow or stop the growth of rapidly dividing cancer cells in the body. Among the various chemotherapeutic drugs, cisplatin, carboplatin, 5-fluorouracil (5-FU), paclitaxel, and docetaxel are most commonly used against OSCC. Cisplatin, or cis-diamminedichloroplatinum (II) (CDDP), a high potency anticancer agent was the first platinum-based anticancer drug developed for clinical purposes. Cisplatin-based chemotherapeutic regimens have been the most widely and frequently used adjuvant treatments against OSCC.[8] Differences in chemotherapy response may be related to inter individual genetic polymorphisms in patient's genes since they can change the expression, or the function of enzymes related to the metabolism of the chemotherapeutic drug offered to the patient. Several previous studies have investigated the influences of the ERCC1 gene on cisplatin response in many cancers including non-small cell lung cancer, esophageal cancer, and ovarian cancer.[3],[4] However, for osteosarcoma, the results have been inconsistent. The aim of this study was to investigate the association between polymorphisms in ERCC1 (C118T and C8092A) genes and the response to cisplatin-based chemotherapy.


   Subjects and Methods Top


Study population

Oral squamous cell carcinoma (OSCC) patients were assessed on the basis of clinical and pathological examinations. This is a hospital-based split mouth study was conducted. All incidents of OSCC cases were newly diagnosed during the study period Ethics Committee approved the study for the benefit of humans in general. The procedures followed were in accordance with the ethical standards of responsible committee of the Institute/Hospital, to participate in a face-to-face interview using a structured questionnaire. A follow-up period for 6 months after last cycle chemotherapy was done.

Inclusion and exclusion criteria

We included all patients with OSCC who were treated with cisplatin-based chemotherapy. Patients of confirmed OSCC who give their consent were included. All patients who refuse to give consent were excluded.

Sampling for the study

Based on the above criteria, tissue samples from 150 OSCC patients as cases as well as 150 normal tissues from the same patients were collected as controls for this study. Sampling was done from Cancer Hospital. Senior pathologists confirmed all diagnoses. We interviewed and collected the data about the patient's demographic factors. We collected the information on age, smoking, and previous cancer diagnoses. Participants were also asked about their family history of cancer, and the clinical information for these cases was obtained from medical records like tumor size, stage, and whether they were receiving chemotherapy. A total of 150 patients were treated with Cisplatin -based chemotherapy. The response to treatment was evaluated according to the World Health Organization criteria.

Collection of tissue samples

Incisional biopsy was done from the representative oral cancer tissue and sent to histopathology lab for diagnosis. All the samples were diagnosed mainly as oral squamous cell carcinoma. Healthy tissue samples were also collected from same patients, which were used as controls.

Genotyping

Tissue samples collected from the patients prior to their treatment in EDTA-anticoagulant tubes. Genomic DNA was extracted from the blood, using the QIAamp DNA MAX Kit (Qiagen, Hilden, Germany). The ERCC1 rs11615 and rs3212986 polymorphisms were assessed using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. The following forward and reverse primers were used in this assay: rs11615: 5'-GCTGTGCACTCCAGTGGTTC-3' and 5'-GTGGAGCTTGTTGAGGAGGT-3'; rs3212986: 5'-CAGAGACAGTGCCCCAAGAG-3' and 5'-GGGCACCTTCAGCTTTCTTT-3'. The restriction enzymes for rs11615 and rs3212986 were TaiI and PvuII, respectively. The PCR conditions were set as follows: initial denaturation at 94°C for 8 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. The DNA fragments were confirmed via electrophoresis on a 3.5% agarose gel, which was subsequently visualized under UV light after staining with ethidium bromide [Figure 1] and [Figure 2].
Figure 1: ERCC1 rs11615, PCR products after restriction digestion with Tai1 on 3% agarose gel. Lane 1 = 50bp DNA Ladder, Lane 1 and 3 = TT genotypes, Lane 4 = CC genotypes and Lane 5 = CT genotype

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Figure 2: ERCC1 rs 3212986PCR products after restriction digestion with MboII on 3% agarose gel. Lane 1 = 50bp DNA Ladder, Lane 2 and 7 = CA genotypes, Lane 1,3,5,6 and 8 = CC genotypes and Lane 4 = AA genotype

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Statistical analysis

The demographic and clinical data were expressed as number (N) and percentage (%). Statistical data analysis was done using Medcalc. Statistical significance was set as P < 0.05. Values were expressed as percentage and mean. Data were compiled according to the genotype and allele frequencies.


   Results Top


Biological characteristics

The distribution patient's biological characteristics and selected risk factors are shown in [Table 1]. Age range for OSCC patients and controls was 9–87 years in males and 27–75 in years in females. However, many of the ages mentioned in case sheets or given by patients were arbitrary, exact age of 150 OSCC patients and controls were (Males 81 and Females 69); hence analysis was carried out with those, mean age at which OSCC identified as 9-87/49.30 ± 15.55 in males and 27-75/84.20 ± 11.26 in females years. To understand the role of gene mutations/polymorphisms in onset of the disease, the patients were divided into 4 categories, <25 years (1.33%), 26 to 45 years (32.00%), 46 to 65 years (54.00%), and above 66 years (20.12%). Highest percentage of OSCC patients was identified between 46 and 65 years. Regarding the primary tumor site, there was a neat predominance on the BM adding up 56 patients (37.33%), followed by tongue adding up 33 patients (22.0%), then mandible, oral cavity, and RMT adding up 12%, 10%, and 7%. In the present study, high percentage was identified in BM patients and low percentage was observed in BOT, FOM maxilla, palate, and lip sites. The stage of a cancer is a descriptor (usually numbers I to IV) of how much the cancer has spread. The stage often takes into account the size of a tumor, In the present study Stage III showed the highest frequency (40%) when compared to Stage II (22%) and Stage IV (31.33%), and other types of tumor grade like Stage I (6.67%) showed very low frequency when compared to other staging groups. The percentage of patients with family history was 5% in oral squamous cell carcinoma patients. In the present study, majority of patients 40.7% had received radiation therapy with adjuvant chemotherapy, 23.3% underwent surgery, followed by surgery with adjuvant chemotherapy 14.0%. 13.3% and 0.7% of the patients received radiation and chemotherapy alone. Surgical excision and/or adequate radiation therapy remain the most effective means of treating the patients with OSCC.
Table 1: Patient and Tumor Characteristics (n=150)

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ERCC1 gene polymorphism in patients with OSCC and controls

[Table 2] shows the genotype frequencies distribution of 150 OSCC patients together with 150 controls. Frequencies of ERCC1 C118C, C118T, and T118T genotypes were 60%, 28%, and 12% in OSCC patients and 78%, 19%, and 3% in the controls, respectively. The C118T and T118T genotype had a 1.69 and 4.97-folds increased risk for OSCC (OR = 1.694; 95% CI = 0.983–2.918; P = 0.057). Frequencies of ERCC1 C8092C, C8092A and A8092A were 78%, 18%, and 4% in the OSCC patients and 89%, 10%, and 1% in the controls, respectively. The C8092A genotype showed a 1.97-fold increased risk for OSCC (OR = 1.97; 95% CI = 1.004–3.887; P = 0.048). Listed in [Table 2] and [Table 3] are the observed frequencies of the ERCC1 118 and ERCC1 8092 polymorphisms among 150 OSCC patients and 150 controls. We found the ERCC1 C118C genotype present among 90 (60%) OSCC cases and 118 (78%) controls, the C118T genotype among 42 (28%) OSCC cases and 28 (19%) controls and the T118T genotype among 18 (12%) OSCC cases and 4 (3%) controls. For ERCC1 8092, the C8092C genotype was observed in 117 (78%) of the OSCC cases and 133 (89%) of the controls, the C8092A genotype was observed in 27 (18%) OSCC cases and 15 (10%) controls and the A8092A genotype among 6 (4%) OSCC cases and 2 (1%) controls. The ERCC1 118T allele frequency was 0.26% in the OSCC patients and 0.14% in the controls, which was statistically significant.
Table 2: Distribution of ERCC1 (rs 11615) polymorphism genotypes in OSCC patients and controls

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Table 3: Distribution of ERCC1 (rs3212986) polymorphism genotypes in OSCC patients and controls

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Correlations of clinical characteristics of OSCC patients with ERCC1 polymorphisms

ERCC1 genotypes were correlated with demographic factors, like age, site of diagnosis, staging, and habitual risks of Oral squamous cell carcinoma patients to see the effect of genetic polymorphism in modulating the risk of developing lung cancer in association with all demographic's factors. In the present study, no significant difference was revealed between the CC + CT frequency and CC frequency of ERCC1 rs11615, and there was also no difference between the CA + AA frequency and CC frequency of ERCC1 rs 3212986 [Table 4].
Table 4: Correlations of clinical characteristics of Oral Squamous Cell Ccarcinoma patients with ERCC1 rs11615, andrs 3212986 polymorphisms

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Association between ERCC1 polymorphisms and response to cisplatin therapy

In our study, at the end of follow-up 69 (46%) patients showed good response and 81 (54%) patients showed poor response to cisplatin-based chemotherapy. Patients carrying CC and CT genotypes of ERCC1 rs11615 were found to be more likely to have had good response to chemotherapy when compared with those carrying TT genotypes; the ORs (95%CIs) were 4.20 (2.01–8.03) and 5.06 (1.53–20.4), respectively [Table 2]. Patients carrying CC & CA genotypes of ERCC1 rs 3212986 were found to be more likely to have had good response to chemotherapy when compared with those carrying AA genotypes; the ORs (95%CIs) were 3.60 (1.61-8.04) and 0.46 (0.19-1.07), respectively [Table 5].
Table 5: Association between ERCC1 polymorphisms and response to Cisplatin therapy

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   Discussion Top


Cisplatin chemotherapy is a commonly used chemotherapeutic agent that exerts its Cytotoxic effect mainly through formation of different kinds of DNA lesions, which could induce inter-strand and intra-strand cross-link generation, as well as DNA-protein cross-links, resulting in inhibition of cell growth and apoptosis of targeted cells unless repair. DNA repair mechanisms may therefore play a very important role in response to cisplatin -based chemotherapy. The nucleotide excision repair (NER) Pathway is highly powerful and sophisticated DNA damage removal pathway. ERCC1 is key rate-limiting enzyme acting in the multistep NER process. ERCC1 is involved in DNA damage recognition. The deficiencies in DNA repair capacity due to mutations or polymorphisms of repair genes, including ERCC1, can lead to genomic instability, which results in chromosomal instability syndromes and play important roles in cancer progression and response to platinum-based chemotherapy.[9]

In the present study, we investigated whether functional ERCC1 rs3213986 G > T SNPs have any cancer risk and its effect on clinical outcomes of OSCC patients in a Telangana population. We found that, the ERCC1 C118T, T118T and C8092A genotypes are associated with an increased risk for oral squamous cell carcinoma. ERCC1 C/T polymorphism at codon 118 was found to influence the level of ERCC1 expression. This may be due to that, although both the AAC and AAT codons encode asparagine, the AAT codon usage is significantly reduced, thereby decreasing ERCC1 translation capability and protein level.

In our study we have investigated the potential predictive and prognostic role of ERCC1 (C118T and C8092A) genetic polymorphisms on either tumors' response to cisplatin chemotherapy in oral squamous cell carcinoma. We found that, ERCC1 codon 118 C/C genotype was significantly associated with higher response rate. ERCC1 C118T polymorphism involved in the regulation of mRNA and protein expression of ERCC1. Therefore, the functional ERCC1 C118T polymorphism may reveal the mechanism to resistance to cisplatin and serve as a useful predictive biomarker. Many studies have investigated the prognostic role of ERCC1 (C118T and C8092A) genetic polymorphisms on either tumors' response to cisplatin chemotherapy or clinical outcomes in many cancers including non-small cell lung cancer, esophageal cancer, and ovarian cancer.[10],[11] Of these studies, many have shown significant associations with some of these variants, but others still revealed contradictory outcomes. The inconsistency results might be due to differences in ethnicities, source of patients, sample size, and by chance.


   Conclusion Top


We conclude that the ERCC1 C118T and C8092A genotypes are associated with an increased risk for oral squamous cell carcinoma. ERCC1 codon 118 and 8092 C/C genotypes were significantly associated with higher response rate. Due to the limitation of unavailable clinical data, we were not able to evaluate the disease-free survival. We were unable to explore the exact mechanism by which ERCC1 SNPs influence OSCC survival. We are planning to confirm current findings in our ongoing study of a larger study population with a longer follow-up time.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
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Kiyohara C, Yoshimasu K. Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis. Int J Med Sci 2007;4:59-71.  Back to cited text no. 2
    
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Park DJ, Zhang W, Stoehlmacher J, Tsao-Wei D, Groshen S, Gil J, et al. ERCC1 gene polymorphism as a predictor for clinical outcome in advanced colorectal cancer patients treated with platinum-based chemotherapy. Clin Adv Hematol Oncol 2003;1:162-6.  Back to cited text no. 3
    
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Ryu JS, Hong YC, Han HS, Lee JE, Kim S, Park YM, et al. Association between polymorphisms of ERCC1 and XPD and survival in non-small-cell lung cancer patients treated with cisplatin combination chemotherapy. Lung Cancer 2004;44:311-6.  Back to cited text no. 4
    
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Yu JJ, Lee KB, Mu C, Li Q, Abernathy TV, Bostick-Bruton F, et al. Comparison of two human ovarian carcinoma cell lines (A2780/CP70 and MCAS) that are equally resistant to platinum, but differ at codon 118 of the ERCC1 gene. Int J Oncol 2000;16:555-60.  Back to cited text no. 5
    
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McGurk CJ, Cummings M, Köberle B, Hartley JA, Oliver RT, Masters JR. Regulation of DNA repair gene expression in human cancer cell lines. J Cell Biochem 2006;97:1121-36.  Back to cited text no. 6
    
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Chen P, Wiencke J, Aldape K, Kesler-Diaz A, Miike R, Kelsey K, et al. Association of an ERCC1 polymor-phism with adult-onset glioma. Cancer Epidemiol Biomarkers Prev 2000;9:843-7.  Back to cited text no. 7
    
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Wenguang X, Hao S, Xiaofeng Q, Zhiyong W, Yufeng W, Qingang H, et al. Prognostic factors of primary intraosseous squamous cell carcinoma (PIOSCC): A retrospective review. PLoS One 2016;11:1-11.  Back to cited text no. 8
    
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Biason P, Hattinger CM, Innocenti F, Talamini R, Alberghini M, Scotlandi K, et al. Nucleotide excision repair gene variants and association with survival in osteosarcoma patients treated with neoadjuvant chemotherapy. Pharmacogenomics J 2012;12:476-83.  Back to cited text no. 9
    
10.
Schena M, Guarrera S, Buffoni L, Salvadori A, Voglino F, Allione A, et al. DNA repair gene expression level in peripheral blood and tumour tissue from non-small cell lung cancer and head and neck squamous cell cancer patients. Dna Repair 2012;11:374-80.  Back to cited text no. 10
    
11.
Rumiato E, Cavallin F, Boldrin E, Cagol M, Alfieri R, Basso D, et al. ERCC1 C8092A (rs3212986) polymorphism as a predictive marker in esophageal cancer patients treated with cisplatin/5-FU-based neoadjuvant therapy. Pharmacogenet Genomics 2013;23:597-604.  Back to cited text no. 11
    

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Correspondence Address:
C K Anulekha Avinash
Professor, Department of Prosthodontics, Kamineni Institute of Dental Sciences, Narketpally, Nalgond, Telangana State
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_964_19

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