Immunohistochemical Expression Patterns of p16 and Ki‐67 in Oral Condyloma Acuminatum: A Case Report

Abstract

Condyloma acuminatum (CA) is a type of benign tumor that is associated with the human papillomavirus (HPV). Although histopathology remains the gold standard for diagnosing CA, immunohistochemical (IHC) biomarkers may be able to provide additional characterization of these lesions. In this case report, we present a 45‐year‐old female who presented with an exophytic papillomatous mass on the lateral aspect of her tongue. The treatment involved an uncomplicated surgical excision of the lesion. The histopathological diagnosis confirmed oral CA. An IHC study was then performed using p16 and Ki67 as IHC markers. The p16 IHC marker demonstrated diffuse positivity in < 5% of epithelial cells at the parabasal level, whereas the Ki67 marker was positive in 60% of parabasal cells. Therefore, it was determined that this corresponded to a squamous lesion exhibiting a profile consistent with low‐risk behavior, with no evidence of dysplasia. Periodic follow‐up showed adequate healing and no recurrence. Surgical excision is an effective therapeutic modality for oral CA. The p16 and Ki‐67 markers are useful tools for distinguishing oral malignancies from nonmalignant oral lesions. Their use also supports the identification and exclusion of premalignant changes as a means of prognosticating oral malignant neoplasms. These markers currently appear to have a low‐risk profile but may undergo further characterization, with eventual incorporation into quantitative PCR or in situ hybridization methods.

1. Introduction

Condyloma acuminatum (CA) is a group of benign lesions associated with low‐risk human papillomavirus (HPV), specifically Genotypes 6 and 11; however, the presence of high‐risk genotypes such as 16 and 18 has been noted in CA coinfections and is associated with an increased risk of dysplasia [1].

In the oral cavity, CA has been reported as an uncommon lesion, with the maxillary lip being the most common site of occurrence [1, 2]. In general, CA is seen more frequently in adults and is most prevalent during the 30s and 40s of life [2].

The diagnosis of oral CA is usually made through histopathological analysis. In traditional immunohistochemistry, there is a trend toward using only broad‐spectrum antibodies to help identify HPV; however, many newer studies are also adding other types of tests, including molecular and immunohistochemical assays (such as p16 and Ki‐67). Biomarker p16 is considered to identify the imbalance in cell cycle regulation and determine oncogenic activity. Similarly, Ki‐67 is a biomarker that shows cell proliferation behavior, allowing the identification of associated dysplastic changes [3, 4].

Although oral CA is not very prevalent, its diversity in phenotypic expression makes the correctly described clinical cases relevant. The aim of this article is to present the clinical, therapeutic, histopathological, and immunohistochemical characteristics of a case of oral CA.

2. Case Report

This case report was prepared in accordance with the CARE guidelines, and the completed CARE checklist is provided as supporting information. A 45‐year‐old male patient came to the clinic complaining of an increase in the size of his tongue, which was causing difficulty in chewing. The patient has not experienced any systemic conditions. Family and physician history do not yield useful information.

The exophytic lesion on the right lateral side of the tongue was identified during the intraoral examination as measuring 0.5 × 0.7 cm. The lesion′s appearance included a papillomatous and verrucous texture along with an irregular surface appearing as a papillary‐digitiform pattern. Upon palpation, the lesion felt soft, had a rough texture, and was of oval configuration, with slight mobility (Figure 1). Based on this clinical presentation, we developed a differential diagnosis for benign epithelial lesions including oral squamous papilloma, verruca vulgaris, and CA; the location, well‐defined shape, and the ability to access the lesion all indicated that no preoperative imaging or laboratory tests were needed.

Figure 1.

Exophytic lesion with a verrucous surface, located on the lateral border of the tongue.

In terms of the treatment plan, complete excision by scalpel and curettage of the area was planned for the next step in the case. The procedure was performed using local anesthesia with articaine 1:100,000 epinephrine. The surgical procedure removed the lesion at a 45° angle from the mucosal surface, oriented longitudinally to the axis of the tongue, and was performed using an inclined incision (including 1 mm of tissue surrounding the excised site as per conventional methods for removal). The suture technique used consisted of simple 6‐0 nylon stitches. At the end of the surgical procedure, amoxicillin was prescribed at a dose of 500 mg every 8 h for 7 days, along with ibuprofen 600 mg every 8 h for 3 days as an analgesic and anti‐inflammatory agent. The sample obtained was fixed in 10% formalin. (Figure 2).

Figure 2.

Excised specimen.

Histological analysis using routine hematoxylin and eosin showed stratified squamous epithelium with koilocytic atypia in the upper third; koilocytes with hyperchromatic nuclei, clumpy chromatin with nuclear membrane indentation, and perinuclear halos were observed. The evaluation outcomes matched with the CA diagnosis (Figures 3, 4, and 5). Immunohistochemistry was also used to assess expression of the markers, p16 and Ki67. The p16 marker showed focal positivity in fewer than 5% of epithelial cells in the parabasal layer (Figures 6 and 7), whereas, Ki67 was positive in 60% of parabasal epithelial cells. Although specific molecular HPV genotyping was not performed, the well‐demarcated immunohistochemical pattern is highly suggestive of a low‐risk oncogenic viral profile, consistent with a benign HPV‐associated squamous lesion (CA), and without evidence of dysplasia (Figure 8). For the immunohistochemical study, 4‐micron tissue sections were obtained from paraffin blocks fixed in 37% formalin. A monoclonal antibody against p16 (p16INKa: clone E6H4, Roche brand, prediluted) and against Ki‐67 (clone MIB‐1, Dako brand, 1:50 dilution) was used. Antigen retrieval was performed using heat with EDTA buffer. The reaction was visualized using the EnVision detection system with diaminobenzene as the chromogen. p16 positivity was defined as diffuse and strong nuclear and cytoplasmic staining (block staining). Negative results were defined as isolated (single) expression (i.e., Ki‐67 5% or less), whereas positive results were defined as Ki‐67 positivity in lesions where, for example; expression was greater than the basal third of the epithelial layer. Of the 13 patients with ocular lesions, only 5% were positive for p16. The p16 staining results must be compared with the number of cells positive for Ki‐67 according to the lower anogenital squamous terminology (LAST) requirement. Therefore, there is a need for confirmation of high‐risk HPV‐positive genital lesions (i.e., based on the p16 expression); diffuse “block” staining is required to identify these lesions as being HPV‐positive.

Figure 3.

Hematoxylin and eosin photomicrograph (10x): stratified squamous epithelium with pseudoepitheliomatous hyperplasia.

Figure 4.

Hematoxylin and eosin photomicrographs at (A) 10x and (B) 40x: stratified squamous epithelium with koilocytic atypia in the upper third. Koilocytes with geometric nuclei, clumpy chromatin membrane with nuclear membrane indentation, and perinuclear halos are observed.

Figure 5.

p16 biomarker (40x): squamous cells in parabasal and intermediate layers with focal nuclear and cytoplasmic positivity.

Figure 6.

p16 biomarker (40x): cells with focal nuclear and cytoplasmic positivity of the middle and upper stratum of the squamous epithelium.

Figure 7.

Ki67 biomarker (10x): Photomicrograph showing a panoramic view of the parabasal layer with nuclear positivity in 60% of the cells.

Figure 8.

Ki67 biomarker (40x): Basal and parabasal cells with 60% positive nuclei.

The amount of p16 staining in lesions indicates the level of cellular proliferation by Ki‐67 staining; 60% of Ki‐67–positive cells were located in the epithelial basal and parabasal layers. The results would be consistent with the biological behavior of oral CA, and no integrated replication pattern or overexpression of p16INK4a protein would be expected. During the 3‐month follow‐up period, adequate healing with optimal soft tissue restoration was observed. However, we emphasize that the patient remains under active, ongoing monitoring.

The patient was satisfied with the therapeutic management and reported that chewing did not cause discomfort in the weeks immediately following the procedure. After the patient was informed that the lesion was benign and that proper follow‐up was critical for the early detection of any potential recurrence, there was no remaining uncertainty regarding the nature of the lesion.

A chronological documentation of the patient′s clinical course, interventions, and follow‐up appointments can be found in Table 1.

Table 1.

Timeline of the case.

Timeframe	Clinical event	Description and findings
4 months prior	Lesion onset and progression	The patient noticed a small, asymptomatic, gradually enlarging growth on the lateral border of the tongue that slowly progressed over several months
Initial presentation	Clinical examination	Intraoral examination revealed a 0.5 × 0.7‐cm exophytic lesion on the right lateral border of the tongue. The lesion had a papillomatous, verrucous appearance with an irregular, papillary, digitiform surface resembling cauliflower. The patient reported difficulty with chewing.
1 week	Surgical intervention	Under local anesthesia, the lesion was completely excised with a No. 15C scalpel, maintaining a 1‐mm peripheral safety margin. Histopathologic analysis was performed, followed by prescriptions for antibiotics and analgesics.
	Pathological diagnosis	The diagnosis of condyloma acuminatum was confirmed by histopathological analysis, supplemented by immunohistochemical profiling with p16 and Ki‐67, which indicated a low‐risk HPV etiology with no evidence of dysplasia.
2 weeks	First follow‐up evaluation	The first postoperative follow‐up was completed, and the sutures were removed. Initial healing was progressing as expected.
3 months	Second follow‐up visit	Adequate and optimal healing was observed, with no signs of recurrence.
Continuous	Long‐term monitoring	The patient remains under strict, continuous clinical monitoring to ensure timely identification of any recurrent lesions.

Abbreviation: HPV, human papillomavirus.

3. Discussion

The incidence rates of HPV‐related oral lesions have dramatically risen in recent years among nonsmokers. As a result, there has been much interest in developing timely and accurate means for diagnosing these lesions. In the oral cavity, clinical diagnosis of CA presents significant challenges because of the phenotypic and histopathological similarities between CA and other verrucous and papillomatous oral lesions, as well as their common predisposition to malignant transformation [1]. Therefore, in order to achieve early and accurate diagnosis of oral CA (which allows for appropriate treatment intervention), it is essential that the clinician has a solid foundation of clinical, histopathological, and immunohistochemical characterization of oral CA.

In most reported clinical cases, the diagnosis of oral CA has been made through histopathological analysis, and few studies have incorporated the use of PCR (polymerase chain reaction) or fluorescent in situ hybridization (ISH) (Table 2). The complementary use of immunohistochemical markers—p16 and Ki‐67—significantly enhances our knowledge concerning the biological behavior of these entities. The diagnostic usage of both of these two biomarkers has been well‐established for anogenital lesions and high‐risk oropharyngeal carcinomas. However, to date, there is very limited research specifically examining this immunohistochemical panel in the setting of benign oral cancers [1]. There are many settings where p16 evaluation is routinely performed, such as urology, otolaryngology, and gynecological oncology; however, p16 overexpression has also been shown to provide information about tumor stratification in different types of malignant neoplasia [3, 4]. In the present study, restricted expression of p16 (less than 5% of epithelial cells at the parabasal level) shows a nontransforming phenotype that points toward a low‐risk viral behavior, significantly reducing the possibility of active oncogenic viral integration. Conversely, Ki‐67 analysis is not routinely used in dermatology hematology, and gynecologic oncology; however, it is implemented in instances where assessing proliferative potential and ruling out premalignant changes is desired [4, 13, 14]. The primary finding of this study was that there was an increased Ki‐67 index solely in the parabasal layer, suggesting that true epithelial dysplasia may be less likely. The use of immunohistochemical test markers can not only facilitate and confirm the diagnosis of HPV‐associated CA of low oncogenic potential but also help clarify the differential diagnosis and prognostic implications.

Table 2.

Reported clinical cases of oral condyloma acuminatum.

Study	Age of patient	Sex	Site	Size	Diagnosis	HPV genotype/subtype	Management/treatment	Follow‐up duration (months)	Recurrence
Lacouture et al. [5]	38	Male	Lateral border of the tongue	NR	Fluorescence in situ hybridization	NR	Mohs micrographic surgery	9	No
Nucci et al. [6]	27	Female	Lateral border of the tongue	NR	In situ DNA hybridization	NR	Topical photodynamic therapy with 5‐aminolevulinic acid	18	No
Dos Reis et al. [7]	27	Female	Tip of the tongue	1 cm	Histopathological	NR	Surgical excision	24	No
Jaiswal et al. [8]	52	Male	Buccal mucosa	2 × 2 cm	Histopathological	NR	Cold scalpel surgical excision	9	No
Percinoto et al. [9]	5	Male	Posterior dorsum of the tongue and hard palate	0.4 cm (tongue) and 0.6 cm (palate)	Histopathological	NR	Surgical excision	12	No
Sen et al. [2]	75	Male	Alveolar ridge, palate, buccal mucosa, floor of the mouth	Variable (multiple lesions)	Histopathological + PCR	HPV 6	Surgical excision	6	No
Chaturvedi and Chaturevedi [10]	45	Male	Right lower lip	NR	Histopathological	NR	Surgical excision	6	No
Dávila Torres and Pacheco Ludeña [11]	9	Female	Right labial commissure, buccal mucosa, lower labial mucosa, tongue	0.5 cm (main injury)	Clinical	NR	Proposed surgical treatment (not performed)	NR	NR
Chérrez Bermejo et al. [12]	7	Female	Palatine raphe	NR	Histopathological + PCR	HPV positive, genotype undetermined	Surgical excision	12	No
Valente et al. [13]	55	Male	Middle dorsum of the tongue	2 mm	Histopathological	NR	Surgical excision	6	No
Present study	45	Male	Right lateral border of the tongue	0.5 × 0.7 × 0.8 cm	Histopathological + immunohistochemical (p16 and Ki67)	NR	Surgical excision	3	No

Abbreviations: HPV, human papillomavirus; NR, not reported; PCR, polymerase chain reaction.

Another important consideration is the cost‐benefit ratio of the available diagnostic tools. Although PCR and ISH are both considered gold standards for definitive HPV genotyping, they are expensive and require substantial resources to implement in clinical practice. Consequently, they are not widely available in many clinical facilities because of their cost and technical complexity. An alternative increasingly used for primary screening in similar clinical settings is the combined use of p16 and Ki‐67 as diagnostic markers. By using these biomarkers to characterize an individual′s low‐risk status and identify dysplasia, clinicians can perform initial risk stratification without incurring the costs associated with DNA genotyping. This immunohistochemical approach is therefore an important diagnostic tool, particularly in areas where advanced molecular diagnostic tests are unavailable or impractical.

Entities in the differential diagnosis of oral CA include verruca vulgaris, squamous papilloma, nonviral verruciform lesions (such as verruciform xanthoma), and verrucous carcinoma. Squamous papillomas usually appear as solitary lesions with limited proliferative potential, presenting as pedunculated or sessile masses. In contrast, lesions that present as verruca vulgaris typically have prominent papillomatous projections and rete pegs oriented toward the center of the wart, with basal changes that differ from the sessile base of oral CA. In addition, differentiating early‐stage verrucous carcinoma from benign verrucous growths is extremely difficult; in this setting, identifying p16 and Ki‐67 coexpression is essential to the analysis. In the present case, the Ki‐67 index was elevated at 60%, although it was restricted to the basal and parabasal layers, with little to no expression in the upper epithelial maturation zones. Knowing where a lesion falls on the anatomical spectrum helps distinguish benign HPV/virus‐associated proliferation from dysplasia and malignancy by the atypical pattern in which abnormal cells ascend to the upper layers in terms of shape and appearance. Newly arising verrucous carcinomas lack evidence of strong cellular instability and abnormal p16 expression in combination with a high Ki‐67 index in the uppermost epithelial layers, whereas benign oral CA histologically shows a well‐defined basal/parabasal cell expression pattern that excludes any evidence of malignancy and indicates strong cellular instability associated with dysplastic change. There are significant limitations to using these protein markers in oral mucosal tissues, and although combined p16/Ki‐67 markers are useful for assessing cell cycle behavior and ruling out malignancies, these two cellular markers only demonstrate the behavior of cellular proliferation and do not specify which HPV genotype is associated with that behavior. This necessitates the use of a molecular test (PCR or ISH) to identify the specific HPV genotype associated with positive cell cycle marker results. Additionally, there is no uniformity in how positivity criteria for p16 are applied to anogenital tract lesions, and thus the criteria used to assess these protein markers in benign disease present significant challenges to establishing standardized applications in larger sample sizes of benign oral lesions.

The treatment of oral CA can utilize a variety of treatment modalities (Table 2) including Mohs micrographic surgery, electrocautery, surgical excision, and photodynamic therapy. In recent years, the trend for treating this condition in the oral cavity has been surgical excision, which involves the complete removal of the lesion, considering safety margins. This technique has certain advantages, such as being a standard technique, requiring less surgical time, being suitable for well‐defined lesions, and being relatively inexpensive, as it does not require intraoperative histopathological evaluation [2, 10–13]. Due to the various qualities of this technique, it was the modality chosen in the present clinical case, which has been successfully implemented in other studies [10, 12, 13]. Due to the potential for CA recurrence and HPV persistence, regular monitoring of long‐term evolution is recommended.

This clinical case has several limitations. Studies with larger samples are needed to validate the immunohistochemical profile of p16 and Ki‐67 in benign oral mucosal lesions. Histological and immunohistochemical findings indicated a low‐risk behavioral profile; however, no molecular tests (PCR, hybridization, and genotyping) were available to corroborate these results. The LAST criteria were developed to aid in the evaluation of anogenital squamous lesions; therefore, there is no universal standard for applying them to benign oral lesions. For this reason, in this case only, the LAST criteria were used for exploratory purposes to help clarify the p16 negativity threshold (< 5%) required to exclude high‐risk, transforming HPV infections. The 3‐month follow‐up showed complete healing and no evidence of recurrence; however, because of the high recurrence rate associated with this condition, it is important to follow patients for a longer period to assess the biological behavior of the lesion and the prognostic value of the biomarkers used.

4. Conclusion

The similarity in histopathological and phenotypic description of oral CA coupled with the difficulties that exist in determining an accurate diagnosis for this rare lesion is the reason for the need for an all‐inclusive evaluation, which includes not only clinical findings but also histopathological and immunohistochemical findings to provide a clear distinction.

Adding a p16 and Ki‐67 immunohistochemical panel provides more precise biological information and supports prognostic assessment. The markers analyzed indicated that the lesion is likely low risk; however, additional testing to establish a definitive diagnosis using either PCR or ISH could provide further clarification. In many settings with limited clinical sites and resources, molecular DNA genotyping is often not feasible. Surgical excision of oral CA tissue is an accepted and safe option for managing this lesion; however, accurate diagnosis, a well‐defined treatment plan, and follow‐up to monitor recurrence are essential for effective management of oral CA.

Author Contributions

Mario Alberto Alarcón‐Sánchez: writing—review and editing, writing—original draft preparation, visualization, validation, supervision, software, resources, project administration, methodology, investigation, formal analysis, data curation, conceptualization. Eder Javier Martínez‐González: visualization, validation, supervision, resources, methodology, investigation, formal analysis. Gustavo Eder González‐Alvarez: writing—review and editing, writing—original draft preparation, visualization, validation, supervision, methodology, formal analysis, conceptualization. Daniel Ortuño‐Sahagún: writing—review and editing, writing—original draft preparation, visualization, validation, supervision, software, resources, project administration, methodology, investigation, formal analysis, data curation, conceptualization. Artak Heboyan: writing—review and editing, writing—original draft preparation, visualization, validation, supervision, software, resources, project administration, methodology, investigation, formal analysis, data curation, conceptualization. Sarah Monserrat Lomelí‐Martínez: writing—review and editing, writing—original draft preparation, visualization, validation, supervision, methodology, formal analysis, conceptualization.

Funding

No funding was received for this manuscript.

Disclosure

All authors have read and approved the final version of the manuscript. Sarah Monserrat Lomelí‐Martínez and Artak Heboyan had full access to all data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis.

Ethics Statement

Written informed consent was obtained from the patient before initiating the treatment.

Consent

Written informed consent was obtained from the patient to publish this case report and any accompanying images.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting Information Additional supporting informatiosn can be found online in the Supporting Information section. CARE checklist (2016) statement. This case report was prepared in accordance with the CARE guidelines, and the completed CARE checklist is provided as supporting information.

Alarcón‐Sánchez, Mario Alberto , Martínez‐González, Eder Javier , González‐Alvarez, Gustavo Eder , Ortuño‐Sahagún, Daniel , Heboyan, Artak , Lomelí‐Martínez, Sarah Monserrat , Immunohistochemical Expression Patterns of p16 and Ki‐67 in Oral Condyloma Acuminatum: A Case Report, Case Reports in Dentistry, 2026, 9289667, 8 pages, 2026. 10.1155/crid/9289667

Data Availability Statement

All data supporting the findings of this study are included in the published article.