Abstract
Background/Aim: Severe nuclear atypia can be associated with condyloma acuminatum. In this study, we investigated nine cases of perianal condyloma acuminatum with severe nuclear atypia and determined whether severe nuclear atypia is sufficient for the diagnosis of high-grade squamous intraepithelial lesion (HSIL). Materials and Methods: The clinical data and pathological features of the nine patients were collected. p16 Immunostaining and human papillomavirus genotyping were also performed. Results: The resected specimens of six men infected with human immunodeficiency virus showed features suggestive of HSIL, including the expansion of basaloid cells, severe nuclear pleomorphism in the lower one-third, bizarre nuclei, mitotic figure in the upper two-thirds, atypical mitosis, block positivity for p16, and high-risk human papillomavirus infection. In contrast, the resected specimens of the remaining three patients did not show any of those HSIL features, even though there were several microscopic foci showing severe nuclear atypia in the upper two-thirds of the papillomatous epithelium. Conclusion: Our observation regarding the occurrence of HSIL involving perianal condyloma acuminatum in human immunodeficiency virus-infected patients suggests that active, complete surgical excision of perianal condyloma acuminatum and a thorough histopathological examination are necessary. The diagnosis of severe nuclear atypia involving the upper two-thirds of the epithelium should be made with great caution.
- High-grade squamous intraepithelial lesion
- condyloma acuminatum
- human immunodeficiency virus
- human papillomavirus
- p16
Human papillomavirus that infects the lower anogenital tract is classified into low- and high-risk types. Low-risk human papillomavirus, such as types 6 and 11, is involved in the development of anogenital condyloma (1). High-risk human papillomavirus, such as types 16, 18, and 31, is implicated in the development of anogenital squamous cell carcinoma and its precursor lesion, as well as condyloma (2). The terminology for human papillomavirus-associated squamous lesions has a long history marked by disparate diagnostic terms derived from multiple specialties. It often does not reflect the current knowledge of human papillomavirus biology and pathogenesis (3). The diagnosis of anogenital squamous intraepithelial lesion and the assessment of its severity have been made by subjective histopathological interpretation. The Lower Anogenital Squamous Terminology Standardization Project was designed to reassess and harmonize the terminology used to describe human papillomavirus-associated squamous lesions of the lower anogenital tract, leading to improved diagnostic reliability and reproducibility (4). The Lower Anogenital Squamous Terminology Standardization Project proposed a single unified, two-tiered nomenclature: low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion (HSIL). Even though the grade of SIL in this system is mainly determined by the topography and the severity of nuclear atypia, p16 immunostaining is recommended to confirm a diagnosis of HSIL when encountering equivocal squamous lesions based on routine histopathological examination (5). The equivocal lesions, i.e., morphologically suspected HSIL, with block positivity for p16 are classified as HSIL, whereas those with patchy p16 expression are classified as benign, non-HSIL lesions such as immature squamous metaplasia and atrophy (6). The unambiguous distinction between premalignant lesions and those without malignant potential would inevitably lead to increased consistency in the interpretation of the management guidelines and the therapeutic options offered to patients.
Condyloma acuminatum is a well-known human papillomavirus-related lesion, which typically exhibits papillary proliferation of the squamous epithelium with low-grade cytopathic features of human papillomavirus infection (3). Although uncommon, severe nuclear atypia can be observed in condyloma acuminatum (7). To the best of our knowledge, the development of severe nuclear atypia involving perianal condyloma acuminatum has not been detailed in any prior reports. As anogenital HSIL is considered to be premalignant, its stigmata in condyloma acuminatum should be recognized. It is also important for pathologists not to overcall the degree of nuclear atypia based on subjective evidence. Nevertheless, the lack of a standardized diagnostic approach to perianal condyloma acuminatum with severe nuclear atypia could potentially lead to the overestimation of low-grade lesions to be high-grade lesions, particularly when the topography of severe nuclear atypia is overlooked. In this study, we investigated nine cases of perianal condyloma acuminatum with severe nuclear atypia and distinguished six cases of HSIL involving condyloma acuminatum from three non-HSIL cases. In addition, we analyzed their clinical features and pathological characteristics using electric medial record review, histopathological examination, immunohistochemical staining, and human papillomavirus genotyping.
Materials and Methods
Case selection. This study (4-2017-0952) was reviewed and approved by the Institutional Review Board at Severance Hospital (Seoul, Republic of Korea). During the study period, 729 patients were diagnosed with perianal condyloma acuminatum from January 2006 to December 2016. Nine patients were diagnosed as having perianal condyloma acuminatum with severe nuclear atypia. The reviewed clinical details included the patient age at the initial diagnosis, sex, human immunodeficiency infection status, comorbidity, CD4 count (cells/μl), multiplicity, gross finding, coexistent anogenital pathology, recurrence, and the time interval between initial diagnosis and development of recurrence/ progression.
Pathological examination. The resected tissues were initially examined by pathologists before fixation in 10% neutral-buffered formalin for 12-24 hours. The tissues were then thoroughly examined macroscopically and sectioned. After processing with an automatic tissue processor (Peloris II; Leica Microsystems, Newcastle Upon Tyne, UK), the sections were embedded in paraffin blocks and 4-μm-thick slices were cut using a rotary microtome (RM2245; Leica Microsystems, Newcastle Upon Tyne, UK). The sections were stained with hematoxylin and eosin using an automatic staining instrument (Ventana Symphony System, Ventana Medical Systems, Tucson, AZ, USA), and covered with a glass coverslip. All available hematoxylin and eosin-stained slides were examined by light microscopy (BX43 System Microscope; Olympus, Tokyo, Japan) and a definite pathological diagnosis was made. The most representative slide for each case was chosen for immunohistochemical staining and human papillomavirus genotyping. Pathological details that were reviewed included the presence of basaloid cell expansion into the upper two-thirds of the epithelium, presence of mitotic figures, presence of atypical mitosis, presence of severe nuclear atypia in the lower one-third, presence of bizarre nuclei, and appearance of the basement membrane beneath areas of HSIL.
Immunohistochemistry. The formalin-fixed, paraffin-embedded tissue blocks were sectioned at 4 μm thickness onto Superfrost Plus glass slides (Thermo Fisher Scientific, Waltham, MA, USA). These sections were deparaffinized in xylene and rehydrated through graded alcohols. Immunohistochemical staining was performed using an automatic immunostaining instrument (Ventana Benchmark XT; Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturer's recommendations (8-15). Antigen retrieval was performed using a Cell Conditioning Solution (CC1; Ventana Medical Systems, Tucson, AZ, USA). The sections were incubated with a p16 primary antibody (prediluted, clone E6H4, Ventana Medical Systems, Tucson, AZ, USA). After chromogenic visualization using an ultraView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, AZ, USA), the slices were counterstained with hematoxylin, dehydrated in graded alcohols and xylene, and then embedded in mounting solution. Appropriate positive and negative controls were concurrently stained to validate the staining method. According to the recommendation of the Lower Anogenital Squamous Terminology Standardization Project, the p16 immunostaining pattern was interpreted as block positive when p16 expression was horizontally continuous and strong, with nuclear or nuclear plus cytoplasmic staining. All other p16 immunostaining patterns, described as focal nuclear staining or wispy, blob-like, puddled, or scattered cytoplasmic staining, were interpreted as patchy positive (3).
Human papillomavirus genotype assay. We performed a polymerase chain reaction (PCR)-based microarray for human papillomavirus genotyping using a commercially available HPV 9G DNA chip (BMT HPV 9G DNA Chip; Biometrix Technology, Chuncheon, Republic of Korea) (15-17). The 9G test examined the presence of 14 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and five low-risk (6, 11, 34, 40, and 42) human papillomavirus types; analyses were performed according to the manufacturer's recommendations. Briefly, the PCR mixture consisted of 10 μl of the extracted target DNA, 10 μl of BMT Primer set (Biometrix Technology, Chuncheon, Republic of Korea), and PCR premix (Biometrix Technology, Chuncheon, Republic of Korea) that contained dNTP and Taq DNA polymerase in an amplification buffer. Amplification was performed using the following steps: predenaturation for 5 min at 94°C; 40, 30 sec denaturation cycles at 94°C; 40, 30 sec annealing cycles at 45°C; 40, 30 sec elongation cycles at 72°C; and a final 5-min elongation step at 72°C. The PCR products were electrophoresed in a 2% agarose gel to confirm successful amplification. Each hybridization chamber of the 9G was covered with a mixture of the hybridization solution (35 μl) and the polymerase chain reaction product (15 μl) and incubated at 23-26°C for 30 min. After washing, array images were scanned and imaged using a fluorescent scanner (ScanArray GX Microarray Scanner; PerkinElmer Life and Analytical Sciences, Waltham, MA, USA).
Results
Clinical features. Table I summarizes the clinical features of nine patients diagnosed as having perianal condyloma acuminatum with severe nuclear atypia. Eight (88.9%) patients were male and the median age was 52 years (range=41-78 years). Six (66.7%) patients were human immunodeficiency virus-infected males; their CD4 count ranged from 25 to 543 cells/μl. Three of the six human immunodeficiency virus-infected patients had systemic diseases including tuberculosis, concurrent viral and fungal infection, and diffuse large B-cell lymphoma. At the time of presentation, seven (77.8%) had coexistent perianal diseases including perianal abscess and/or fistula (5/7) and hemorrhoids (4/7). Two (22.2%) had a history of perianal condyloma acuminatum before the diagnosis of condyloma acuminatum with severe nuclear atypia. Grossly, the lesions were mainly located within 3 cm of the anal verge. The greatest dimension ranged from 0.5 to 3.0 cm (median, 1.0 cm). They presented as single or multiple, white-to-gray/tan, solid, cauliflower-like, warty, or polypoid masses. The outer surfaces were smooth, verrucous, or ulcerated. All patients underwent complete surgical excision.
Clinical features of nine patients originally diagnosed as having perianal condyloma acuminatum with severe nuclear atypia.
The follow-up period ranged from 25-84 months (median, 54 months). Two (22.2%) of the nine patients with HSIL developed recurrent perianal masses (case 2), with disease progression to invasive carcinoma (case 3). One patient (case 2) presented with several tan/gray cauliflower-like masses 50 months postoperatively. The other patient (case 3) presented with a 4 cm-sized, gray/white, fungating mass with verrucous surface 54 months postoperatively. The remaining 7/9 (77.8%) patients did not experience recurrence or disease progression after surgical excision.
Pathological features. Table II summarizes the histopathological features, immunostaining results, and human papillomavirus genotypes of the 10 specimens originally diagnosed as condyloma acuminatum with severe nuclear atypia. First, all specimens demonstrated typical condyloma acuminatum morphology characterized by papillomatosis, acanthosis, and koilocytosis (Figure 1A). In the superficial epithelium, the squamous cell nuclei exhibited anisonucleosis, angulated contour, irregular membrane, hyperchromasia, and inconspi-cuous nucleoli (Figure 1B). Binucleated and occasionally multinucleated cells were also noted. The cytoplasm was pale-to-weakly eosinophilic. The basal and parabasal cells showed a regular arrangement and occasional mitoses (Figure 1C). They possessed a densely eosinophilic cytoplasm. Some areas of condyloma acuminatum were associated with mixed inflammatory infiltrate, of which the degree varied with location and was most prominent in the superficial layer. In addition, all specimens showed several microscopic foci indicating severe nuclear atypia involving the upper two-third of the papillomatous epithelium. The nuclei of these foci were markedly angulated, irregularly enlarged, and hyperchromatic, with coarse chromatin and inconspicuous nucleoli (Figure 1D).
Pathological features of 10 specimens originally diagnosed as perianal condyloma acuminatum with severe nuclear atypia.
In addition to the above-mentioned histopathological features, seven (70.0%) specimens displayed the expansion of small, undifferentiated cells involving the full thickness of the papillomatous and acanthotic epithelium (Figure 2A). The morphological features of these cells were different from those of condyloma acuminatum. These basaloid cells were ovoid-to-elongated, had hyperchromatic nuclei and a scant, densely eosinophilic cytoplasm, resulting in a high nuclear-cytoplasmic ratio (Figure 2B). Nucleoli were inconspicuous. These features were consistent with HSIL. The proportion of surface epithelium involved in HSIL ranged from 5-20%. In 4/7 specimens, proliferating basaloid cells were bordered by a linear, clearly delineated basement membrane. These cells showed no severe nuclear pleomorphism in the lower one-thirds, atypical mitosis, or bizarre nuclei (Figure 2C). In contrast, 3/7 specimens exhibited marked irregular acanthosis with infiltrative, endophytic borders extending into the superficial dermis, resembling those of pseudoepitheliomatous hyperplasia (Figure 2D and 2E). In these specimens, severe nuclear pleomorphism in the lower one-third and mitotic figures in upper two-thirds were observed. Enlarged bizarre nuclei and multinucleated giant cells were also identified in two of the three specimens (Figure 2F). One specimen had atypical mitosis (Figure 2G). The remaining 3/10 (30.0%) specimens did not show the expansion of basaloid cells, severe nuclear pleomorphism in the lower one-thirds, bizzare nuclei, mitotic figure in the upper two-thirds, or atypical mitosis.
Histopathological features of perianal condyloma acuminatum. A: Condyloma acuminatum consisted of a papillary proliferation of squamous epithelium with acanthosis and koilocytosis. B: The superficially located squamous cell nuclei exhibited anisonucleosis, angulated contour, irregular membrane, and hyperchromasia. C: In contrast, in the lower one-third, the basal and parabasal cells showed a regular arrangement, minimal nuclear atypia, and occasional mitosis. D: There were several microscopic foci showing severe nuclear atypia involving the upper two-thirds of the papillomatous epithelium. The nuclei of these foci were markedly angulated, irregularly enlarged, and hyperchromatic with coarse chromatin.
Immunohistochemical staining revealed that the 7/10 specimens with the features of HSIL exhibited a horizontally continuous and strong p16 expression in the nuclei and cytoplasm (block positivity for p16; Figure 3A), whereas the remaining 3/10 specimens displayed patchy p16 expression (Figure 3B). Human papillomavirus genotype was analyzed using the HPV DNA chip assay. Low-risk human papillomavirus was detected in 8/10 (80.0%) specimens. High-risk human papillomavirus was detected in all the seven specimens with features of HSIL. The genotype was variable; type 16 was detected in 5/7 (71.4%) specimens, and type 18 (14.3%) and type 31 (14.3%) were detected in one specimen for each. Results of p16 immunostaining and human papillomavirus genotyping confirmed that the 7/10 specimens showing morphological features suggestive of HSIL, block p16 positivity, and high-risk human papillomavirus infection were HSIL involving perianal condyloma acuminatum.
The pathological diagnosis of a recurrent lesion in case 2 was HSIL involving perianal condyloma acuminatum. The pathological diagnosis of progressive disease in case 3 was well-differentiated squamous cell carcinoma.
Histopathological features and p16 immunostaining results of perianal condyloma acuminatum showing features of high-grade squamous intraepithelial lesion (HSIL). A: Small, undifferentiated (basaloid) cells occupied the full thickness of the papillomatous and acanthotic epithelium. B: These basaloid cells were ovoid-to-elongated, possessed hyperchromatic nuclei and a scant densely eosinophilic cytoplasm, and displayed an increased nuclear-cytoplasmic ratio. C-E: Two morphological patterns of HSIL occurring in association with perianal condyloma acuminatum were observed: C: the proliferation of basaloid cells with a linear, clearly delineated basement membrane, and D and E: those with an irregular basement membrane resembling that of pseudoepitheliomatous hyperplasia. F-G: The pseudoepitheliomatous-like pattern demonstrated severe nuclear pleomorphism in the lower one-third, F: enlarged bizarre nuclei, and G: atypical mitosis.
p16 Immunostaining results. A: Areas of HSIL showed continuous and strong p16 expression in the nuclei and cytoplasm (block positivity). B: In contrast, areas of severe nuclear atypia involving the upper two-thirds of the papillomatous epithelium displayed patchy p16 expression.
Discussion
Severe nuclear atypia associated with perianal condyloma acuminatum was observed along the upper two-thirds of the epithelium. These specimens did not exhibit any features suggestive of HSIL. The Lower Anogenital Squamous Terminology Standardization Project recommended that the criteria for diagnosis of HSIL was the expansion of basaloid cells, severe nuclear pleomorphism, bizzare nuclei, mitotic figure in the upper two-thirds, and/or atypical mitosis (6). Despite severe nuclear atypia the upper two-thirds, a lack of HSIL features, patchy p16 expression, and negative high-risk human papillomavirus oppose the diagnosis of HSIL. In a previous study, severe nuclear atypia in the superficial layer was observed in 12% of condylomatous lesions with low-risk human papillomavirus infection (18). Association of severely atypical cells with mixed inflammatory infiltrate, which was most prominent in the superficial layer, further support the possibility that the atypia is reactive in nature.
The histopathological features of 7/10 specimens satisfied at least one of the HSIL criteria. The expansion of basaloid cells into the upper two-thirds or full thickness of the epithelium was consistently observed. Three of the seven specimens showed three or more HSIL features. Block p16 positivity and high-risk human papillomavirus infection further support the diagnosis of HSIL. Five of the seven specimens showed concurrent infection with low- and high-risk human papillomavirus, raising the possibility that superimposed high-risk human papillomavirus infection is at least partly involved in the development of HSIL in patients with condyloma acuminatum. In addition, our observation that only a small proportion of surface epithelium is involved in HSIL indicates that the biopsy does not accurately reflect the actual pathology of the entire specimen. We suggest that the most appropriate way to diagnose HSIL, accompanying a perianal condyloma acuminatum, is to increase the possibility of detection through multiple biopsies or through complete excision of the condyloma acuminatum lesion. A combination of p16 immunostaining and human papillomavirus genotyping may aid in the interpretation of condyloma acuminatum cases with severe nuclear atypia that are insufficient for the diagnosis of HSIL.
It is noteworthy that all (6/6) patients with HSIL involving perianal condyloma acuminatum were human immunodeficiency virus-infected males. Human immunodeficiency virus infection is an well-known risk factor for perianal squamous cell carcinoma (19). In this study, we observed that 2/6 human immunodeficiency virus-infected patients developed recurrent HSIL and squamous cell carcinoma, respectively. This finding suggests that human immunodeficiency virus infection is implicated in the development of HSIL within perianal condyloma acuminatum. Conversely, the diagnosis of HSIL in perianal condyloma acuminatum should be made with great caution if the patient is immunocompetent.
We observed two morphological patterns of HSIL occurring in association with perianal condyloma acuminatum: the proliferation of basaloid cells with a linear, clearly delineated basement membrane and those with an irregular basement membrane resembling that of pseudoepitheliomatous hyperplasia. Severe nuclear atypia in the lower one-third of the epithelium, enlarged bizarre nuclei, and mitotic figures in the upper two-thirds were frequently observed in cases with the pseudoepitheliomatous-like pattern, whereas the former pattern was not accompanied by any of the features suggestive of HSIL. Interestingly, the HSILs in the two patients who developed recurrent HSIL and squamous cell carcinoma predominantly possessed the pseudoepitheliomatous-like pattern. HSIL with a pseudoepitheliomatous pattern can sometimes exhibit nests of highly atypical squamous epithelium in the dermis, raising the suspicion of invasive squamous cell carcinoma in patients with HSIL. The lack of a desmoplastic stromal reaction and individually infiltrating atypical cells can be helpful to exclude the possibility of malignancy.
In conclusion, we demonstrated a series of cases of HSIL occurring in association with perianal condyloma acuminatum. High-risk human papillomavirus infection was found to be associated with the presence of HSIL in human immunodeficiency virus-infected, immunocompromised patients. The gross distinction between HSIL and perianal condyloma acuminatum is almost impossible when both appear simultaneously. Our observation regarding the occurrence of HSIL involving perianal condyloma acuminatum suggests that active, complete surgical excision of the perianal condyloma acuminatum and a thorough histopathological examination are necessary. A combination of p16 immunostaining and human papillomavirus genotyping may aid in the interpretation of HSIL in cases showing insufficient morphological evidence for the diagnosis of HSIL.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B4007704) and by the Ministry of Education (2016R1D1A1B03935584), and a Faculty Research Grant of Yonsei University College of Medicine (6-2017-0036).
- Received December 11, 2017.
- Revision received January 1, 2018.
- Accepted January 3, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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