- Email: [email protected]
Genetic alterations of the PIK3CA oncogene in human oral squamous cell carcinoma in an Indian population
Accepted Manuscript Genetic Alterations of the PIK3CA Oncogene in Human Oral Squamous Cell Carcinoma in an Indian population Sejal Shah, Siddharth Shah, Harish Padh, Kiran Kalia PII:
S2212-4403(15)01134-7
DOI:
10.1016/j.oooo.2015.08.003
Reference:
OOOO 1269
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 1 February 2015 Revised Date:
25 July 2015
Accepted Date: 4 August 2015
Please cite this article as: Shah S, Shah S, Padh H, Kalia K, Genetic Alterations of the PIK3CA Oncogene in Human Oral Squamous Cell Carcinoma in an Indian population, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2015), doi: 10.1016/j.oooo.2015.08.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title : Genetic Alterations of the PIK3CA Oncogene in Human Oral Squamous Cell Carcinoma in an Indian population.
2.
Head and Neck Oncosurgeon E N T department P. S. Medical College, Karamsad - 388325 Gujarat, India.
Corresponding author
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Dr. Kiran Kalia Professor in Biochemistry BRD School of Biosciences, Sardar Patel University, Vallabh Vidhyanagar - 388 120 Gujarat, India. Email – [email protected] (M) - +919824335881
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BRD School of Biosciences, Sardar Patel University, Vallabh Vidhyanagar - 388 120 Gujarat, India.
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1.
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Sejal Shah1, Siddharth Shah2, Harish Padh1, Kiran Kalia1*
*Present address
Dr. Kiran Kalia Director, National Institute of Pharmaceuticals Education and Research (NIPER) C/o B.V.Patel PERD Centre, S.G.Highway Thaltej, Ahmedabad -380 054 Gujarat, India Email – [email protected] (M) - +919824335881
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Word count Abstract-191; Manuscript-3406 Number of figures/tables - 8 Number of references - 30
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Financial support for this work: Department of Science and Technology Women Scientist Scheme-A, New Delhi, India. Sanction no: -SR/WOS-A/LS/511/2011-G dated on 24/5/2012
Disclosure– This work has been recently presented in the form of a poster at “American
U.S.A., at the time when the manuscript was under review.
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Conflict of interest - None.
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Association of Cancer Research Annual Meeting 2015” held from 18th-22nd April, Philadelphia,
Abstract Objective
The phosphatidylinositol 3-kinase (PI3K) genes, which code for heterodimeric lipid kinases,
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consist of a catalytic subunit, p110α (PIK3CA), which regulates cell proliferation, apoptosis and metastasis. Recently, a high frequency of somatic mutations was observed in the PIK3CA gene
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in various cancer types, including oral squamous cell carcinoma (OSCC). The study was aimed to determine the frequency of oncogenic hotspot mutations in exon 9 and 20 of the PIK3CA gene
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and its correlation with the clinical characteristics of OSCC patients in an Indian population. Study Design
We analyzed exon 9 and 20 of the PIK3CA gene using PCR and the direct genomic sequencing of 50 OSCC primary tumors.
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Results We observed two hot spot oncogenic mutations (E542K, E545K) in exon 9 and two synonymous
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mutations (A994A, T1025T) in exon 20. Moreover, we identified two SNPs, rs114587137 (C>T) and rs17849071 (T>G), in intron 9 of the PIK3CA gene. Both oncogenic hotspot mutations were reported primarily in patients with advanced stage cancer of the buccal mucosa.
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Conclusions
We have observed a 4% oncogenic mutation frequency of the PIK3CA gene, which plays a
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minor role in the development of OSCC in an Indian population. Keywords
OSCC, PI3K-AKT pathway, PIK3CA, mutation, Indian population, SNP
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Abbreviations OSCC - Oral squamous cell carcinoma
PI3K/AKT - Phosphatidylinositol 3-kinase/Akt
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PIK3CA - Phosphoinositide3-kinase catalytic α
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SNP – Single Nucleotide Polymorphism
1. Introduction
Oral squamous cell carcinoma (OSCC), a subset of head and neck cancer, is the eighth most common cancer worldwide and is the leading type of cancer in the India. Annually, ~128,000 deaths and 260,000 new cases of OSCC are recorded1. It is the most common cancer in men and the third most common cancer among women in India2. The prevalence of oral cancer is
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discrepant throughout the world due to variations in specific etiological factors and the predominance of carcinogenic compounds. Tobacco chewing and smoking are major primary causes of OSCC3. The western part of India (Gujarat) is a prime tobacco-growing region in
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India. People living in this area have higher use of tobacco in diverse forms of chewing and smoking, consequently have an increased occurrence of OSCC. In the western part of India, OSCC patients commonly have a habit of chewing tobacco in the form of betel-quid comprising
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of tobacco, areca nut, slaked lime with or without betel leaf.
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Cancer is a multistep and multifactorial genetic disease. The activation of oncogenes, such as phosphoinositide3-kinase catalytic α (PIK3CA), rat sarcoma (RAS), epidermal growth factor receptor (EGFR) and the deactivation of tumor suppressor genes, such as phosphatase-tensin homolog (PTEN), p53, and cyclin D1, ensue uncontrolled cell proliferation. The phosphatidylinositol 3-kinase-Akt (PI3K-AKT) pathway is one of the highly deregulated
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pathways in OSCC. This impairs the regulation of various cellular processes. The cell growth, proliferation, mortality, and apoptosis are affected due to the gain or loss of function of PIK3CA, PTEN and AKT 4. Thus, the PI3K-AKT pathway is associated with the development of OSCC as
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well as with the potential response of a tumor to cancer therapeutics5.
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The PI3K gene is located on chromosome 3q26.32, which codes for a heterodimeric lipid kinase consisting a catalytic subunit, p110α (PIK3CA), and a regulatory subunit, p85α. PIK3CA had a role in the regulation of cell proliferation, apoptosis, and metastasis, thus reported as an oncogene due to its elevated kinase activity and genomic amplification. Recently, a high frequency of somatic mutations has been observed in the PIK3CA gene in numerous cancer types 6-8.
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It has been reported that 80% of somatic mutations are clustered in the helical domain coded by exon 9 and in the kinase domain coded by exon 20 of the PIK3CA gene. The canonical mutations, E542K, E545K, and H1047R, have been proven to have higher oncogenic potential4and lead to the subsequent activation of the downstream AKT signaling pathway. This finding
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directly designates the role of mutated PIK3CA in the oncogenic activation of the PI3K–AKT
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pathway in cancer development.
Therefore, we explored the mutation profile of exon 9 and exon 20 of the PIK3CA gene in fifty
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primary OSCC tumors. The study would determine the frequency of oncogenic mutations and its correlation if any, between PIK3CA gene mutations and the clinical characteristics of OSCC patients in the western part of India (Gujarat). 2. Subjects and Methods
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2.1 Subjects
The present study was approved by the Human Research Ethics Committee of P.S. Medical College and H M Patel Centre for Medical Care and Education, Karamsad, Gujarat. Informed
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consent was obtained from all of the patients included in the study. The patients were personally
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interviewed according to the structured questionnaire. The demographic history, type of tobacco used, daily frequency of tobacco consumed, and duration of tobacco use were recorded. Between May 2012 and February 2014, 50 primary tumors were obtained from 50 OSCC patients who underwent surgery at the Department of Otorhinolaryngology, P.S. Medical College.
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2.2 Inclusion criteria a) Patients with lesions in the oral cavity that includes buccal mucosa, tongue, lip, hard palate,
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Gingival-buccal complex, and alveolus. b) Patients: Biopsy confirmed patients of squamous cell carcinoma who underwent a wide excision for tumour clearance were selected. The samples for the study were collected from the
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primary tumour at the time of tumour clearance. 50 cases were taken for the study.
c) Controls: 34 controls were taken from the histopathologically normal surrounding tissue of the
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patients who had undergone wide excision (paired tumor samples), as 16 patients did not give consent for control tissue. We have included unpaired tumor samples of those patients to investigate their mutational profile, to increase the statistical significance of the data that in turn may help to evaluate our results more precisely.
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d) The patients and controls were demographically restricted to the Gujarati population [non migrant population; born and brought up in Gujarat (a state in the western part of India)].
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2.3 Exclusion criteria
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a) OSCC patients undergoing/ undergone chemotherapy b) OSCC patients undergoing/undergone radiotherapy c) OSCC recurrence patients. 2.4 Biopsy and histopathological examination The incisional or excisional biopsy was carried out under utmost aseptic conditions. The site of biopsy for OSCC was selected by clinical examination and intravital staining using toluidine
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blue solution followed by immediate fixation in 10% formalin and carried to pathology department, where the histopathological study was conducted by a team of pathologists. The histological diagnosis, pathological staging and grading of each tumor were verified on formalin-
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fixed, paraffin-embedded, hematoxylin-eosin stained slides using the criteria established according to the guidelines laid down by American Joint Committee on Cancer (AJCC) 201011
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for oral cavity cancers. 2.5 Sample collection
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Primary tumors, as well as control tissue, were collected at the time of surgery. The tissue samples were washed thrice with 1X phosphate-buffered saline (pH 7.2) and stored in RNA later® (Qiagen) at -20°C until further downstream analysis.
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2.6 DNA Isolation
Each tissue sample was microdissected, and the genomic DNA was isolated from the tumor
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tissue as well as control tissue using a DNeasy® blood and tissue mini kit (Qiagen) following the manufacturer’s tissue protocol. The quantity and quality of DNA were analyzed by measuring
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the UV absorption at 260nm and 280nm, and by 0.8% agarose gel electrophoresis, respectively. Samples with an OD260/OD280 ratio ≥1.8 were used for further downstream analysis, including PCR amplification and DNA sequencing. 2.7 Screening of mutations The amplification of PIK3CA gene exon 9 and exon 20 was performed by PCR using oligonucleotides reported previously4. The sequence of oligonucleotides used for the PCR, their
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corresponding amplicon size, and the melting temperature (Tm) of the reactions are presented in Table 1. PCR amplification of both exon 9 and exon 20 was carried out. The 25 µl reaction mixtures was containing Taq 2X master mix (New England Biolabs), 400nmol forward and
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reverse primers, 80ng of DNA following the manufacturer’s protocol. The reaction mixture was placed in a thermo cycler (ABS verity 96) and according to the Taq 2X master mix manufacturer’s protocol, cycling conditions were: 5 min at 95°C; 30 cycles of denaturation for
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25 s at 95°C, annealing for 30 s at 58°C and extension for 25 s at 68°C followed by the final step of extension for 5 minutes at 68°C and a hold at 4°C. An aliquot of PCR products was separated
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by 1.2% agarose gel electrophoresis to ensure the integrity of the targeted amplification before sequencing. The DNA sequencing of PCR products was carried out by Macrogen, Inc. (Korea) using forward primers. Observed mutations were verified by reverse sequencing of the PCR products. Then, the mutations were further confirmed by sequencing a second PCR product
2.8 Statistical Analysis
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derived independently from the original template.
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The presence of reported somatic mutations is indicated as a percentage of the total number of samples screened. Fisher’s exact test was used to check correlations between PIK3CA oncogenic
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mutations and clinicopathological characteristics of patients. A P-value less than 0.05 was defined as being statistically significant. 3. Results
3.1 Clinical profile of OSCC patients The clinicopathological features of OSCC patients included in this study have been shown in Table 5. The age of the patients was ranged from 27 to 70 (mean; 47.14) years. Among 50 OSCC
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patients, 36 (72%) were males and 37(74%) patients were having the history of tobacco chewing of minimum ten years, where the frequency of chewing tobacco was ranging from 5-8times/day. The 4(8%) patients have history of tobacco chewing and smoking, while one patient (2%) have
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history of tobacco smoking only.” The majority of patients, 66% and 76%, presented the larger (T3+T4) tumors and regional lymph node involvement respectively. Most of the patients (84%) were at the advanced stage (III & IV). Half of the patients (52%) showed well-differentiated
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OSCC. Buccal mucosa (64%) was the most commonly affected site and the rest of the affected
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sites are shown in Table 5.
We investigated the genomic DNA of 50 OSCC primary tumors via the direct sequencing of the PCR products of PIK3CA exon 9 and exon 20. We found two somatic, oncogenic, hot spot missense mutations in exon 9. Moreover, we observed two synonymous mutations in exon 20
illustrated in Figure 1.
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and two single nucleotide polymorphisms (SNPs) in intron 9 of the PIK3CA gene, which are
3.2 Mutational spectrum of the PIK3CA gene
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We analyzed 50 primary OSCC tumor tissue samples to detect mutations in exon 9 and exon 20 of the PIK3CA gene, which codes for the helical domain and the kinase domain respectively. As
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the preponderance of the mutations is found in these clustered regions, we have restricted our study particular to helical and the kinase domain. We observed two hot spot oncogenic mutations, G1624A and G1633A, in exon 9 of the PIK3CA gene in two different patients with a corresponding amino acid change of E542K and E545K. Both the mutations were found heterozygous mutated and did not observe in the relevant control tissue of the respective patient. Therefore, these missense mutations were somatic in nature (Figure 2(a & b), Table 2). Our
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study has reported a 4% oncogenic mutation frequency in 50 OSCC primary tumor samples. We did not observe its significant association with gender, tumor site, disease stage, and histological
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grading (Table 4). Moreover, two synonymous mutations, C2985T and C3075T, were observed in exon 20 of the PIK3CA gene. C2985T and C3075T were identified in one and fourteen primary OSCC tumors
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respectively, with no amino acid change at the 994 position and 1025 position (Figure 2 (c & d), Table 3). We did not observe mutation at the 1047 position in exon 20 in any of our patients
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though other studies have reported this position as mutation hotspot4,6,9,10. We did not find any missense mutations in exon 20 of the PIK3CA gene. Additionally, we identified rs114587137 (C>T) and rs17849071 (T>G) SNPs in intron 9 of the PIK3CA gene in fifteen and three OSCC patients respectively.
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4. Discussion
Although genetic abnormalities are frequent the PI3K/AKT pathway and play a fundamental role in tumorigenesis, this pathway has not yet been investigated for OSCC in the population of the
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western part of India (Gujarat), who were originated from Ancestral North Indian population, genetically divergent from rest of India29. PIK3CA gene has been found frequently mutated in
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colorectal cancer (32%), brain cancer (27%), gastric cancer (25%) and at relatively low frequency in breast cancer (8%) and lung cancer (4%) 12. PIK3CA mutation was first reported in OSCC by Qui et al. in 2006 with a 10.8% frequency in the American population7. Other studies from various ethnicities have analyzed a total of 738 OSCC subjects and observed a 5.1% mutational rate all over the world. It includes Greece, Japan, Israel, the U.S.A., the southern part of India, Thailand, Vietnam and Germany (Figure 3)12. Qui et al. reported the highest mutational
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frequency of 20.8% in OSCC patients in an American population9, whereas, the absence of hot spot mutations were reported in Greek, Vietnamese and German populations6,13,14. We have found 4% oncogenic mutation frequency and did not observe its significant correlation with the
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clinicopathological characteristics of OSCC patients. In contrast, Kozaki et al. reported a 7.4% oncogenic mutation rate and found a statistically significant association between the mutation frequency and the stage of disease4. A recent study in Taiwan has reported PIK3CA mutations in
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11/ 79 cases (13.92%) of OSCC, including a rare mutation, Q546P27.
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In the present study, we reported two missense mutations in the helical domain encoded by exon 9 of the PIK3CA gene. Interestingly, both of the somatic mutations were observed at the same organ site from the buccal mucosa of different patients with advanced stage of OSCC. It may be due to more than ten years of tobacco chewing history of both of the patients. These findings confirmed previous observations in both Asian and Caucasian populations in which a lower
Western countries1,
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oncogenic mutation frequency was observed in Asian OSCC compared to that observed in . A similar study by Murugan et al., in the south Indian population
identified a 10.8% (2/19) oncogenic hot spot mutation frequency in OSCC patients6. They
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observed E545K and frameshift mutation in helical and kinase domain respectively6. On the
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contrarily we found E542K and E545K in helical domain. Moreover, they found T1025T mutation/polymorphism in 47% of patients6, while we found it as synonymous mutation in 28% of patients.
The observed somatic mutations of PIK3CA gene, E542K and E545K, were in the helical domain, and we did not observe missense mutations in the kinase domain. The mechanism for these two mutations revealed that the resulting amino acid changes occurred at the interface between the helical domain of P110α and the nSH2 domain of P85α. Biochemical studies
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showed that Glu542 and Glu545 interact with Lys379 and Arg340 in the nSH2 domain of P85α8. Thus, mutated residues modify the inhibitory activity of P85α on the catalytic subunit and disrupt the P110α/85α complex16. Alterations in P110α enzymatic activity lead to PIK3CA oncogenic
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activation with a subsequent downstream constitutive activation of the AKT signaling pathway, which has a role in OSCC. Recent studies have shown a 5.7% prevalence of oncogenic mutations in Caucasian populations, including mutations such as E542K, E545K, Q546R, M1043I, and
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H1047R in six OSCC tissues and one cell line15. Functional studies have shown that the presence of any one of the three oncogenic hotspot mutations was able to induce the transformation in
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chicken embryo fibroblasts cultures. Therefore, increase the lipid kinase activity of the protein, which leads to the subsequent constitutive activation of the AKT signaling pathway17,18. Previous studies have shown the higher level of circulating PI3K P110α in OSCC patients compared to control subjects and concluded that PI3K P110α is over expressed in OSCC tissues compared to
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control tissues19. The observed PIK3CA oncogenic mutations in two patients may be responsible for over expression. The other OSCC patients may have some other genetic mutations such as changes in RAS, which is located upstream of the PIK3CA gene and may trigger the whole
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pathway3. The xenobiotic metabolizing genes of the patients with tobacco chewing history may be involved in the development of OSCC in the population of this area. A previous study showed
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that the GSTM1 (glutathione-S-transferase M1) null genotype is a risk factor for OSCC among the Indian tobacco-addicted population2. Likewise, a recent study in the south Indian population reported the increased prevalence of GSTM1 null polymorphism as the severity of the lesions progressed from oral leukoplakia to OSCC30. Both the studies related to GSTM1 null polymorphism in an Indian subsets suggested its role mainly in tobacco addicted OSCC patients.
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Hence, GSTM1 null polymorphism may play an immense role in the development of OSCC in tobacco habituated studied population.
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Although somatic mutations were not a frequent event in OSCC in our study, the detection of mutations is imperative to support that notion that the PI3K/AKT pathway is involved in oral tumorigenesis. The role of the PIK3CA gene mutation may be minor in our population, but the
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knowledge of PIK3CA involvement is critical. The particular kinase inhibitor could be considered as a personalized therapeutic option in OSCC patients with PIK3CA mutations. It is
chemotherapy in OSCC
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believed that a small selective inhibitor molecule has tremendous potential as novel cancer . The diversity in the results of various studies suggests that cultural
habits, sample size, and ethnicities may influence the mutation frequency. Future screening studies will uncover the roles of various genes that play a significant role in the development of
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OSCC to establish their clinical relevance in the population of the western part of India Our study has reported two synonymous mutations in the kinase domain of PIK3CA gene encoded by exon 20. One of these observed mutation (C>T) is located at the 3075 position with
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no amino acid change at the 1025 position and was found in fourteen OSCC samples. This finding was previously reported in the COSMIC database. Previous findings in an Indian ethnic
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population confirmed our results, as Murugan et al. studied nineteen primary OSCC tumors and found nine identical T1025T silent mutations in the people of the southern part of India6. Likewise, Kozaki et al. have reported T1025T in 4/58 OSCC tumors in a Thai population4, similarly it was also observed in breast cancer and ovarian cancer in an American population8. In contrast, this mutation has been found in pituitary tumors with a corresponding amino acid change of T1025A in a Chinese population21. The other observed synonymous mutation, C2985T, with no corresponding amino acid change at 994 position in exon 20 of PIK3CA gene.
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Though the frequency of this synonymous mutation is very less (1/50), it is not reported earlier in OSCC. We are the first to report C2985T synonymous mutation in OSCC.
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Moreover, we identified two SNPs, rs114587137 (C>T) and rs17849071 (T>G), in intron 9 of the PIK3CA gene, in fifteen and three OSCC patients, respectively. None of the patients harbored both of the polymorphisms simultaneously. The clinical significance and the role of
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SNP rs114587137 (C>T) is still unknown, and it has not been previously reported in OSCC. As we have identified SNP rs114587137 (C>T) in 30% OSCC patients, a future study with healthy
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individuals as a control will be warranted to establish its role and susceptibility. SNP rs17849071 (T>G) was reported previously in OSCC in a Greek population13. In contrast, a recent study has reported that SNP rs17849071 (T>G) is inversely associated with follicular thyroid cancer and PIK3CA amplification28. We found that both polymorphisms were found heterozygous mutated and further study with healthy individuals as control may reveal the role of the observed
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polymorphisms in the development of OSCC.
The limitation of our study was to follow up the OSCC patients. Moreover, a number of genes
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Conclusion
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may play role in development of OSCC, which are not covered in the present study.
Within the limitation of the study, the following conclusions were strained: •
The prevalence of oncogenic mutations in the PIK3CA gene is not a frequent event in the population of the western part of an India. Oncogenic mutations in PIK3CA have a minor role in the development of OSCC in a western Indian population; however, their role in personalized combination therapeutics cannot be ignored.
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•
The kinase inhibitors therapy may not be used in the current population due to the lower frequency of PIK3CA mutations; however, patients harboring PIK3CA mutations may be treated with pathway-specific kinase inhibitor-based combinational therapy instead of
•
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conventional treatment.
The performed study is cross sectional primary study. The longitudinal study with follow
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up of patients can provide more precise findings in the similar ethnic population. Acknowledgments
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We acknowledge the Dean and medical staff of the Pramukh Swami Medical College and Hospital, Karamsad, for the clinical diagnosis and collection of cancerous as well as healthy tissues for the present study. The authors thank to Dr. Anuja Kedia for her guidance in sample collection. Sejal Shah has been awarded as a woman scientist in this project.
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27. Chang YS, Hsu HT, KO YC, Yeh KT. Combined mutational analysis of RAS, BRAF, PIK3CA, and TP53 genes in Taiwanese patients with oral squamous cell carcinoma. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology. 2014;18(1):110–116
28. Xing JC, Tufano RP, Murugan AK, Liu D, Wand G, et al. Single Nucleotide Polymorphism rs17849071 G/T in the PIK3CA Gene Is Inversely Associated with Follicular Thyroid Cancer and PIK3CA Amplification. PloS One. 2012;7(11):e49192.
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29. Reich D, Thangaraj K, Patterson N, Price AL, and Singh L. Reconstructing Indian population history. Nature. 2009; 461(7263): 489-494 30. Tanwar R, Iyengar AR, Nagesh KS, Patil S, and Subhash BV. Prevalence of glutathione
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S-transferase M1 null polymorphism in tobacco users, oral leukoplakia and oral squamous cell carcinoma patients in South Indian population: A polymerase chain
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reaction study. Contemporary clinical dentistry. 2015;6(Suppl 1):S59-64.
Figure Captions
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Figure 1
PIK3CA MAP: Exon-intron organization of the PIK3CA gene. The location of the observed somatic missense mutations E542K and E545K in exon 9, the synonymous mutations A994A
PIK3CA gene. Figure 2
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and T1025T in exon 20 andrs114587137 (C>T) and rs17849071 (T>G) in intron 9 of the
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SEQUENCE ANALYSIS: Sequence chromatogram of genetic mutations and SNPs as determined by automated sequence analysis shows: (a) G1624A (E542K) of exon 9 (1/50) of the
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PIK3CA gene; (b) G1633A (E545K) of exon 9 (1/50) of the PIK3CA gene; (c) C2985T (A994A) of exon 20 (1/50) of the PIK3CA gene; (d) C3075T (T1025T) of exon 20 (14/50) of the PIK3CA gene;(e) SNP rs114587137 (C>T) (15/50) in intron 9 of the PIK3CA gene; (f) SNP rs17849071 (T>G) (3/50) in intron 9 of the PIK3CA gene. Figure 3
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MUTATION FREQUENCY OF THE PIK3CA GENE: The mutation frequencies of the PIK3CA gene in oral cancer patients in the western part of India compared with the mutation
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frequencies of other populations. [4, 6, 7, 15, 22-27]
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Sense Primer
Antisense Primer
Product size
Tm
Exon 9
5’-GATTGGTTCTTTCCTGTCTCTG-3’
5’-CCACAAATATCAATTTACAACCATTG-3’
487bp
58°C
Exon20
5’-TGGGGTAAAGGGAATCAAAAG-3’
5’-CCTATGCAATCGGTCTTTGC-3’
525bp
58°C
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PIK3CA Gene
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Age
Gender
Mutation
Mutation type
Tumor site
Classification Stage T
N
M
15
31
M
E542K
G:C>A:T
BM
4
1
0
56
38
M
E545K
G:C>A:T
BM
4
1
0
IV
Tobacco chewing >10 yrs Tobacco chewing >10 yrs
IV
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BM- buccal mucosa
Habit
Differentiation
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Sample
Well
Well
Table 3: Summary of PIK3CA synonymous mutations in Oral Squamous Cell Carcinoma
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Mutation
Codon Change
Amino Acid position
Mutation Type
Status of Mutation
Domain
3
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
8
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
12
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
17
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
19
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
30
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
31
20
2985
GCC/GCT
994
Synonymous
Heterozygous
Kinase
32
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
33
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
36
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
46
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
52
20
3075
1025
Synonymous
Heterozygous
Kinase
57
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
58
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
60
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
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ACC/ACT
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Sample no.
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Total number (%)
2(4%)
48(96%)
Age (years) [mean]
34.5(31-38)
48.02(27-70)
2 0
Tumor site Buccal Mucosa Others*
2 0
Disease stage I-III IV
0 2
34 14
30 18
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Gender Male Female
Histological grading Well Moderate Poor
1 1 0
16 32
25 12 11
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* Others include tumor site at the tongue, lip, hard palate, GB complex and alveolus p values were calculated using Fisher’s exact test
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p-value
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Observed mutation
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Characteristic
p<0.5
p<0.4
p<0.4
p<1.0
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n=50
Frequency (%)
n=34 (tumor control pair)
72 28
27 7
Gender
Male Female
36 14
Age
Mean Range
47.14 47.14
Risk Habits
Tobacco Chewing Smoking Chewing + smoking None
37 1 4 8
Tumor Site*
Buccal Mucosa Tongue Lip Hard Palate GB complex Alveolus
32 6 1 2 6 3
Tumor Size
T1 &T2 T3 & T4
Lymph node Metastasis
Negative Positive
TNM Stage
Tumor Differentiation
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Variable
45.74 45.74
74 2 8 16
28 2 1 3
64 12 2 4 12 6
19 5 1 2 6 1
17 33
34 66
13 21
12 38
24 76
8 26
Early (I&II) Late (III&IV)
8 42
16 84
3 31
Well Moderate Poor
26 13 11
52 26 22
23 6 5
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*The study includes patients with SCC of left or right buccal mucosa, three, two and one of
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the tongue lesions were taken from right lateral border of tongue, left lateral border of the tongue and central part of the tongue respectively. One patient with chronic tobacco chewing history >15 years developed lower lip stage IVa OSCC (mucosal extent of lip) (CT4 N2c Mo), two patients with left side hard palate. Four lesions with right lower GB complex, two lesions from left lower GB complex, two alveolar lesions from lower alveolar left side and one sample from upper alveolar left side included in the study.
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Statement of Clinical relevance
“PIK3CA gene may be a prognostic biomarker and the knowledge of the involvement of
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kinase inhibitors along with conventional therapy”.
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PIK3CA gene is important for the patients harboring such mutations and will be benefited with
S2212-4403(15)01134-7
DOI:
10.1016/j.oooo.2015.08.003
Reference:
OOOO 1269
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 1 February 2015 Revised Date:
25 July 2015
Accepted Date: 4 August 2015
Please cite this article as: Shah S, Shah S, Padh H, Kalia K, Genetic Alterations of the PIK3CA Oncogene in Human Oral Squamous Cell Carcinoma in an Indian population, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2015), doi: 10.1016/j.oooo.2015.08.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title : Genetic Alterations of the PIK3CA Oncogene in Human Oral Squamous Cell Carcinoma in an Indian population.
2.
Head and Neck Oncosurgeon E N T department P. S. Medical College, Karamsad - 388325 Gujarat, India.
Corresponding author
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Dr. Kiran Kalia Professor in Biochemistry BRD School of Biosciences, Sardar Patel University, Vallabh Vidhyanagar - 388 120 Gujarat, India. Email – [email protected] (M) - +919824335881
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BRD School of Biosciences, Sardar Patel University, Vallabh Vidhyanagar - 388 120 Gujarat, India.
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1.
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Sejal Shah1, Siddharth Shah2, Harish Padh1, Kiran Kalia1*
*Present address
Dr. Kiran Kalia Director, National Institute of Pharmaceuticals Education and Research (NIPER) C/o B.V.Patel PERD Centre, S.G.Highway Thaltej, Ahmedabad -380 054 Gujarat, India Email – [email protected] (M) - +919824335881
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Word count Abstract-191; Manuscript-3406 Number of figures/tables - 8 Number of references - 30
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Financial support for this work: Department of Science and Technology Women Scientist Scheme-A, New Delhi, India. Sanction no: -SR/WOS-A/LS/511/2011-G dated on 24/5/2012
Disclosure– This work has been recently presented in the form of a poster at “American
U.S.A., at the time when the manuscript was under review.
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Conflict of interest - None.
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Association of Cancer Research Annual Meeting 2015” held from 18th-22nd April, Philadelphia,
Abstract Objective
The phosphatidylinositol 3-kinase (PI3K) genes, which code for heterodimeric lipid kinases,
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consist of a catalytic subunit, p110α (PIK3CA), which regulates cell proliferation, apoptosis and metastasis. Recently, a high frequency of somatic mutations was observed in the PIK3CA gene
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in various cancer types, including oral squamous cell carcinoma (OSCC). The study was aimed to determine the frequency of oncogenic hotspot mutations in exon 9 and 20 of the PIK3CA gene
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and its correlation with the clinical characteristics of OSCC patients in an Indian population. Study Design
We analyzed exon 9 and 20 of the PIK3CA gene using PCR and the direct genomic sequencing of 50 OSCC primary tumors.
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Results We observed two hot spot oncogenic mutations (E542K, E545K) in exon 9 and two synonymous
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mutations (A994A, T1025T) in exon 20. Moreover, we identified two SNPs, rs114587137 (C>T) and rs17849071 (T>G), in intron 9 of the PIK3CA gene. Both oncogenic hotspot mutations were reported primarily in patients with advanced stage cancer of the buccal mucosa.
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Conclusions
We have observed a 4% oncogenic mutation frequency of the PIK3CA gene, which plays a
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minor role in the development of OSCC in an Indian population. Keywords
OSCC, PI3K-AKT pathway, PIK3CA, mutation, Indian population, SNP
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Abbreviations OSCC - Oral squamous cell carcinoma
PI3K/AKT - Phosphatidylinositol 3-kinase/Akt
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PIK3CA - Phosphoinositide3-kinase catalytic α
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SNP – Single Nucleotide Polymorphism
1. Introduction
Oral squamous cell carcinoma (OSCC), a subset of head and neck cancer, is the eighth most common cancer worldwide and is the leading type of cancer in the India. Annually, ~128,000 deaths and 260,000 new cases of OSCC are recorded1. It is the most common cancer in men and the third most common cancer among women in India2. The prevalence of oral cancer is
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discrepant throughout the world due to variations in specific etiological factors and the predominance of carcinogenic compounds. Tobacco chewing and smoking are major primary causes of OSCC3. The western part of India (Gujarat) is a prime tobacco-growing region in
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India. People living in this area have higher use of tobacco in diverse forms of chewing and smoking, consequently have an increased occurrence of OSCC. In the western part of India, OSCC patients commonly have a habit of chewing tobacco in the form of betel-quid comprising
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of tobacco, areca nut, slaked lime with or without betel leaf.
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Cancer is a multistep and multifactorial genetic disease. The activation of oncogenes, such as phosphoinositide3-kinase catalytic α (PIK3CA), rat sarcoma (RAS), epidermal growth factor receptor (EGFR) and the deactivation of tumor suppressor genes, such as phosphatase-tensin homolog (PTEN), p53, and cyclin D1, ensue uncontrolled cell proliferation. The phosphatidylinositol 3-kinase-Akt (PI3K-AKT) pathway is one of the highly deregulated
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pathways in OSCC. This impairs the regulation of various cellular processes. The cell growth, proliferation, mortality, and apoptosis are affected due to the gain or loss of function of PIK3CA, PTEN and AKT 4. Thus, the PI3K-AKT pathway is associated with the development of OSCC as
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well as with the potential response of a tumor to cancer therapeutics5.
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The PI3K gene is located on chromosome 3q26.32, which codes for a heterodimeric lipid kinase consisting a catalytic subunit, p110α (PIK3CA), and a regulatory subunit, p85α. PIK3CA had a role in the regulation of cell proliferation, apoptosis, and metastasis, thus reported as an oncogene due to its elevated kinase activity and genomic amplification. Recently, a high frequency of somatic mutations has been observed in the PIK3CA gene in numerous cancer types 6-8.
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It has been reported that 80% of somatic mutations are clustered in the helical domain coded by exon 9 and in the kinase domain coded by exon 20 of the PIK3CA gene. The canonical mutations, E542K, E545K, and H1047R, have been proven to have higher oncogenic potential4and lead to the subsequent activation of the downstream AKT signaling pathway. This finding
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10
directly designates the role of mutated PIK3CA in the oncogenic activation of the PI3K–AKT
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pathway in cancer development.
Therefore, we explored the mutation profile of exon 9 and exon 20 of the PIK3CA gene in fifty
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primary OSCC tumors. The study would determine the frequency of oncogenic mutations and its correlation if any, between PIK3CA gene mutations and the clinical characteristics of OSCC patients in the western part of India (Gujarat). 2. Subjects and Methods
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2.1 Subjects
The present study was approved by the Human Research Ethics Committee of P.S. Medical College and H M Patel Centre for Medical Care and Education, Karamsad, Gujarat. Informed
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consent was obtained from all of the patients included in the study. The patients were personally
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interviewed according to the structured questionnaire. The demographic history, type of tobacco used, daily frequency of tobacco consumed, and duration of tobacco use were recorded. Between May 2012 and February 2014, 50 primary tumors were obtained from 50 OSCC patients who underwent surgery at the Department of Otorhinolaryngology, P.S. Medical College.
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2.2 Inclusion criteria a) Patients with lesions in the oral cavity that includes buccal mucosa, tongue, lip, hard palate,
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Gingival-buccal complex, and alveolus. b) Patients: Biopsy confirmed patients of squamous cell carcinoma who underwent a wide excision for tumour clearance were selected. The samples for the study were collected from the
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primary tumour at the time of tumour clearance. 50 cases were taken for the study.
c) Controls: 34 controls were taken from the histopathologically normal surrounding tissue of the
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patients who had undergone wide excision (paired tumor samples), as 16 patients did not give consent for control tissue. We have included unpaired tumor samples of those patients to investigate their mutational profile, to increase the statistical significance of the data that in turn may help to evaluate our results more precisely.
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d) The patients and controls were demographically restricted to the Gujarati population [non migrant population; born and brought up in Gujarat (a state in the western part of India)].
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2.3 Exclusion criteria
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a) OSCC patients undergoing/ undergone chemotherapy b) OSCC patients undergoing/undergone radiotherapy c) OSCC recurrence patients. 2.4 Biopsy and histopathological examination The incisional or excisional biopsy was carried out under utmost aseptic conditions. The site of biopsy for OSCC was selected by clinical examination and intravital staining using toluidine
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blue solution followed by immediate fixation in 10% formalin and carried to pathology department, where the histopathological study was conducted by a team of pathologists. The histological diagnosis, pathological staging and grading of each tumor were verified on formalin-
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fixed, paraffin-embedded, hematoxylin-eosin stained slides using the criteria established according to the guidelines laid down by American Joint Committee on Cancer (AJCC) 201011
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for oral cavity cancers. 2.5 Sample collection
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Primary tumors, as well as control tissue, were collected at the time of surgery. The tissue samples were washed thrice with 1X phosphate-buffered saline (pH 7.2) and stored in RNA later® (Qiagen) at -20°C until further downstream analysis.
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2.6 DNA Isolation
Each tissue sample was microdissected, and the genomic DNA was isolated from the tumor
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tissue as well as control tissue using a DNeasy® blood and tissue mini kit (Qiagen) following the manufacturer’s tissue protocol. The quantity and quality of DNA were analyzed by measuring
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the UV absorption at 260nm and 280nm, and by 0.8% agarose gel electrophoresis, respectively. Samples with an OD260/OD280 ratio ≥1.8 were used for further downstream analysis, including PCR amplification and DNA sequencing. 2.7 Screening of mutations The amplification of PIK3CA gene exon 9 and exon 20 was performed by PCR using oligonucleotides reported previously4. The sequence of oligonucleotides used for the PCR, their
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corresponding amplicon size, and the melting temperature (Tm) of the reactions are presented in Table 1. PCR amplification of both exon 9 and exon 20 was carried out. The 25 µl reaction mixtures was containing Taq 2X master mix (New England Biolabs), 400nmol forward and
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reverse primers, 80ng of DNA following the manufacturer’s protocol. The reaction mixture was placed in a thermo cycler (ABS verity 96) and according to the Taq 2X master mix manufacturer’s protocol, cycling conditions were: 5 min at 95°C; 30 cycles of denaturation for
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25 s at 95°C, annealing for 30 s at 58°C and extension for 25 s at 68°C followed by the final step of extension for 5 minutes at 68°C and a hold at 4°C. An aliquot of PCR products was separated
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by 1.2% agarose gel electrophoresis to ensure the integrity of the targeted amplification before sequencing. The DNA sequencing of PCR products was carried out by Macrogen, Inc. (Korea) using forward primers. Observed mutations were verified by reverse sequencing of the PCR products. Then, the mutations were further confirmed by sequencing a second PCR product
2.8 Statistical Analysis
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derived independently from the original template.
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The presence of reported somatic mutations is indicated as a percentage of the total number of samples screened. Fisher’s exact test was used to check correlations between PIK3CA oncogenic
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mutations and clinicopathological characteristics of patients. A P-value less than 0.05 was defined as being statistically significant. 3. Results
3.1 Clinical profile of OSCC patients The clinicopathological features of OSCC patients included in this study have been shown in Table 5. The age of the patients was ranged from 27 to 70 (mean; 47.14) years. Among 50 OSCC
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patients, 36 (72%) were males and 37(74%) patients were having the history of tobacco chewing of minimum ten years, where the frequency of chewing tobacco was ranging from 5-8times/day. The 4(8%) patients have history of tobacco chewing and smoking, while one patient (2%) have
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history of tobacco smoking only.” The majority of patients, 66% and 76%, presented the larger (T3+T4) tumors and regional lymph node involvement respectively. Most of the patients (84%) were at the advanced stage (III & IV). Half of the patients (52%) showed well-differentiated
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OSCC. Buccal mucosa (64%) was the most commonly affected site and the rest of the affected
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sites are shown in Table 5.
We investigated the genomic DNA of 50 OSCC primary tumors via the direct sequencing of the PCR products of PIK3CA exon 9 and exon 20. We found two somatic, oncogenic, hot spot missense mutations in exon 9. Moreover, we observed two synonymous mutations in exon 20
illustrated in Figure 1.
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and two single nucleotide polymorphisms (SNPs) in intron 9 of the PIK3CA gene, which are
3.2 Mutational spectrum of the PIK3CA gene
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We analyzed 50 primary OSCC tumor tissue samples to detect mutations in exon 9 and exon 20 of the PIK3CA gene, which codes for the helical domain and the kinase domain respectively. As
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the preponderance of the mutations is found in these clustered regions, we have restricted our study particular to helical and the kinase domain. We observed two hot spot oncogenic mutations, G1624A and G1633A, in exon 9 of the PIK3CA gene in two different patients with a corresponding amino acid change of E542K and E545K. Both the mutations were found heterozygous mutated and did not observe in the relevant control tissue of the respective patient. Therefore, these missense mutations were somatic in nature (Figure 2(a & b), Table 2). Our
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study has reported a 4% oncogenic mutation frequency in 50 OSCC primary tumor samples. We did not observe its significant association with gender, tumor site, disease stage, and histological
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grading (Table 4). Moreover, two synonymous mutations, C2985T and C3075T, were observed in exon 20 of the PIK3CA gene. C2985T and C3075T were identified in one and fourteen primary OSCC tumors
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respectively, with no amino acid change at the 994 position and 1025 position (Figure 2 (c & d), Table 3). We did not observe mutation at the 1047 position in exon 20 in any of our patients
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though other studies have reported this position as mutation hotspot4,6,9,10. We did not find any missense mutations in exon 20 of the PIK3CA gene. Additionally, we identified rs114587137 (C>T) and rs17849071 (T>G) SNPs in intron 9 of the PIK3CA gene in fifteen and three OSCC patients respectively.
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4. Discussion
Although genetic abnormalities are frequent the PI3K/AKT pathway and play a fundamental role in tumorigenesis, this pathway has not yet been investigated for OSCC in the population of the
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western part of India (Gujarat), who were originated from Ancestral North Indian population, genetically divergent from rest of India29. PIK3CA gene has been found frequently mutated in
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colorectal cancer (32%), brain cancer (27%), gastric cancer (25%) and at relatively low frequency in breast cancer (8%) and lung cancer (4%) 12. PIK3CA mutation was first reported in OSCC by Qui et al. in 2006 with a 10.8% frequency in the American population7. Other studies from various ethnicities have analyzed a total of 738 OSCC subjects and observed a 5.1% mutational rate all over the world. It includes Greece, Japan, Israel, the U.S.A., the southern part of India, Thailand, Vietnam and Germany (Figure 3)12. Qui et al. reported the highest mutational
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frequency of 20.8% in OSCC patients in an American population9, whereas, the absence of hot spot mutations were reported in Greek, Vietnamese and German populations6,13,14. We have found 4% oncogenic mutation frequency and did not observe its significant correlation with the
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clinicopathological characteristics of OSCC patients. In contrast, Kozaki et al. reported a 7.4% oncogenic mutation rate and found a statistically significant association between the mutation frequency and the stage of disease4. A recent study in Taiwan has reported PIK3CA mutations in
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11/ 79 cases (13.92%) of OSCC, including a rare mutation, Q546P27.
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In the present study, we reported two missense mutations in the helical domain encoded by exon 9 of the PIK3CA gene. Interestingly, both of the somatic mutations were observed at the same organ site from the buccal mucosa of different patients with advanced stage of OSCC. It may be due to more than ten years of tobacco chewing history of both of the patients. These findings confirmed previous observations in both Asian and Caucasian populations in which a lower
Western countries1,
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oncogenic mutation frequency was observed in Asian OSCC compared to that observed in . A similar study by Murugan et al., in the south Indian population
identified a 10.8% (2/19) oncogenic hot spot mutation frequency in OSCC patients6. They
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observed E545K and frameshift mutation in helical and kinase domain respectively6. On the
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contrarily we found E542K and E545K in helical domain. Moreover, they found T1025T mutation/polymorphism in 47% of patients6, while we found it as synonymous mutation in 28% of patients.
The observed somatic mutations of PIK3CA gene, E542K and E545K, were in the helical domain, and we did not observe missense mutations in the kinase domain. The mechanism for these two mutations revealed that the resulting amino acid changes occurred at the interface between the helical domain of P110α and the nSH2 domain of P85α. Biochemical studies
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showed that Glu542 and Glu545 interact with Lys379 and Arg340 in the nSH2 domain of P85α8. Thus, mutated residues modify the inhibitory activity of P85α on the catalytic subunit and disrupt the P110α/85α complex16. Alterations in P110α enzymatic activity lead to PIK3CA oncogenic
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activation with a subsequent downstream constitutive activation of the AKT signaling pathway, which has a role in OSCC. Recent studies have shown a 5.7% prevalence of oncogenic mutations in Caucasian populations, including mutations such as E542K, E545K, Q546R, M1043I, and
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H1047R in six OSCC tissues and one cell line15. Functional studies have shown that the presence of any one of the three oncogenic hotspot mutations was able to induce the transformation in
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chicken embryo fibroblasts cultures. Therefore, increase the lipid kinase activity of the protein, which leads to the subsequent constitutive activation of the AKT signaling pathway17,18. Previous studies have shown the higher level of circulating PI3K P110α in OSCC patients compared to control subjects and concluded that PI3K P110α is over expressed in OSCC tissues compared to
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control tissues19. The observed PIK3CA oncogenic mutations in two patients may be responsible for over expression. The other OSCC patients may have some other genetic mutations such as changes in RAS, which is located upstream of the PIK3CA gene and may trigger the whole
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pathway3. The xenobiotic metabolizing genes of the patients with tobacco chewing history may be involved in the development of OSCC in the population of this area. A previous study showed
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that the GSTM1 (glutathione-S-transferase M1) null genotype is a risk factor for OSCC among the Indian tobacco-addicted population2. Likewise, a recent study in the south Indian population reported the increased prevalence of GSTM1 null polymorphism as the severity of the lesions progressed from oral leukoplakia to OSCC30. Both the studies related to GSTM1 null polymorphism in an Indian subsets suggested its role mainly in tobacco addicted OSCC patients.
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Hence, GSTM1 null polymorphism may play an immense role in the development of OSCC in tobacco habituated studied population.
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Although somatic mutations were not a frequent event in OSCC in our study, the detection of mutations is imperative to support that notion that the PI3K/AKT pathway is involved in oral tumorigenesis. The role of the PIK3CA gene mutation may be minor in our population, but the
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knowledge of PIK3CA involvement is critical. The particular kinase inhibitor could be considered as a personalized therapeutic option in OSCC patients with PIK3CA mutations. It is
chemotherapy in OSCC
20
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believed that a small selective inhibitor molecule has tremendous potential as novel cancer . The diversity in the results of various studies suggests that cultural
habits, sample size, and ethnicities may influence the mutation frequency. Future screening studies will uncover the roles of various genes that play a significant role in the development of
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OSCC to establish their clinical relevance in the population of the western part of India Our study has reported two synonymous mutations in the kinase domain of PIK3CA gene encoded by exon 20. One of these observed mutation (C>T) is located at the 3075 position with
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no amino acid change at the 1025 position and was found in fourteen OSCC samples. This finding was previously reported in the COSMIC database. Previous findings in an Indian ethnic
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population confirmed our results, as Murugan et al. studied nineteen primary OSCC tumors and found nine identical T1025T silent mutations in the people of the southern part of India6. Likewise, Kozaki et al. have reported T1025T in 4/58 OSCC tumors in a Thai population4, similarly it was also observed in breast cancer and ovarian cancer in an American population8. In contrast, this mutation has been found in pituitary tumors with a corresponding amino acid change of T1025A in a Chinese population21. The other observed synonymous mutation, C2985T, with no corresponding amino acid change at 994 position in exon 20 of PIK3CA gene.
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Though the frequency of this synonymous mutation is very less (1/50), it is not reported earlier in OSCC. We are the first to report C2985T synonymous mutation in OSCC.
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Moreover, we identified two SNPs, rs114587137 (C>T) and rs17849071 (T>G), in intron 9 of the PIK3CA gene, in fifteen and three OSCC patients, respectively. None of the patients harbored both of the polymorphisms simultaneously. The clinical significance and the role of
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SNP rs114587137 (C>T) is still unknown, and it has not been previously reported in OSCC. As we have identified SNP rs114587137 (C>T) in 30% OSCC patients, a future study with healthy
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individuals as a control will be warranted to establish its role and susceptibility. SNP rs17849071 (T>G) was reported previously in OSCC in a Greek population13. In contrast, a recent study has reported that SNP rs17849071 (T>G) is inversely associated with follicular thyroid cancer and PIK3CA amplification28. We found that both polymorphisms were found heterozygous mutated and further study with healthy individuals as control may reveal the role of the observed
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polymorphisms in the development of OSCC.
The limitation of our study was to follow up the OSCC patients. Moreover, a number of genes
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Conclusion
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may play role in development of OSCC, which are not covered in the present study.
Within the limitation of the study, the following conclusions were strained: •
The prevalence of oncogenic mutations in the PIK3CA gene is not a frequent event in the population of the western part of an India. Oncogenic mutations in PIK3CA have a minor role in the development of OSCC in a western Indian population; however, their role in personalized combination therapeutics cannot be ignored.
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•
The kinase inhibitors therapy may not be used in the current population due to the lower frequency of PIK3CA mutations; however, patients harboring PIK3CA mutations may be treated with pathway-specific kinase inhibitor-based combinational therapy instead of
•
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conventional treatment.
The performed study is cross sectional primary study. The longitudinal study with follow
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up of patients can provide more precise findings in the similar ethnic population. Acknowledgments
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We acknowledge the Dean and medical staff of the Pramukh Swami Medical College and Hospital, Karamsad, for the clinical diagnosis and collection of cancerous as well as healthy tissues for the present study. The authors thank to Dr. Anuja Kedia for her guidance in sample collection. Sejal Shah has been awarded as a woman scientist in this project.
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Figure Captions
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Figure 1
PIK3CA MAP: Exon-intron organization of the PIK3CA gene. The location of the observed somatic missense mutations E542K and E545K in exon 9, the synonymous mutations A994A
PIK3CA gene. Figure 2
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and T1025T in exon 20 andrs114587137 (C>T) and rs17849071 (T>G) in intron 9 of the
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SEQUENCE ANALYSIS: Sequence chromatogram of genetic mutations and SNPs as determined by automated sequence analysis shows: (a) G1624A (E542K) of exon 9 (1/50) of the
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PIK3CA gene; (b) G1633A (E545K) of exon 9 (1/50) of the PIK3CA gene; (c) C2985T (A994A) of exon 20 (1/50) of the PIK3CA gene; (d) C3075T (T1025T) of exon 20 (14/50) of the PIK3CA gene;(e) SNP rs114587137 (C>T) (15/50) in intron 9 of the PIK3CA gene; (f) SNP rs17849071 (T>G) (3/50) in intron 9 of the PIK3CA gene. Figure 3
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MUTATION FREQUENCY OF THE PIK3CA GENE: The mutation frequencies of the PIK3CA gene in oral cancer patients in the western part of India compared with the mutation
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frequencies of other populations. [4, 6, 7, 15, 22-27]
ACCEPTED MANUSCRIPT Table 1: Primers used for the PCR amplification of PIK3CA exon 9 and exon 20.
Sense Primer
Antisense Primer
Product size
Tm
Exon 9
5’-GATTGGTTCTTTCCTGTCTCTG-3’
5’-CCACAAATATCAATTTACAACCATTG-3’
487bp
58°C
Exon20
5’-TGGGGTAAAGGGAATCAAAAG-3’
5’-CCTATGCAATCGGTCTTTGC-3’
525bp
58°C
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PIK3CA Gene
ACCEPTED MANUSCRIPT Table 2: Summary of PIK3CA oncogenic missense mutations in Oral Squamous Cell Carcinoma in Indian population.
Age
Gender
Mutation
Mutation type
Tumor site
Classification Stage T
N
M
15
31
M
E542K
G:C>A:T
BM
4
1
0
56
38
M
E545K
G:C>A:T
BM
4
1
0
IV
Tobacco chewing >10 yrs Tobacco chewing >10 yrs
IV
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BM- buccal mucosa
Habit
Differentiation
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Sample
Well
Well
Table 3: Summary of PIK3CA synonymous mutations in Oral Squamous Cell Carcinoma
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Mutation
Codon Change
Amino Acid position
Mutation Type
Status of Mutation
Domain
3
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
8
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
12
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
17
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
19
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
30
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
31
20
2985
GCC/GCT
994
Synonymous
Heterozygous
Kinase
32
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
33
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
36
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
46
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
52
20
3075
1025
Synonymous
Heterozygous
Kinase
57
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
58
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
60
20
3075
ACC/ACT
1025
Synonymous
Heterozygous
Kinase
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ACC/ACT
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Sample no.
ACCEPTED MANUSCRIPT Table 4: Correlations between PIK3CA oncogenic mutations and the clinicopathological characteristics of OSCC patients No mutation
Total number (%)
2(4%)
48(96%)
Age (years) [mean]
34.5(31-38)
48.02(27-70)
2 0
Tumor site Buccal Mucosa Others*
2 0
Disease stage I-III IV
0 2
34 14
30 18
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Gender Male Female
Histological grading Well Moderate Poor
1 1 0
16 32
25 12 11
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* Others include tumor site at the tongue, lip, hard palate, GB complex and alveolus p values were calculated using Fisher’s exact test
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p-value
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Observed mutation
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Characteristic
p<0.5
p<0.4
p<0.4
p<1.0
ACCEPTED MANUSCRIPT Table 5. Demographic and clinicopathological characteristics of OSCC patients included in the study.
n=50
Frequency (%)
n=34 (tumor control pair)
72 28
27 7
Gender
Male Female
36 14
Age
Mean Range
47.14 47.14
Risk Habits
Tobacco Chewing Smoking Chewing + smoking None
37 1 4 8
Tumor Site*
Buccal Mucosa Tongue Lip Hard Palate GB complex Alveolus
32 6 1 2 6 3
Tumor Size
T1 &T2 T3 & T4
Lymph node Metastasis
Negative Positive
TNM Stage
Tumor Differentiation
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Variable
45.74 45.74
74 2 8 16
28 2 1 3
64 12 2 4 12 6
19 5 1 2 6 1
17 33
34 66
13 21
12 38
24 76
8 26
Early (I&II) Late (III&IV)
8 42
16 84
3 31
Well Moderate Poor
26 13 11
52 26 22
23 6 5
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*The study includes patients with SCC of left or right buccal mucosa, three, two and one of
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the tongue lesions were taken from right lateral border of tongue, left lateral border of the tongue and central part of the tongue respectively. One patient with chronic tobacco chewing history >15 years developed lower lip stage IVa OSCC (mucosal extent of lip) (CT4 N2c Mo), two patients with left side hard palate. Four lesions with right lower GB complex, two lesions from left lower GB complex, two alveolar lesions from lower alveolar left side and one sample from upper alveolar left side included in the study.
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Statement of Clinical relevance
“PIK3CA gene may be a prognostic biomarker and the knowledge of the involvement of
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kinase inhibitors along with conventional therapy”.
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PIK3CA gene is important for the patients harboring such mutations and will be benefited with