Abstract
Background: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma is biologically and clinically distinct from conventional tobacco- and alcohol-related head and neck squamous cell carcinoma. Indian data remain heterogeneous because oral cavity and oropharyngeal tumors are often analyzed together despite their different etiologic patterns. This study evaluated p16INK4a (p16) expression and high-risk HPV DNA detection in oral cavity and oropharyngeal squamous cell carcinoma and assessed their association with clinicopathologic variables.
Materials and methods: This cross-sectional observational study included 100 adult patients with biopsy-proven squamous cell carcinoma of the oral cavity or oropharynx treated at a tertiary care center in Surat, Gujarat, India. Demographic characteristics, tobacco-related habits, tumor site, clinical presentation, and histopathological findings were recorded. All cases underwent hematoxylin and eosin examination and p16 immunohistochemistry. Fresh tissue was available for high-risk HPV DNA testing in 89 cases and was analyzed for HPV genotypes 16, 18, and 45 using multiplex real-time polymerase chain reaction. Associations were tested using the chi-square test or Fisher's exact test, as appropriate, and effect size was estimated using Cramer's V.
Results: p16 positivity was identified in six of 100 cases (6.0%), and HPV DNA was detected in five of 89 tested cases (5.6%). All p16-positive and HPV DNA-positive patients were male. p16 positivity was significantly more frequent in oropharyngeal tumors than in oral cavity tumors (5/24, 20.8% vs. 1/76, 1.3%; p=0.0028). The base of the tongue was the most common p16-positive subsite (4/6, 66.7%). p16 expression was significantly associated with HPV DNA detection, with five of six p16-positive tumors showing HPV DNA positivity and no HPV DNA detection among p16-negative tumors (Fisher's exact p < 0.001). Most p16-positive cases had no documented history of tobacco chewing, smoking, or alcohol consumption.
Conclusion: p16 expression and HPV DNA detection were uncommon in this Western Indian cohort, likely reflecting the predominance of oral cavity tumors and traditional risk-factor-associated disease. Biomarker-positive cases were concentrated in male patients with oropharyngeal tumors, particularly tumors involving the base of the tongue. Because HPV DNA testing was performed in 89 cases and was limited to genotypes 16, 18, and 45, these findings should be interpreted as a site-aware assessment of common high-risk HPV types rather than a comprehensive estimate of all HPV-driven disease. Combining p16 expression with HPV DNA testing may provide a more balanced assessment of HPV involvement than either test alone, particularly in resource-limited settings.
Keywords: human papillomavirus, immunohistochemistry, oral squamous cell carcinoma, oropharyngeal squamous cell carcinoma, p16ink4a, real-time polymerase chain reaction
Introduction
Squamous cell carcinoma of the oral cavity and oropharynx is a major public health problem, but its etiologic profile varies across regions. In India, oral cavity squamous cell carcinoma remains closely associated with smokeless tobacco, betel leaves, and areca nut use, bidi or cigarette smoking, and alcohol consumption. High-risk human papillomavirus (HPV), especially HPV-16, is also an established cause of a subset of oropharyngeal squamous cell carcinomas [1-5].
Distinguishing HPV-driven disease from conventional carcinogen-associated disease is clinically relevant because HPV-associated oropharyngeal carcinoma differs in patient profile, molecular mechanism, treatment response, prognosis, and staging implications. HPV-positive oropharyngeal tumors generally show better treatment responsiveness and survival than HPV-negative tobacco-associated tumors [3].
Reported HPV positivity rates in Indian head and neck squamous cell carcinoma vary widely. This heterogeneity is likely related to differences in anatomical site selection, sample types, tissue preservation, HPV detection method, p16 scoring criteria, risk-factor exposure, and sample size. Earlier and more recent Indian studies using PCR-based approaches have also reported variable HPV detection in oral cancers and potentially malignant disorders [6,7]. Local institutional data remain useful in regions where tobacco-related oral cancer is common.
p16INK4a (p16) immunohistochemistry is widely used as a surrogate marker of transcriptionally active HPV in oropharyngeal carcinoma because viral E7-mediated retinoblastoma pathway disruption leads to p16 overexpression. However, p16 is not a perfect standalone marker, particularly outside the oropharynx, where non-viral mechanisms may also produce p16 expression [8,9]. HPV DNA testing by polymerase chain reaction is analytically sensitive for viral genome detection but does not, by itself, confirm transcriptionally active oncogenesis. A combined clinicopathologic, immunohistochemical, and molecular approach is therefore more informative than reliance on either method alone [8,10].
The primary objective of this study was to assess p16INK4a expression and HPV DNA detection in oral cavity and oropharyngeal squamous cell carcinoma in a tertiary care setting in Western India. Secondary objectives were to correlate biomarker status with demographic, behavioral, anatomical, and histopathological variables and to assess concordance between p16 immunohistochemistry and HPV DNA detection.
Materials and methods
This cross-sectional observational study was conducted at a tertiary care center in Surat, South Gujarat, India, from March 2022 to October 2024. The study was approved by the Institutional Ethics Committee (SMIMER/IEC/2522), and written informed consent was obtained from all participants.
A total of 100 consecutive adult patients with histopathologically confirmed squamous cell carcinoma of the oral cavity or oropharynx were included. Clinical and demographic data, including age, sex, tobacco-related habits, anatomical site, and presenting symptoms, were obtained from clinical records and structured questionnaires after informed consent. The inclusion and exclusion criteria are summarized in Table 1.
Table 1. Inclusion and exclusion criteria .
| Inclusion Criteria | Exclusion Criteria |
| Adult patients aged ≥18 years | Recurrent tumors |
| Histopathologically confirmed squamous cell carcinoma | Metastatic lesions to the head and neck region |
| Primary tumor involving the oral cavity or oropharynx | Non-squamous malignancies |
| Availability of adequate biopsy tissue for histopathological evaluation | Synchronous or metachronous second primary malignancies |
| Availability of clinical and demographic data | Patients previously or currently treated with chemotherapy & radiotherapy, or both. |
| Written informed consent obtained from the participant | Incomplete clinical records or missing key study variables |
As this was a pilot observational study conducted in a resource-limited setting, the sample size was limited to 100 eligible cases. p16INK4a immunohistochemistry was performed in all cases. HPV DNA analysis was performed in 89 cases for which an additional fresh tissue specimen of adequate quantity and quality was available and met the predefined criteria for molecular testing.
Biopsy specimens were fixed in 10% neutral-buffered formalin, routinely processed, and embedded in paraffin wax. Sections measuring four micrometers were stained with hematoxylin and eosin and reviewed by experienced pathologists. Tumors were graded as well-differentiated, moderately differentiated, or poorly differentiated squamous cell carcinoma according to the World Health Organization Classification of Head and Neck Tumours [11].
Immunohistochemical staining for p16INK4a was performed on formalin-fixed, paraffin-embedded tissue sections using a commercially available monoclonal mouse anti-human p16 antibody. Heat-induced epitope retrieval was performed using citrate buffer at pH 6.0, followed by staining with a standard peroxidase-based detection system according to the manufacturer's instructions.
p16 immunohistochemistry was interpreted according to guidance from the College of American Pathologists. Tumors with strong, diffuse nuclear and cytoplasmic staining in 70% or more of viable tumor cells were considered p16-positive. Tumors with absent, focal, patchy, weak, or less than 70% staining were considered p16-negative [8]. Histopathological and immunohistochemical evaluations were performed independently by two pathologists who were blinded to HPV DNA status.
Fresh tissue suitable for molecular analysis was tested using the Hi-PCR Human Papillomavirus Genotyping Multiplex Probe PCR Kit (HiMedia Laboratories, India), which detects HPV genotypes 16, 18, and 45. DNA was extracted from frozen tissue samples using a validated column-based extraction method and analyzed on a real-time PCR platform according to the manufacturer's instructions. Multiplex real-time polymerase chain reaction was performed for qualitative HPV DNA detection. Positive controls and non-template negative controls were included in each assay run.
Data were entered in Microsoft Excel and analyzed using IBM SPSS Statistics for Windows, Version 29 (Released 2022; IBM Corp., Armonk, New York, United States). Categorical variables were summarized as frequencies and percentages. Associations between HPV DNA status, p16 expression, and clinicopathological parameters were assessed using Pearson’s chi-square test or Fisher’s exact test, as appropriate. Fisher’s exact test was applied when expected cell counts were small. Cramer’s V was used to estimate the strength of association. A two-tailed p-value of less than 0.05 was considered statistically significant.
Results
The study included 100 patients with histopathologically confirmed squamous cell carcinoma of the oral cavity or oropharynx. p16 immunohistochemistry was performed in all cases, and HPV DNA testing was performed in 89 cases for which adequate fresh tissue was available for molecular analysis.
p16 expression was detected in six of 100 cases (6.0%), and HPV DNA was detected in five of 89 tested cases (5.6%). The most common age group was 41-50 years, followed by 31-40 years. Oral cavity tumors accounted for 76 cases, while 24 tumors arose in the oropharynx. Among the six p16-positive cases, three were in the 31-40-year age group, two were in the 51-60-year age group, and one was in the 21-30-year age group. There was no significant age-wise difference in p16 expression or HPV DNA status in this cohort. The age-wise distribution of p16 expression and HPV DNA detection is shown in Table 2.
Table 2. Association of p16 expression and HPV DNA detection with the age group.
HPV: human papillomavirus; DNA: deoxyribonucleic acid
| Age group (years) | p16 positive n (%) | p16 negative n (%) | HPV DNA positive n (%) | HPV DNA negative n (%) |
| 21-30 | 1 (16.7) | 9 (9.5) | 0 (0.0) | 9 (10.7) |
| 31-40 | 3 (50.0) | 28 (29.8) | 3 (60.0) | 25 (29.8) |
| 41-50 | 0 (0.0) | 38 (40.4) | 0 (0.0) | 34 (40.5) |
| 51-60 | 2 (33.3) | 19 (20.2) | 2 (40.0) | 16 (19.0) |
| Total | 6 (100.0) | 94 (100.0) | 5 (100.0) | 84 (100.0) |
The study population included 66 male patients and 34 female patients, with a male-to-female ratio of 1.9:1. All six p16-positive cases and all five HPV DNA-positive cases occurred in male patients. Sex was not significantly associated with p16 expression, χ²(1) = 3.29, p = 0.070, Cramer’s V = 0.18, indicating a weak association. Similarly, sex was not significantly associated with HPV DNA status, χ²(1) = 2.31, p = 0.129, Cramer’s V = 0.16, indicating a weak association. The association of sex with p16 expression and HPV DNA detection is shown in Table 3.
Table 3. Association of sex with p16 expression and HPV DNA detection.
Fisher’s exact test was used to assess the association of sex with p16 expression and HPV DNA detection.
For p16 expression: χ²(1)=3.29, Fisher’s exact p=0.070, Cramer’s V=0.18.
For HPV DNA detection: χ²(1)=2.31, Fisher’s exact p=0.129, Cramer’s V=0.16. HPV: human papillomavirus; DNA: deoxyribonucleic acid
| Sex | p16 positive n (%) | p16 negative n (%) | HPV DNA positive n (%) | HPV DNA negative n (%) |
| Male | 6 (100.0) | 60 (63.8) | 5 (100.0) | 57 (67.9) |
| Female | 0 (0.0) | 34 (36.2) | 0 (0.0) | 27 (32.1) |
| Total | 6 (100.0) | 94 (100.0) | 5 (100.0) | 84 (100.0) |
Oropharyngeal tumors showed a significantly higher frequency of p16 positivity than oral cavity tumors. p16 expression was observed in five of 24 oropharyngeal tumors (20.8%) compared with one of 76 oral cavity tumors (1.3%). Among the six p16-positive cases, four involved the base of the tongue, one involved the tonsil, and one involved the buccal mucosa. The association between the anatomical site and p16 expression was statistically significant by Fisher’s exact test (p = 0.0028). The corresponding Pearson chi-square result was χ²(1) = 12.32, with a moderate effect size as indicated by Cramer’s V = 0.35. The association between the anatomical site and p16 expression is shown in Table 4.
Table 4. Association of the anatomical site with p16INK4a expression.
Fisher’s exact test was used because of small cell count which showed a statistically significant association between anatomical site and p16INK4a expression
(χ² = 12.32, df = 1, Fisher’s exact p = 0.0028). Cramer’s V was 0.35, indicating a moderate association.
| Site | p16 positive n (%) | p16 negative n (%) | Total n (%) |
| Oral cavity | 1 (1.3) | 75 (98.7) | 76 (100.0) |
| Oropharynx | 5 (20.8) | 19 (79.2) | 24 (100.0) |
| Total | 6 (6.0) | 94 (94.0) | 100 (100.0) |
Traditional risk factors, including tobacco chewing, cigarette or bidi smoking, and alcohol consumption, were documented in 87 of 100 patients. p16 positivity was observed in five of 13 patients without a documented traditional risk-factor history compared with one of 87 patients with at least one traditional risk factor. In contrast, 86 of 94 p16-negative patients had one or more traditional risk-factor exposures. Risk-factor history was significantly associated with p16 expression by Fisher’s exact test (p < 0.001). The corresponding Pearson chi-square analysis showed a strong association, χ²(1) = 27.92, with a large effect size as indicated by Cramer’s V = 0.53. The association between traditional risk-factor history and p16 expression is shown in Table 5.
Table 5. Association between traditional risk-factor history and p16 expression.
Fisher’s exact test showed a statistically significant association between traditional risk-factor history and p16 expression
(χ² = 27.92, df = 1, Fisher’s exact p < 0.001). Cramer’s V was 0.53, indicating a strong association.
| Risk-factor history | p16 positive n (%) | p16 negative n (%) | Total n (%) |
| Present | 1 (1.1) | 86 (98.9) | 87 (100.0) |
| Absent | 5 (38.5) | 8 (61.5) | 13 (100.0) |
| Total | 6 (6.0) | 94 (94.0) | 100 (100.0) |
The distribution of histopathological grade according to the anatomical site and p16 expression is shown in Table 6. Among oral cavity tumors, the only p16-positive case was a well-differentiated squamous cell carcinoma. In contrast, p16 positivity among oropharyngeal tumors was observed in five cases, comprising three moderately differentiated and two poorly differentiated squamous cell carcinomas. Among p16-negative oral cavity tumors, well-differentiated squamous cell carcinoma was the most frequent histological subtype, followed by moderately differentiated squamous cell carcinoma. These findings suggest that p16-positive cases in this study were more commonly observed in oropharyngeal tumors with moderate or poor differentiation.
Table 6. Histopathological grade according to the anatomical site and p16 expression.
SCC: squamous cell carcinoma
| Histopathological diagnosis | Oral cavity p16 positive | Oral cavity p16 negative | Oropharynx p16 positive | Oropharynx p16 negative |
| Well-differentiated SCC | 1 | 36 | 0 | 2 |
| Moderately differentiated SCC | 0 | 34 | 3 | 15 |
| Poorly differentiated SCC | 0 | 5 | 2 | 2 |
| Total | 1 | 75 | 5 | 19 |
Representative H&E-stained sections demonstrated the histopathological spectrum of squamous cell carcinoma, ranging from well-differentiated tumors with keratinization to poorly differentiated tumors with marked nuclear atypia and minimal squamous differentiation in Figure 1.
Figure 1. Hematoxylin and eosin-stained sections of squamous cell carcinoma. (A) Well-differentiated squamous cell carcinoma showing keratin pearl formation and limited cellular atypia (10x). (B) Moderately differentiated squamous cell carcinoma showing atypical squamous cells with mitotic activity and individual cell dyskeratosis (45x). (C) Poorly differentiated squamous cell carcinoma showing marked nuclear pleomorphism, high nuclear-to-cytoplasmic ratio, and minimal squamous differentiation (45x).
Among the 100 cases evaluated by p16 immunohistochemistry, six cases (6.0%) showed p16 positivity. Histologically, these cases included one well-differentiated, three moderately differentiated, and two poorly differentiated squamous cell carcinomas. The positive cases demonstrated nuclear and cytoplasmic immunoreactivity in tumor cells. Focal or patchy p16 immunoreactivity was observed in tumor cell nests with intervening unstained areas, while strongly positive areas showed diffuse nuclear and cytoplasmic staining. Of the six p16-positive cases, five were located in the oropharynx and one was located in the oral cavity. Representative p16 immunohistochemical staining patterns are shown in Figure 2.
Figure 2. Representative p16 immunohistochemical staining in squamous cell carcinoma. (A) Low-power view showing focal/patchy p16 immunoreactivity in tumor cell nests with intervening unstained areas (10x). (B) High-power view showing diffuse nuclear and cytoplasmic p16 immunoreactivity in tumor cells (45x).
HPV DNA PCR was performed in 89 cases, of which five were HPV-positive and 84 were HPV-negative. Among the six p16-positive cases, five were HPV DNA-positive and one was HPV DNA-negative. The association between p16 immunohistochemistry and HPV DNA PCR was statistically significant by Fisher’s exact test (p < 0.001). Because of the small cell counts, Fisher’s exact test was considered the primary statistical analysis. The corresponding Pearson chi-square test also demonstrated a significant association between p16 expression and HPV DNA status, χ²(1) = 73.28, p < 0.001, with a strong effect size as indicated by Cramer’s V = 0.91. Concordance between p16 immunohistochemistry and HPV DNA detection is shown in Table 7.
Table 7. Concordance between p16 immunohistochemistry and HPV DNA .
Fisher’s exact test was used as the primary test because of small cell counts. χ² = 73.28; df = 1; Fisher’s exact p < 0.001; Cramer’s V = 0.91.
HPV: human papillomavirus; DNA: deoxyribonucleic acid; IHC: immunohistochemistry.
| p16 IHC status | HPV positive n (%) | HPV negative n (%) | Total n (%) |
| Positive | 5 (83.3) | 1 (16.7) | 6 (100.0) |
| Negative | 0 (0.0) | 83 (100.0) | 83 (100.0) |
| Total | 5 (5.6) | 84 (94.4) | 89 (100.0) |
Discussion
This study found a low frequency of p16 expression and HPV DNA detection in oral cavity and oropharyngeal squamous cell carcinoma in Western India. p16 positivity was present in 6.0% of cases, and HPV DNA was detected in 5.6% of tested samples. These results support the continued predominance of conventional carcinogen-associated disease in this population, while also identifying a small but clinically recognizable HPV-associated subgroup. The prevalence of p16 positivity and HPV DNA detection observed in the present study was lower than that reported in several Indian and international studies. Published estimates of HPV positivity in head and neck squamous cell carcinoma vary widely across studies and populations, reflecting differences in anatomical site distribution, HPV detection methodology, sample size, biomarker definitions, and regional tobacco-use patterns [12-14].
The low prevalence of p16 expression and HPV DNA detection in the present study may be attributable to the predominance of oral cavity tumors, which accounted for 76% of the cohort. HPV-driven carcinogenesis is generally uncommon in true oral cavity squamous cell carcinoma, whereas it is more strongly linked to oropharyngeal squamous cell carcinoma. This interpretation is consistent with the multicenter study by Lingen et al., in which high-risk HPV E6/E7 expression was detected in only 5.9% of oral cavity squamous cell carcinoma cases [5]. Furthermore, the study population demonstrated a high prevalence of conventional risk factors, including tobacco chewing, smoking, and alcohol consumption, which continue to represent the principal etiological factors for oral cavity squamous cell carcinoma in India. An interesting observation was the differing risk-factor profiles between p16-positive and p16-negative cases. Most p16-negative tumors were associated with one or more conventional risk factors, whereas most p16-positive tumors occurred in patients without documented exposure to these factors. Fisher's exact test demonstrated a significant inverse association between traditional risk-factor exposure and p16 positivity (p<0.001). Although the small number of p16-positive cases precludes definitive conclusions, this finding is consistent with previous reports suggesting that HPV-associated oropharyngeal squamous cell carcinoma may arise through a distinct carcinogenic pathway separate from the traditional tobacco- and alcohol-related pathway [2,7,9,15].
Despite the low overall positivity rate, p16 expression showed a clear anatomical pattern. Oropharyngeal tumors were significantly more likely to be p16-positive than oral cavity tumors, and most p16-positive tumors involved the base of the tongue. This distribution is consistent with the established predilection of HPV-associated head and neck carcinogenesis for the oropharynx, particularly lymphoepithelial-rich subsites such as the palatine tonsil and base of the tongue. D'Souza et al. [2] demonstrated a strong association between HPV exposure and oropharyngeal cancer, while Ang et al. [3] showed that HPV-positive oropharyngeal tumors represent a clinically distinct group with better survival. Similarly, Gillison et al. [4] emphasized that HPV-positive head and neck squamous cell carcinoma is epidemiologically concentrated in oropharyngeal subsites rather than the oral cavity. Therefore, the predominance of p16 positivity in the base of the tongue and other oropharyngeal tumors in the present study supports the expected site-specific distribution of HPV-associated disease, although p16 results should be interpreted alongside HPV DNA or RNA testing.
The HPV DNA findings also require site-aware interpretation. HPV DNA testing was performed in 89 cases rather than the full cohort, and the PCR panel targeted HPV-16, HPV-18, and HPV-45 only. Therefore, infections caused by other high-risk genotypes would not have been detected. Sampling variation may also contribute to false-negative molecular results when the tested tissue contains limited tumor, necrosis, or degraded DNA. Finally, p16 immunohistochemistry and HPV DNA testing address related but distinct biological questions: p16 indicates pathway-level dysregulation, whereas PCR confirms viral DNA but does not necessarily demonstrate transcriptionally active oncogenesis [8-10].
Histologically, p16-positive oropharyngeal tumors in this study were mostly moderately or poorly differentiated squamous cell carcinomas. This is compatible with the broader observation that HPV-associated oropharyngeal tumors may show non-keratinizing or less differentiated morphology [4,12]. However, morphology alone cannot establish HPV-driven disease, and isolated or low-level p16 staining in oral cavity tumors should not be overinterpreted as evidence of HPV-mediated carcinogenesis. Current pathology guidance supports site-specific interpretation of p16 and HPV-specific testing, particularly in oropharyngeal carcinoma [8,9].
In the tested subset, p16 immunohistochemistry and HPV DNA detection showed concordant results in most p16-positive cases. Five of six p16-positive tumors were HPV DNA-positive, while none of the p16-negative tumors demonstrated detectable HPV DNA. This supports the practical value of p16 immunohistochemistry as a screening method in resource-limited settings, particularly for oropharyngeal squamous cell carcinoma. This interpretation is consistent with the College of American Pathologists guideline by Lewis et al. [8], which recommends p16 immunohistochemistry as the primary approach for HPV testing in newly diagnosed oropharyngeal squamous cell carcinoma, while emphasizing more limited routine use in non-oropharyngeal head and neck carcinomas. However, because only a small number of biomarker-positive cases were identified, larger studies are required before diagnostic performance can be estimated with confidence.
From a practical diagnostic perspective, these findings suggest the potential utility of a tiered workflow in resource-limited settings. p16 immunohistochemistry is relatively inexpensive, technically feasible, and suitable for routine pathology laboratories, whereas HPV DNA testing requires molecular infrastructure, contamination control, and validated assay systems. A reasonable approach is to use p16 screening for oropharyngeal squamous cell carcinoma and HPV-specific molecular testing when confirmation would affect staging, prognosis, trial eligibility, or treatment decisions [8,10]. For oral cavity tumors, HPV attribution should be made cautiously and preferably with confirmatory molecular evidence, particularly when p16 staining is focal or sporadic [8,9].
This study has limitations. It was a single-center pilot study with a relatively small sample size and few biomarker-positive cases. The cohort was enriched for oral cavity tumors, which may have reduced the overall observed HPV-related fraction. HPV DNA testing was not available for all cases and was limited to genotypes 16, 18, and 45. Risk-factor exposure was based on available clinical records and structured questionnaires and may be affected by recall error, incomplete documentation, or underreporting. Tissue adequacy also limited molecular testing in a subset of cases, and variation in tumor content or nucleic acid quality may have influenced HPV DNA detection. Although two pathologists independently reviewed the histopathology and p16 immunohistochemistry, formal interobserver concordance statistics were not calculated. The study did not include HPV E6/E7 RNA testing, detailed sexual behavior or HPV vaccination data, or long-term clinical outcomes. Further multicenter studies using standardized anatomic site classification, broader HPV testing, transcriptional assays, formal reproducibility metrics, and outcome follow-up are needed to clarify the epidemiological and prognostic significance of HPV-associated disease in this population.
Conclusions
In this cross-sectional study from Western India, p16 expression and high-risk HPV DNA detection were uncommon in oral cavity and oropharyngeal squamous cell carcinoma, likely reflecting the predominance of oral cavity tumors and traditional tobacco- and alcohol-related cancers in this study. Most biomarker-positive tumors occurred in male patients without documented traditional risk factors and were concentrated in the oropharynx, particularly the base of the tongue, suggesting a possible HPV-associated subset; however, the small number of positive cases limits definitive conclusions. Because HPV DNA testing was performed in 89 cases and was limited to genotypes 16, 18, and 45, the molecular findings should be interpreted within this testing scope. p16 immunohistochemistry may serve as a practical and cost-effective initial screening tool in resource-limited settings, while HPV DNA testing provides molecular confirmation. Larger multicenter studies with broader HPV genotyping, standardized p16 interpretation, transcriptional HPV assays, reproducibility assessment, and clinical outcome data are needed.
Disclosures
Human subjects: Informed consent for treatment and open access publication was obtained or waived by all participants in this study. Surat Municipal Institute of Medical Education and Research, Institutional Ethics Committee issued approval 2522.
Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.
Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following:
Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.
Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.
Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
Author Contributions
Concept and design: Hardik Jain, Ashish Surana, Sudha Jain, Gunjan Jain
Acquisition, analysis, or interpretation of data: Hardik Jain, Akruti Patel
Drafting of the manuscript: Hardik Jain, Ashish Surana, Sudha Jain, Akruti Patel, Gunjan Jain
Supervision: Hardik Jain, Ashish Surana, Sudha Jain, Gunjan Jain
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