|Year : 2021 | Volume
| Issue : 1 | Page : 12-18
Human papilloma virus testing in oral squamous cell carcinoma in Southern India: A case–control study
Jennifer Prince Kingsley1, Pranay Gaikwad1, Priya Abraham2, John Chandrakumar Muthusami3
1 Department of Surgery Unit 1 - General and Head and Neck Surgery, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Virology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Surgery, Oman Medical College, Sohar, Oman
|Date of Submission||21-Jun-2020|
|Date of Decision||04-Aug-2020|
|Date of Acceptance||14-Aug-2020|
|Date of Web Publication||13-Jan-2021|
Dr. Pranay Gaikwad
Department of Surgery Unit 1 - Head and Neck Oncosurgery, Christian Medical College, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Oral cancer is a major public health burden in India ranking third among cancers; about 90% of all oral cancers are oral squamous cell carcinomas (OSCCs). Human papillomavirus (HPV) is a well-established oncogenic agent in the causation of cervical squamous cell carcinoma (SCC) and an important risk factor for oropharyngeal SCC, but the link between HPV and OSCC is inconsistent. Knowledge of HPV profile in the OSCC has positive implications for treatment and prevention strategies. Methods: A case–control study was conducted to determine the prevalence of HPV status in 40 newly diagnosed OSCC cases (24 males; 16 females). Age- and gender-matched controls were recruited from the outpatient clinic of a large teaching hospital in Southern India. A questionnaire was used to ascertain the risk factors for OSCC. Using a cytobrush, epithelial cells were obtained from oral cancer lesions or normal mucosa in cases and controls, respectively. The HPV detection and genotyping were done using a real-time polymerase chain reaction technique. Results: Chewing tobacco was strongly associated with OSCC (odds ratio: 3.6; 95% confidence interval: 1.4–9.7), adjusted for potential confounding factors including smoking and alcohol consumption. All the OSCC cases and controls tested negative for HPV DNA. Conclusions: There is an inconsistency in the prevalence of HPV infection in OSCC in India. The prevalence of HPV in OSCC was nil, so the testing for HPV may be unnecessary. The inconsistency of the positivity of HPV in OSCC may be due to a wide palette of geographic and sociocultural differences that exist in the subcontinent. Further studies are, therefore, needed to evaluate the prevalence of HPV in OSCC in the subcontinent and plan optimal therapeutic strategies.
Keywords: Human papillomavirus, oral cancer, squamous cell carcinoma
|How to cite this article:|
Kingsley JP, Gaikwad P, Abraham P, Muthusami JC. Human papilloma virus testing in oral squamous cell carcinoma in Southern India: A case–control study. Curr Med Issues 2021;19:12-8
|How to cite this URL:|
Kingsley JP, Gaikwad P, Abraham P, Muthusami JC. Human papilloma virus testing in oral squamous cell carcinoma in Southern India: A case–control study. Curr Med Issues [serial online] 2021 [cited 2021 Apr 20];19:12-8. Available from: https://www.cmijournal.org/text.asp?2021/19/1/12/306926
| Introduction|| |
Oral cancers are defined as the cancers involving the oral cavity beginning at the lips and extending to the anterior pillar of the fauces and include cancers of the lip, buccal mucosa, floor of the mouth and tongue., Globally, oral cancer ranks sixth among cancers., Oral cancer ranks third among cancers in India following breast and cervical cancer. Oral squamous cell carcinoma (OSCC) accounts for >90% of all oral cancers., The primary risk factors for OSCC in India include tobacco smoking or chewing, betel quid chewing, and regular consumption of alcoholic beverages. However, 20% of oral cancers develop without definite risk factors suggesting the role of other risk factors.
Human papillomavirus (HPV), a DNA virus, is a sexually-transmitted infection. It is a “necessary cause” of cervical cancer, 70% of which are squamous cell carcinoma (SCC)., Of more than 100 HPV genotypes, 15 are classified as “high-risk” as they induce lesions progressing to cancer. The HPV 16 and HPV 18 are the most common high-risk genotypes in cervical cancer prevalent in 70% of cervical cancer.
Oral sexual practices have the potential to transmit HPV to the oral cavity. In the last decade, HPV has emerged as an important risk factor for oropharyngeal (pharynx, the base of the tongue, and tonsil) SCC, a subgroup for head-and-neck SCC. On the other hand, the role of HPV in OSCC is less clear. The International Agency for Research on Cancer (IARC) noted wide variations in HPV prevalence among OSCC from different countries ranging from 2% to 74%. A review of HPV in head-and-neck SCC worldwide (60 studies from 26 countries) found a significantly lower prevalence of HPV in OSCC (23.5% of 2642 cases; 95% confidence interval [CI] = 21.9–25.1) or laryngeal SCC (24.0% of 1435 cases; 95% CI = 21.8–26.3) as compared to oropharyngeal SCC (35.6% of 969; 95% CI = 32.6–38.7).
Most data on HPV and OSCC are from developed countries, while there is a paucity of research in South Asia. Knowledge of the HPV profile in OSCC has important implications for management. The HPV-positive tumors tend to be more radiosensitive, and the HPV vaccine used in cervical cancer prevention may benefit squamous cell cancers at other sites, including OSCC.
This study attempts to evaluate the HPV prevalence among OSCC patients hailing from all over India and the neighboring countries attending the outpatient department of a major teaching hospital in Southern India.
| Methods|| |
A hospital-based prospective case–control study was conducted in the Department of General Surgery-Head and Neck of a tertiary-care medical college hospital from south India. The study was approved by the Institutional Review Board.
The study included all the newly-diagnosed histologically confirmed cases of OSCC that presented to the outpatient clinic. The subsites of the oral cavity included the tongue, gum, cheek, oor of the mouth, palate, and other parts of the mouth and mucosa of the lip. Cases with previous cancers, more than one primary cancer, and previous history of radiation or chemotherapy were excluded. For each case subject, a control subject who also attended the General Surgery outpatient clinic at the hospital without oral cancer, any other type of cancer, leukoplakia, or chronic infection was selected matched by age (±5 years) and gender. Written informed consent was obtained for all patients.
At enrollment, a questionnaire was administered at a dedicated space provided at the clinic to elicit information on sociodemographic and potential lifestyle risk factors for oral cancer.
Biological specimen collection
After rinsing the mouth once with drinking water, exfoliated oral squamous cells were collected using a conical cytobrush. For both cases and controls, buccal mucosa scrapings were collected in the following areas: right and left buccal mucosa; right and left upper gingiva buccal sulcus; right and left lower gingival sulcus; right and left lateral borders of the tongue; the floor of the mouth; and the posterior third of the tongue. For the confirmed cases of oral cancer, samples were obtained from the oral lesions as well. Cells collected were mixed with a transport medium, a prepacked product from Hybribio®. The specimens were then stored at -70°C until laboratory analysis.
Real-time polymerase chain reaction
Real-time polymerase chain reaction (RT-PCR) was performed using the Hybribio extraction kit® (Hybribio Ltd., Hong Kong), a gene array kit used for DNA amplification and flow-through hybridization technique. If HPV was found to be positive, there was a provision to identify HPV subtypes, including both high-risk (16, 18, 33, 35) and the low-risk types (6, 11). The test utilized L1 consensus primers to simultaneously amplify 21 HPV genotypes, followed by flow-through hybridization with immobilized genotype-specific probes.
Unconditional logistic regression was used to assess univariate associations between each risk factor and OSCC, estimating crude odds ratios (ORcrude) and corresponding 95% CIs. Variables having biologically plausible associations with OSCC, irrespective of their significance in univariate analysis, were assessed further in multivariate logistic regression analysis estimating adjusted ORs (ORadjusted). Analyses were performed using the IBM SPSS Statistics 25.0 (IBM Corp, 2017).
| Results|| |
A total of 40 histologically-confirmed OSCC cases and 40 controls were recruited in this case-control study. The cases and controls were matched for age (±5 years) and gender. The mean age (± standard deviation) of cases and controls was 52 years (±11) years and 50 years (±10 years), respectively. A male preponderance was noted among OSCC cases (males, 60%; females, 40%) [Table 1].
|Table 1: Univariate and multivariate logistic regression models for selected risk factors and oral squamous cell carcinoma, crude and adjusted odds ratios and 95% confidence intervals|
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In univariate analyses, tobacco chewing was the only risk factor that was statistically associated with OSCC (ORcrude = 3.7, 95% CI = 1.4–9.5). The cases and controls were similar in smoking patterns, alcohol consumption, diabetes, hypertension, or dental disorders.
The strength of the association of tobacco chewing with OSCC remained unchanged after adjustment of covariates, including age, gender, educational level, smoking, and alcohol consumption (OR adjusted = 3.6, 95% CI = 1.4–9.7). Further, tobacco chewing was categorized into three groups based on the duration of chewing tobacco. The subjects with <10 years of usage were considered as the referent group and the OR compared to those with 10–20 years (OR of 1.7 (95% CI: 0.4, 3.2) and more than 20 years of usage OR of 6.9 (95% CI: 1.5, 35.5).
[Table 2] presents the clinicopathological features of oral cancer lesions among cases (n = 40). The lesions were most commonly located in the buccal mucosa (n = 19, 47.5%) followed by the tongue (n = 15, 37.5%). In 21 cases (52.5%), the lesions were 2–4 cm in size, and in 17 cases (42.5%), the size was 4 cm or more. Lymph nodes were present in 24 of 40 cases (60%) and were predominantly single and <3 cm in size. The tumors were moderately differentiated in 24 cases (60%), and well-differentiated tumors in 14 patients (35%); only two cases (5%) were poorly differentiated. With regard to the TNM staging, 18 cases (45%) presented in Stage III and 11 cases (27.5%) presented in Stage IV. Eight cases (20%) presented in Stage II, and three cases (7.5%) presented with Stage I disease.
|Table 2: Clinicopathological features of oral squamous cell carcinoma lesions|
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All of the 80 subjects included in the study (cases, n = 40; controls, n = 40) were tested for HPV DNA, but none of the subjects from either group tested positive.
| Discussion|| |
This hospital-based prospective case-control study evaluated the association between a marker for HPV infection and OSCC at a tertiary care hospital setting in southern India. Forty cases who were histologically-confirmed OSCC, and with their age- and gender-matched controls (n = 40), were included in the study. A questionnaire ascertained information on the risk factors. The HPV testing was done using the highly sensitive RT-PCR, with provision for genotyping. The most glaring finding of this study was that all of the OSCC cases and the controls tested negative for HPV DNA.
This assay was found to be highly comparable to the Roche's LA Assay. The PCR amplification used primers PGMY 09/11 (target size 450 bp). The housekeeping genes were beta-globin (target size 230 bp). The test has a sensitivity and specificity of >95%.
Although the procedure is similar to standard PCR, there are several advantages to RT-PCR. The RT-PCR can identify amplified fragments during the PCR process and measure the amount of the product during the exponential phase, while the standard PCR measures product during the plateau phase that may not indicate the quantity of starting material. Another advantage is that the entire process from amplification to analysis is performed in the same tube, minimizing contamination, whereas, in standard PCR, the product is moved and manipulated into other formats.
The negative test results of HPV DNA in this study do raise some doubts about the validity of the results, especially when there is data showing HPV positivity in OSCC in studies from India. This allowed us the opportunity to re-examine our methods.
The biosamples were obtained from oral mucosal scrapings have shown to be a highly sensitive and reliable method to make cytological diagnoses of oral neoplasia. The samples were collected, transported to the laboratory, stored, and analyzed following a standard protocol that included rigorous quality assurance/quality control procedures. All the biospecimens were collected by a single person trained in the collection, and all samples were analyzed at the hospital Virology Laboratory, a World Health Organization-accredited research center. Adequacy of each sample was confirmed by measuring β-globin. The HPV DNA viral load was measured with RT-PCR, a technique that is considered to be the most sensitive test for DNA viruses. The use of PCR techniques and liquid-phase, immunocaptured hybridization eliminated the severe misclassification of HPV status by the first generation of molecular epidemiology studies., The PCRs were confirmed using housekeeping genes with both positive and negative controls. All of the control subjects tested negative. Further, the results of similar RT-PCR testing done for HPV in cervical mucosa in the same laboratory showed high detection rates in cervical cancer. Thus, it may be safely concluded that the results of the HPV DNA testing in this study were accurate.
The findings of the present study are consistent with other, relatively recent (published between 2011 and 2018), studies from India [Table 3]. The single common result of all these studies was that they were unable to show any HPV-positive test among the OSCC cases with the number of cases tested ranging from 40 to 350.,,,, Of the five studies, three were conducted in South India.,, One of the abovementioned three studies is the largest study to date in India with 350 OSCC cases. This hospital-based case-control study was conducted in South India, biospecimens were collected by oral brushings, and samples tested by PCR, features that are similar to our study. The prevalence of HPV DNA was 0%; the investigators concluded that “there is no role for HPV in OSCC in Southern India.” HPV negativity has also been reported in other countries such as Bangladesh, the Netherlands, and China.,, Taken together, the above studies suggest that HPV negativity is plausible in OSCC.
|Table 3: Studies of human papilloma virus status and oral squamous cell carcinoma from India according to human papilloma virus status|
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HPV negativity in OSCC may be explained by a low prevalence of high-risk sexual behavior. The HPV is detected more commonly among patients with more than one sexual partner and from those who practiced oral sex. Data from the WHO on sexual health behavior indicators in India are scarce. The few available studies in India suggest that oral sex is not a popular practice due to the cultural bias against such behaviors. Other risk factors for increased risk of HPV infection such as premarital sex and having multiple partners are also less prevalent in India (15%–20%) when compared to the prevalence of these factors noted in Western countries (75%).,, Due to the culturally sensitive nature of questions and the potential ensuing psychological risk compounding the psychological trauma due to cancer diagnosis, it was not possible to elicit a detailed history of sexual behavior among the study group. It has been reported that a relatively high proportion of subjects, particularly cases, had missing data on questions on oral sex. Among those who responded, 47% reported a history of oral sex, and 90% of cases and controls reported as having only one-lifetime partner. There was no difference in OSCC distribution among subjects who chose not to provide information on oral sex versus those who reported a negative history of oral sex. Such patterns of low-risk sexual behavior stemming from cultural and religious norms have a low potential for HPV transmission to the oral cavity.
In contrast to the negative studies, several studies from India report HPV positivity among OSCC ranging from 15% to 74%., It is interesting to note that except for one study, all these studies that showed a high incidence of HPV in OSCC were reported before 2011, while the test was consistently negative in the studies reported after 2011. This is indeed a matter of further investigation with regards to the evolving diagnostic techniques over the last three decades and raises a question if the results predating 10 years or more can be considered valid today. Moreover, the findings from two studies (including the present study) that reported nil incidence of HPV in OSCC could be considered at best Level III evidence (case-control studies) as compared to all the studies that reported positive HPV test in OSCC which would qualify for Level IV evidence (case series).
The reason for this wide variation of presence of HPV infection in the population from a single country but different geographic areas is not clear. However, multiple etiopathogenic factors, as well as the accumulation of several aberrations in a normal cell, are required for the transformation of the normal cell into a malignant phenotype. Geographically, two of the studies with a high incidence of HPV in OSCC were reported from southern India, while the same number of studies, including the present study with a nil incidence, were also conducted in southern India., Although the reason for this difference is unclear, the present study recruited the patients coming from different parts of India and some neighboring countries, making it a more heterogeneous study population that may not have been predisposed to the HPV infection. However, analysis of pooled data from different countries limited to the studies with >40 cases and using PCR for HPV testing revealed an HPV prevalence ranging from 4% to 80%.
Molecular evidence provides support for certain types of HPV in the pathogenesis of OSCC. The IARC in their evaluation of HPV and OSCC concluded that there is “sufficient evidence in humans for the carcinogenicity of HPV 16 in the oral cavity,” a conclusion based on strong evidence in exposed humans and sufficient evidence of carcinogenicity in experimental animals. They also concluded that there was “limited evidence in humans for the carcinogenicity of HPV 18 in the oral cavity,” a conclusion based on limited evidence in humans and sufficient evidence of carcinogenicity in experimental animals. Others have reported that HPV 16 accounted for a larger majority of HPV-positive oropharyngeal SCC (86.7%; 95% CI = 82.6–90.1) compared with HPV-positive OSCC (68.2%; 95% CI = 64.4–71.9) and laryngeal SCC (69.2%; 95% CI = 64.0–74.0). Conversely, HPV18 was rare in HPV-positive oropharyngeal SCC (2.8%; 95% CI = 1.3–5.3) compared with OSCC (34.1%, 95% CI = 30.4–38.0) or laryngeal SCC (17.0%, 95% CI = 13.0–21.6). Besides HPV16 and HPV18, other oncogenic HPV types were rarely detected in HNSCC. It was noted that tumor site-specific HPV prevalence was higher among studies from North America compared with Europe or Asia. As noted above, the frequency of high-risk sexual behaviors is high in western countries.
The male-to-female ratio of 1.5:1 is comparable with previous studies on OSCC, which found a male predilection. Men are more likely to have a higher frequency of established lifestyle risk factors for OSCC compared to women. National data for the 15–49 years population subgroups in India found that men were 5.2 times more likely to use some form of tobacco compared to women (57% vs. 11%).
In this study, a statistically significant strong association was found between tobacco chewing and OSCC, with the risk increasing as the duration of exposure increased, especially beyond 20 years. Other than tobacco chewing, there were no significant differences in the distribution of other established risk factors among cases and controls in this study. This significance seems to be independent of the effects of smoking tobacco and consumption of alcohol. As per the IARC, tobacco smoking or chewing and regular consumption of alcoholic beverages are strongly associated with OSCC, with attributable fractions of about 90%. Tobacco chewing was found to be an independent causative factor for the incidence of OSCC., The relationship stays significant even when the OR is adjusted for smoking tobacco and consuming alcohol. This shows that there is minimal confounding in the causative factors. According to the only systematic review reported on the subject, there is no conclusive evidence to demonstrate an association of smoking and alcohol consumption with HPV-associated oral cancer.
Small sample size and heterogeneity of the patient population were the limiting factors for this study, but when taken along with other pooled data from 2011 onwards, it certainly contributes to the understanding of the relationship between HPV and OSCC.
| Conclusions|| |
The male preponderance and tobacco chewing are the established risk factors for OSCC. Most of the recent studies from India have not found enough evidence to link the positive RT-PCR test result for HPV and oral squamous cell cancer. Conversely, most studies that have reported HPV positivity were reported before 2011. These wide variations in HPV prevalence in OSCC in India may be attributable to geographic differences in the socio-cultural practices that may either facilitate or impede HPV transmission to the oral cavity. There is a need to evaluate HPV prevalence among OSCC in local settings. Gathering such data will enable targeted educational, treatment, and preventive strategies tailored to local needs.
Research quality and ethics statement
The authors of this manuscript declare that this scientific work complies with reporting quality, formatting and reproducibility guidelines set forth by the EQUATOR Network. The authors also attest that this clinical investigation was determined to require Institutional Review Board/Ethics Committee review, and the corresponding protocol/approval number is IRB Min. No. 7096. We also certify that we have not plagiarized the contents in this submission and have done a Plagiarism Check.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]