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| ORIGINAL ARTICLE |
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| Year : 2022 | Volume
: 14
| Issue : 2 | Page : 107-113 |
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Emerging Trend of Oral and Oropharyngeal Squamous Cell Carcinoma in Patients Less than 40 Years: A Molecular Analysis of Role of HPV in Cases with No Known Risk Factors
Priyanka Nair1, Usha Hegde1, Sreeshyla Huchanahalli Sheshanna1, Sunila Ravi2
1 Department of Oral Pathology and Microbiology, JSS Dental College & Hospital, JSS AHER, Mysuru, India
2 Department of Pathology, JSS Medical College, JSS AHER, Mysuru, India
| Date of Submission | 09-Mar-2022 |
| Date of Decision | 07-Oct-2022 |
| Date of Acceptance | 20-Oct-2022 |
| Date of Web Publication | 10-Jan-2023 |
Correspondence Address:
Dr. Usha Hegde
Department of Oral Pathology and Microbiology, JSS Dental College & Hospital, Mysuru, 570015
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/jofs.jofs_72_22
Introduction: Traditional risk factors causing oral and oropharyngeal cancers have been extensively studied in elderly populations. But recent evidences have shown it to be rising in young individuals with no known risk factors. Human Papilloma virus (HPV) is a proven etiologic factor for cervical cancer and has been suggested in oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC). Hence the objective of the study was to evaluate the role of HPV as a risk factor in OSCC and OPSCC patients of less than 40 years of age without known risk factors. Material and Methods: Fifteen years of retrospective data were used for recording the age, gender, site, and various risk factors in patients ≤40 years of age diagnosed histopathologically with primary OSCC and OPSCC. Role of HPV in patients without any known risk factors were evaluated with p16 Immunohistochemistry (IHC) and polymerase chain reaction (PCR). Results: Ninety eight patients aged ≤40 years of age were obtained with known and no known risk factors. Among the habitual risk factors, alcohol did not prove to be a significant factor. Female patients with mean age of 34 years were more prone, though they were not subjected to any of the known risk factors. Tongue and buccal mucosa were the most common sites affected. Role of HPV as a risk factor was found positive. All cases of PCR positive were IHC positive and IHC showed 100% sensitivity and 68.4% specificity. Conclusion: A strong correlation of HPV with OSCC and OPSCC in adults ≤40 years of age was established in the present study.
Keywords: Human Papilloma virus, oral squamous cell carcinoma (OSCC), oropharyngeal squamous cell carcinoma (OPSCC), p16, polymerase chain reaction, South Indian population, young
How to cite this article:
Nair P, Hegde U, Sheshanna SH, Ravi S. Emerging Trend of Oral and Oropharyngeal Squamous Cell Carcinoma in Patients Less than 40 Years: A Molecular Analysis of Role of HPV in Cases with No Known Risk Factors. J Orofac Sci 2022;14:107-13 |
How to cite this URL:
Nair P, Hegde U, Sheshanna SH, Ravi S. Emerging Trend of Oral and Oropharyngeal Squamous Cell Carcinoma in Patients Less than 40 Years: A Molecular Analysis of Role of HPV in Cases with No Known Risk Factors. J Orofac Sci [serial online] 2022 [cited 2023 Jun 8];14:107-13. Available from: https://www.jofs.in/text.asp?2022/14/2/107/367449 |
| Introduction | |  |
In 1959, Dalitsch and Vazirami examined increasing incidence of oral cancer and stated “The disease of our century is cancer.”[1] Oral cancer remains a fatal and constant problem which accounts for 4% of cancers worldwide.[2] It is the fifth most common cancer globally.[3] Markedly higher prevalence of oral cancer is found in some Asian populations, mainly in South Asian countries including India and Pakistan.[4] Squamous cell carcinoma (SCC) accounts for more than 90% of oral cancers.[5] Oral cancer is rare in young patients and has reached an incidence of 0.4% to 3.6% in patients younger than 40 years.[6] The recent evidence suggests an absence of traditional factors in a significant proportion of younger patients. Moreover, the time span for carcinogens such as tobacco and alcohol to exert a detrimental effect in these younger patients is relatively short. It is also important to examine other potential risk factors, such as environmental carcinogens, stress, previous viral infections, and familial episodes of cancer.[7] Some studies have suggested that these patients may exhibit a predisposition to genetic instability.[8]
Young adults most commonly develop primary tumor in oropharynx and oral cavity and less frequently in the larynx compared to older patients.[9],[10] It has even been suggested that oral cancer in the young may be a disease distinct from that occurring in older patients with a different etiology and disease progression.[7]
Human Papilloma viruses (HPV), members of the papillomaviridae family, consists of more than 100 types which are further categorized into low-risk (LR) and high-risk (HR) types according to their potential for causing SCC.[11] Examples of LR-HPV genotypes are HPV-6, 11, 42, 43, 44 and of HR-HPV genotypes are HPV-16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59.[12]
Over the past 15 years, HPV, the necessary cause of cancer of the cervix,[13] has also been etiologically linked with a subset of head and neck squamous cell carcinomas (HNSCC). Some of the high-risk types have been found in the oral cavity and oropharynx of cancer-free adults and in cancer biopsy specimens from HNSCC patients. HPV16, the most prevalent HPV type in cervical SCCs, is also the most common type present in HPV-positive HNSCCs.[14] In the absence of any known risk factors, HPV has been consistently implied as a causative element in recently emerging evidences.[15] It is time to focus on the prevention and early detection for the survival of young people who form a huge part of the future of the nation. Hence, the present study was undertaken to evaluate the different risk factors causing oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) in young adults ≤40 years of age and also to assess the association of HPV in those patients without any known risk factors.
| Material and Methods | | |
Ethical approval for this study (Ethical committee Ref No: JSS/ACP/Ethical/2012-13) was provided by the Institutional Ethical Committee of JSS Dental College & Hospital, Mysuru, on January 31, 2013. Fifteen years of retrospective data from records of patients ≤40 years of age diagnosed with primary OSCC and OPSCC from our institution were taken. The study group was divided into two groups − group 1: Cases with known risk factors and group 2: Cases with no known risk factors. As the pathophysiologic behavior of lip cancer is believed to be substantially different from the oral cavity sites, cancers originating in the lip were not included in the study. Cases with underlying immunocompromised conditions and cases with metastasis to oral and oropharyngeal areas from other sites were excluded.
All details were studied carefully and the age, gender, site, and various risk factors implicated in OSCC and OPSCC were recorded. Formalin fixed paraffin embedded (FFPE) samples from group 2 were further taken and p16 Immunohistochemistry (IHC) marker and polymerase chain reaction (PCR) were performed.
HPV screening
5-µm thick sections from FFPE blocks were cut and mounted on poly-l-lysine coated slides. IHC staining was carried out using polymer labeling technique. Sections were dewaxed, washed in alcohol, and antigen retrieval was carried out in pressure cooker with 10 mM citrate buffer solution for 15 minutes. Endogenous peroxidase was blocked by using 0.3% hydrogen peroxide in methanol at room temperature for 10 minutes. Slides were washed twice with Tris buffer (TBS) briefly and incubated with primary antibody (Biogenex − Anti-p16[INK 4], monoclonal, anti-mouse) for 60 minutes. Sections were washed with phosphate buffer solution (PBS) and incubated with the secondary antibodies for 30 minutes. Sections were then washed with PBS and DAB was used as the chromogen solution in hydrogen peroxide for 10 minutes. Sections were then counterstained with hematoxylin and studied. The sections were considered positive if cytoplasm of tumor cells was stained brown, excluding the cells surrounding the blood vessel.
HPV detection
Detection of HPV was done using DNA amplification with Consensus Primers. Twenty μm slice of paraffin wax embedded tissue was collected, dewaxed, and then rehydrated. After rehydration, samples were digested with proteinase K (20 mg/mL) at 55°C for 1 to 3 hours. Total genomic DNA from the retrieved tissue was isolated by N-Cetyl- N, N, N-trimethyl-ammonium bromide (CTAB) method using extraction buffer. Once the quality and quantity of DNA was checked the samples were further considered for further PCR analysis with HPV 16 designed using Primer3 Plus software and the designed oligonucleotides synthesized in Sigma Corporation USA. PCR reaction mixture and PCR temperature profiling prior to Agarose gel electrophoresis was done. Once the sample was run sufficiently, the gel was removed using gloves and visualized under UV light, photographed and documented using UV trans-illuminator.
Descriptive statistics, mean and standard deviation was done to assess age, site, and gender. Chi-square test was done to find the association between risk of developing cancer and other variables used in the study. Chi-square test was also used to test the difference between negative and positive cases of IHC and PCR and also to find an association between IHC and PCR methods, independent “t” test to analyze the difference between both groups (group 1 and group 2) and mean age, one way Analysis of variance (ANOVA) to compare mean age with groups (group 1 and group 2) and gender.
| Results | | |
From the 15 years of retrospective data obtained from the records, a total of 98 cases of ≤40 years of age, diagnosed with primary OSCC and OPSCC were obtained based on the inclusion and exclusion criteria considered in this study. The age, gender, and site of lesion were available for all cases. However, the detailed history regarding the risk factors/tobacco habits were unavailable for 26 cases. Though all cases could be subjected to statistical analysis with respect to age, gender, and site, only 72 cases could be assessed for known risk factors of which 40 cases gave a history of known risk factors and 32 did not. On statistical analysis we found that from the overall sample of 98 cases, mean age was 36.28 ± 4.58 years with mean age of males and females being 36.84 ± 2.99 and 35.81 ± 5.58 years, respectively. Though statistical significance was not drawn with respect to gender, more number of cases were recorded in females compared to that of males. Most common site affected was tongue followed by buccal mucosa and these results were statistically significant.
When group 1 and 2 were analyzed separately, the mean age recorded was statistically significant [Table 1] with increased risk seen with advancing age. With regard to gender and site [Table 2] and [Table 3] respectively], no significant results were drawn except in group 1 with regard to gender, 71.9% males had chance to develop cancer when exposed to known risk factors. There is a thin line of difference in the site predilection between tongue and buccal mucosa in both the groups. Strong correlation was obtained with tobacco, smoking, and arecanut [Table 4], but not with alcohol habituers [Table 5] or with synergistic effects of tobacco, alcohol, and smoking. | Table 4 Table showing comparison of significant habits and risk of developing cancer
Click here to view |
 | Table 5 Table showing comparison of alcohol and risk of developing cancer
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For performing IHC and PCR, 25 cases were chosen from group 2 (FFPE blocks were not available for two cases and insufficient amplification was noted in five cases). On IHC, 13 samples were positive and on PCR, six samples were positive with 850 bp amplicon. On statistical analysis, PCR showed statistical significance (P value = 0.009) but IHC did not (P value = 0.715). Statistical significance was found between IHC and PCR comparison [Table 6]. In IHC 48% cases were positive as against only 24% in PCR, implying low specificity with IHC.
| Discussion | | |
Though OSCC has a worldwide increase in incidence and mortality, variations do occur in respect to the demographics or lifestyle and habits within different populations. Higher prevalence is consistently found in some Asian populations, mainly in South Asian countries including India, Pakistan, Srilanka, and Bangladesh.[16] Although it is known that HNSCCs usually occurs in patients older than sixth decade of life, incidence is rising in younger age groups and have been reported from different parts of the world. Among them, there is a subgroup of population who reported little or no exposure to the major risk factors.[17] Also there is rising incidence of OPSCC in the absence of rise in smoking and alcohol consumption.[18] These led to the search for additional analytical data on risk factors in such younger age group patients. HPV, a known cause of cervical cancer, has been studied extensively as a strong etiological factor for development of OSCC and OPSCC. In the present study, from the data available, only a marginal difference was found in the number of cases with known risk factors and without risk factors (group 1 and 2) indicating emerging rise in development of cancer in young individuals without prior habitual or personal history.
The mean age of patients ≤40 years of age among both the genders in group 1 and 2 was in the range of 34 to 37 years. The mean female age with no risk factors was 34 years which makes young women in the middle age to be more prone to SCC inspite of no personal or habitual history. Age average in cases registered in literature as young bearers of SCC ranges from 30.8 to 34.2 years.[19] Studies in Taiwan and Northern Thailand showed mean ages to be 36 and 39 years respectively.[8],[20] In the present study, males and females had a risk of 28.1% and 57.5% to develop SCC with females to be at a higher risk in group 2. In group 1, men were affected more than females which had a correlation to numerous studies.[21],[22]
With regard to site, previous studies have shown a predilection between tongue[8],[10],[23],[24] and buccal mucosa.[8],[21] Countries like USA, Australia, Brazil, and Denmark reported tongue to be the most common site.[25],[26] On the contrary, other Asian countries such as Taiwan, Malaysia, Papua New Guinea, Bangladesh, and India have found that buccal mucosa was the most common location.[27],[28],[29] But in the young, OSCC of the buccal mucosa was rare, possibly because of the reducing habit in this age group, while the older population continues to practise the habit.[30] Our study showed tongue and buccal mucosa to be the most common sites in both group 1 and group 2. Buccal mucosa was more affected than tongue in the group 1, but was vice versa in group 2. In group 2, though number of cases was equal in tongue and buccal mucosa, tongue had 57.1% chances of developing SCC compared to buccal mucosa which was 36.4%.
Various risk factors like tobacco,[31],[32] smoking,[33] arecanut,[34],[35] and alcohol[36],[37] have been extensively studied. A definite correlation of these factors with OSCC and OPSCC has been established. In our study, significant results were obtained with risk factors such as chewable tobacco, smoking, and arecanut. Results were not significant in cases of alcohol and other synergistic effects with tobacco, alcohol, and smoking.
In a review on risk factors in young people, Llewellyn et al. have discussed in detail regarding the other risk factors like occupation, immune defence, viral infection, diet, genetic, and familial factors.[6] Studies have been done on anemia[38] and trauma[39] as risk factors of oral cancers. An inverse association between coffee and cancer risk have also found plausible biological support, since coffee beans contain several phenolic compounds with antioxidant properties, such as caffeic acid and chlorogenic acid.[40] In the present study, inspite of thoroughly checking the data available, no other risk factors were reported.
Different methods have been employed for the detection of HPV.[41],[42] Numerous studies have been done using p16 IHC and PCR for detection of HPV in tissues.[43],[44],[45],[46] Most studies were done in the past on association of HPV with OPSCC,[47],[48],[49],[50],[51],[52] but in our study, most cases belonged to the oral rather than oropharyngeal SCCs as those diagnosed of OPSCC were most with habitual history or relevant case sheets were missing to record the known risk factors and could not be considered for further study.
Pannone et al. conducted a study to demonstrate the reliability of triple method applied to detect HPV in HNSCC originating from OSCC and OPSCC using p16 IHC, in-situ hybridization (ISH), and consensus PCR. All the HNSCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC. The IHC results showed a very high level of sensitivity (100% in both OSCC and OPSCC) but lower specificity (74% in OSCC and 93% in OPSCC).[43] Our findings were in agreement with their study, wherein the comparative analysis between IHC and PCR showed sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 68.42%, 50%, and 100% respectively.
Many studies have been done to establish association between OSCC and OPSCC with HPV but very few studies have been done to establish this association in cases with no known risk factors, especially in Indian population, which is the strength of the present study. However, the present study does have few limitations. A prospective study conducted for a longer period of time would give better clarity of data of the patients with a definite personal and habitual history. Further, fresh frozen tissues would be available for HPV confirmation with PCR which gives better results. Thus the present study indicates a strong HPV correlation with OSCC and OPSCC, thereby suggesting it as one of the causative factor for head and neck cancers and as the young population, especially young females, is under threat to develop OSCC and OPSCC without any prior exposure to earlier proven risk factors, prophylactic vaccines against HPV would help in a long-term betterment in these individuals.
Authors’ contribution
PN − Data search, Methodology, Manuscript preparation,
UH − Concept design, Data analysis, Review
SHS − Literature search, Manuscript editing, Proof
SR − Concept design, Review
Acknowledgments
The authors sincerely thank JSSDC&H, JSS AHER, Mysuru for the opportunity to retrieve all data and materials needed for the study, M R Ambedkar Dental College and Hospital, Bangalore, and Credora Life Sciences, Bangalore for their technical support.
Financial support and sponsorship
Nil.
Conflicts of interest
Authors declare no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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