p16 Immunoreactivity Correlates With Morphologic Diagnosis of HPV-associated Anal Intraepithelial Neoplasia: A Study of 1000 Biopsies.

p16 is the most useful diagnostic marker for human papillomavirus (HPV)-associated anogenital lesions. In the cervix, the pattern of p16 immunoreactivity generally correlates with lesion severity. p16 expression in anal intraepithelial neoplasia (AIN) is far less studied. Whether such correlation holds true has to be determined. We correlated the degree and pattern of p16 immunohistochemistry (IHC) results with morphologic diagnoses of 1000 anal squamous and transitional zone biopsy specimens. Using the Lower Anogenital Squamous Terminology criteria, p16 IHC results were classified as block staining, partial staining, or negative. Among 150 samples without morphologic evidence of AIN, p16 was negative in 85% and partial staining in 15%. AIN 1 (n=400) revealed diverse results: 28% negative, 35% partial, and 37% block staining. Among AIN 2 (n=298), 89% were block, 9% partial staining, and 2% negative. AIN 3 (n=152) revealed block (95%) or partial staining (5%). For the detection of AIN 2/3, p16 block staining yielded 91% sensitivity, 73% specificity, 80% positive predictive value, 91% negative predictive value, and a Youden Index of 0.64. Combining block staining and partial staining slightly increased sensitivity (99%) and negative predictive value (98%), but significantly decreased specificity (43%), positive predictive value (59%) and Youden Index (0.42, P<0.001). As with the cervix, p16 immunoreactivity correlates with morphologic diagnoses of AIN. Block staining offers the optimal diagnostic value for AIN 2/3. Caution is required since AIN 1 frequently exhibits block staining; the prognostic value of p16 warrants further investigation.

Clinicopathologic Features of Anal and Perianal Squamous Cell Carcinomas and Their Relationship to Human Papillomavirus.

Anal squamous cell carcinomas (ASCCs) frequently harbor human papillomavirus (HPV), most commonly high-risk (HR-) HPV type 16. While p16 immunohistochemistry (IHC) is typically used as a surrogate for HR-HPV status in the oropharynx and cervix, its overexpression can also occur as a result of oncogenic stress and sometimes prove nonspecific. There have been recent investigations into the use of HPV RNA in situ hybridization (RISH) assays as an alternative method, which have shown robust results for squamous cell carcinomas of the oropharynx and cervix. Our study evaluated HPV RISH and p16 IHC in 50 ASCCs, as well as the clinicopathologic features of ASCC relative to HPV status. We found that HPV RISH and p16 IHC were closely in agreement with 96% concordance. Using the 2 methodologies, 78% of ASCCs were HR-HPV positive, 10% were low-risk HPV positive, and 12% were HPV-negative. None of our cases showed co-infection across HR-HPV and low-risk HPV. ASCCs that were not related to HR-HPV were more likely to have a typical keratinizing morphology (P=0.05) and more likely to involve the perianal area (P=0.006). HPV-negative cases were particularly aggressive with high rates of metastases and patient death within 2 years of diagnosis. Overall, HPV RISH appears to be a reliable methodology for testing, and HPV status may have implications for prognostication of ASCCs.

Presence or Absence of Significant HPVE4 Expression in High-grade Anal Intraepithelial Neoplasia With p16/Ki-67 Positivity Indicates Distinct Patterns of Neoplasia: A Study Combining Immunohistochemistry and Laser Capture Microdissection PCR.

Progression of anal intraepithelial neoplasia (AIN) involves transition from productive to transforming human papillomavirus (HPV) infection. Grading aims to distinguish productive low-grade AIN from high-grade anal intraepithelial neoplasia (HGAIN) with risk of cancer. We describe immunohistochemical patterns in AIN adding a novel marker for initiation of the productive phase of the HPV life cycle (panHPVE4) to those for cell cycle activity (Ki-67) and transforming activity of HPVE7 gene (p16). We studied 67 anal biopsies for suspected anal neoplasia (17 normal, 15 AIN1, 20 AIN2, 15 AIN3) from 54 men who have sex with men at New York Presbyterian Hospital, USA. Two pathologists generated consensus AIN and immunogrades. Whole tissue and laser capture microdissection samples from multiple HPV-infected biopsies were tested for HPV with SPF10-PCR-DEIA-LiPA25, version 1. (Para)basal Ki-67 expression distinguished normal from AIN (≥lower-third Ki-67) with sensitivity 0.92 and specificity 1.0. Ki-67 did not distinguish grades of AIN. Null/patchy p16 versus diffuse ≥lower-third patterns discriminated HGAIN (sensitivity, 1.0; specificity, 0.84). There was marked heterogeneity in E4 expression within HGAIN. Most AIN2 (14/20) was E4 versus 0/15 AIN3 (sensitivity, 0.70; specificity 1.0). HPV was detected in 63 (94%) biopsies, with 49 (77.8%) high-risk HPV. HPV16 was the most frequent (13%). Multiple HPV genotypes were found in 15 (24%) biopsies and laser capture microdissection -polymerase chain reaction confirmed specific HPV types in E4 +/- AIN. Although Ki-67 discriminated AIN and p16 HGAIN, E4/p16 staining shows that most AIN2 is different from transformed AIN3 in showing both entry into productive HPV infection and transforming activity.

Malignant transformation of a chronic anorectal fistula.

Up to date, only a small number of carcinomas arising from a chronic anorectal fistula have been described in medical literature, especially in patients without Chron's disease. A 72-year-old man with a 6-year history of discharging perianal sinus without Crohn's disease arrived at our institution. He had previously undergone three surgical procedures in other institutions for incision and drainage of recurrent perianal abscesses. Our therapeutical approach was to drain the two abscess cavities, perform a fistulectomy, and biopsy the fistula tissue. Anatomopathological examination of the specimen revealed a mucosecerrnig adenocarcinoma arising from the fistula tract. We decided to perform an abdominal perineal resection. The two-year oncological follow-up is negative. In conclusion, it is clear that the diagnosis of mucinous adenocarcinoma occurring in perianal fistula is difficult, particularly in patients without any risks or predisposing factors. Wide resection of the tumor with Miles's procedure still represents the surgical treatment of choice and may provide a good long term outcome in localized disease KEY WORDS: Cronic anorectal fistula, Malignant transformation, Mucinous adenocarcinoma.

No additional prognostic value for MRE11 in squamous cell carcinomas of the anus treated with chemo-radiotherapy.

BACKGROUND: The majority of anal cancers (84-95%) are driven by infection with human papillomavirus (HPV). HPV-positive tumours show significantly better responses to chemo-radiotherapy when compared with HPV-negative tumours. HPV infection is linked to alterations in DNA damage response proteins, including MRE11. MRE11 is a potential predictive biomarker for response to radiotherapy in muscle-invasive bladder cancer and may hold predictive power in other cancers. METHODS: Using a previously reported cohort, we evaluated the levels of MRE11 in anal cancer and assessed its predictive value in this disease. RESULTS: We found no association between the level of MRE11 and relapse-free survival following chemo-radiotherapy. CONCLUSIONS: MRE11 has no predictive value in the analysis of relapse-free survival after chemo-radiotherapy in anal cancer and does not add to the prognostic value of p16 and tumour-infiltrating lymphocyte scores. Further investigation into the role of DNA repair proteins in anal cancer is required.

HR-HPV E6/E7 mRNA In Situ Hybridization: Validation Against PCR, DNA In Situ Hybridization, and p16 Immunohistochemistry in 102 Samples of Cervical, Vulvar, Anal, and Head and Neck Neoplasia.

Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types ("HR-RISH," aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases.

Syringocystadenocarcinoma Papilliferum In Situ-Like Changes in Extramammary Paget Disease: A Report of 11 Cases.

The authors report 11 cases of extramammary Paget disease (EMPD), all of which also demonstrated a combination of histological changes highly reminiscent of syringocystadenocarcinoma papilliferum in situ. In addition to the classical features of EMPD, characterized by the intraepidermal spread of individually dispersed neoplastic cells with ample cytoplasm, many of which contained mucin, there were areas of acanthosis with the substitution of spinous layer keratinocytes by neoplastic cells, whereas the native basal cell layer was intact. In addition to acanthosis (and sometimes papillomatosis), the dermal papillae showed a prominent infiltrate of plasma cells, completing the resemblance to syringocystadenocarcinoma papilliferum in situ; this similarity was further enhanced in 2 cases, which showed conspicuous gland formation. One additional case showed multifocal dermal proliferations compatible with eccrine syringofibroadenoma (syringofibroadenomatous hyperplasia). The changes described herein seem to be relatively rare in EMPD, and they can represent a diagnostic pitfall, as evidenced by 2 cases that were originally misinterpreted as syringocystadenocarcinoma papilliferum in situ. Clinically, these microscopic changes sometimes corresponded to nodular lesions, which were specifically noted to have a papillated erosive surface.

Basaloid Squamous Cell Carcinoma of the Anus Revisited.

Basaloid squamous cell carcinoma (SCC) of the anus, previously called cloacogenic carcinoma, is a subtype of SCC. There are very few data on the morphologic variation within basaloid SCC of the anus, which may contribute to misdiagnosis. We retrospectively evaluated cases originally diagnosed as basaloid SCC for histologic characterization. We retrieved and reviewed cases of basaloid SCC from 1994 to 2013. Ten (27%) cases were reclassified after review, including basal cell carcinoma (n=6), melanoma (n=2), and neuroendocrine carcinoma (n=2). The final group of basaloid SCC (n=27) showed a female predominance (median age=60 y; range, 42 to 92 y). Morphologically, basaloid SCC could be categorized into 4 groups: transitional carcinoma like (n=10), basaloid with peripheral palisade (n=13), adenoid cystic carcinoma like (n=3), and mucinous microcystic (n=1). In 19 cases the histologic patterns were pure and were mixed in the remainder. CK5/6, p16, and high-risk HPV were positive in all cases (n=27). SOX2 was positive in 18/22 cases. Clinical follow-up was available on 60% of cases; 9 patients (53%) developed local recurrence or metastasis, and 5 (29%) died of disease. Basaloid SCC of the anus is characterized by 4 major histologic patterns and is consistently HPV driven.

Papillary Immature Metaplasia of the Anal Canal: A Low-grade Lesion That Can Mimic a High-grade Lesion.

In a natural history study of anal human papillomavirus (HPV) infection and HPV-related lesions among homosexual men in Sydney, Australia, we identified 15 examples of papillary immature metaplasia (PIM) in anal biopsy samples. PIM has previously been described in the cervix, but not in the anal canal. PIM is a form of exophytic low-grade squamous intraepithelial lesion (eLSIL) also known as condyloma. In contrast to the maturing keratinocytes and koilocytosis seen in conventional eLSIL, the slender papillary structures of PIM have a surface population of immature squamous cells. In our anal samples PIM was characterized by close proximity to conventional eLSIL, was negative for p16 (p16) expression, and revealed the presence of a single low-risk HPV genotype (either 6 or 11) in laser capture microdissected lesions. The clinical significance of recognizing PIM lies in preventing misdiagnosis as high-grade squamous intraepithelial lesion, (the presumed precursor to anal cancer), due to the morphologic immaturity of the cell population. In routine practice, awareness of anal canal PIM and p16 immunostaining will prevent this. Further study of the natural history of anal canal PIM is needed.

Role of the Biomarker p16 in Downgrading -IN 2 Diagnoses and Predicting Higher-grade Lesions.

In 2012, the College of American Pathologists and American Society for Colposcopy and Cervical Pathology published the "LAST" recommendations for histopathology reporting of human papilloma virus-related squamous lesions of the lower anogenital tract, including the use of a 2-tier nomenclature (low-grade squamous intraepithelial lesion/high-grade squamous intraepithelial lesion [LSIL/HSIL]) and expanded use of the biomarker p16 to classify equivocal lesions as either precancer (HSIL) or low-grade lesions (LSIL)/non-human papilloma virus changes. We aimed to determine (1) the frequency with which the poorly reproducible diagnosis of intermediate-grade (-IN 2) lesion in the lower anogenital tract would be downgraded on the basis of p16 results, and (2) whether p16 status was predictive of subsequent higher-grade lesions. A total of 200 specimens diagnosed as an intermediate-grade (-IN 2) lesion of the cervix (168), vagina (2), vulva (2), and anus (28) were reviewed and immunostained for p16. Slides were independently reviewed by 2 pathologists, with discrepant p16 interpretations adjudicated by a third pathologist. Of the 200 cases, 32% were negative for p16. Among the 166 patients with subsequent pathology (including 131 excisions), 26.2% of p16-positive cases versus 4.4% of p16-negative cases were associated with a subsequent diagnosis of HSIL (-IN 3) or worse (P=0.002). Reproducibility of the biopsy diagnosis was fair, with no significant difference with the addition of p16 or using 2 versus 3 tiers. In 11.5% of cases, there was discordance in p16 interpretation (κ 0.735, good agreement). The results indicate that using the Lower Anogenital Squamous Terminology recommendations would result in approximately one third of equivocal (-IN 2) diagnoses being downgraded to LSIL over 1 year in a busy academic practice. The significant association of p16 expression with a higher risk for HSIL on a subsequent specimen suggests that use of p16 to adjudicate equivocal (-IN 2) diagnoses in lower anogenital tract specimens as either LSIL or HSIL would likely predict lesion grade more accurately and avoid unnecessary excisional procedures.

Immunohistochemistry and in situ hybridization for the diagnosis and classification of squamous lesions of the anogenital region.

Distinguishing anogenital squamous intraepithelial lesions from benign conditions and mimics may be problematic. Immunohistochemistry for surrogate markers of HPV infection, such as Ki-67, p16, and ProEx™ C, may aid the diagnosis in equivocal cases. The main diagnostic pitfall in the diagnosis of LSIL is the occurrence of "pseudokoilocytes" in benign squamous mucosa, which may lead to overdiagnosis. When interpreted correctly, Ki-67 is a sensitive and specific marker for dysplasia in mature squamous epithelium and is therefore useful for confirmation of LSIL and condyloma. A Ki-67 positive result is defined as the presence of a cluster of at least two strongly stained epithelial nuclei in the upper two-thirds of the epithelial thickness. With such a definition, there is almost complete concordance between consensus diagnosis of LSIL/condyloma confirmed by detection of HPV DNA and positive Ki-67. A related proliferation marker, ProEx™ C, has similar staining patterns and utility for the diagnosis of low grade dysplasia. The differential diagnosis of HSIL includes atypical immature squamous metaplasia and atrophy. A marker with high sensitivity and specificity for the detection of HSIL in cervical, vulvar, and anal mucosa is p16. A 2-tier scoring system is used to evaluate p16 staining. No staining or a discontinuous, patchy nuclear and cytoplasmic staining pattern is considered as a negative result. A positive result is defined as diffuse and strong staining of cells of the basal and parabasal layers of the squamous epithelium, with or without staining of superficial cell layers. New markers that are undergoing evaluation for their clinical utility include stathmin-1, phosphorylated S6, and SOX2. Confirmation of the diagnosis of dysplasia by HPV detection in tissue sections using HPV capsid protein immunohistochemistry, HPV DNA or HPV RNA in situ hybridization offers lower sensitivity as compared to immunohistochemistry for surrogate markers and therefore has more limited utility in this context.

Human papillomavirus-related squamous cell carcinoma of the anal canal with papillary features.

Human papillomavirus (HPV) related squamous cell carcinoma (SCC) involving the anal canal is a well-known carcinoma associated with high-risk types of HPV. HPV-related SCC with papillary morphology (papillary SCC) has been described in the oropharynx. We describe, for the first time, a case of anal HPV-related squamous carcinoma with papillary morphology. The tumor arose from the anal mucosa. The biopsies revealed a superficially invasive SCC with prominent papillary features and associated in situ carcinoma. The tumor cells were positive for p16 and were also positive for high-risk types of HPV using chromogenic in situ hybridization. The findings are consistent with a HPV-related SCC of the anal canal with papillary features. This tumor shows histologic features similar to a papillary HPV-related SCC of the oropharynx. Additional studies are needed to characterize these lesions.

KIT genetic alterations in anorectal melanomas.

BACKGROUND: Mucosal melanomas (MM) represent a heterogeneous tumour population that exhibits site-specific molecular profiles. AIMS: In a multicentre retrospective study, we investigated KIT aberrations in primary anorectal (AR) melanomas compared with melanoma metastatic to the gastrointestinal (GI) tract. METHODS: Primary AR MM (n=31) and GI metastatic melanoma (n=27) were studied for KIT mutations on exons 11, 13, 17 and 18 by high-resolution melting analysis, direct sequencing and c-KIT expression by immunohistochemistry. Selected cases were also investigated for increased KIT gene copy number by fluorescent in situ hybridisation. RESULTS: Functional KIT mutations were demonstrated in 11/31 (35.5%) of AR melanomas and in 1/26 (3.8%) of GI melanoma metastases (p=0.004). A significant difference emerged between primary and metastatic MM with regards to KIT-positive immunostaining (p=0.002). Immunohistochemical c-KIT protein overexpression did not correlate with KIT mutational status. Increased KIT copy number was demonstrated in 5/20 AR primary cases. CONCLUSIONS: The rate of functional mutations in KIT is significantly higher in AR MM than in GI metastatic melanoma. KIT protein overexpression does not correlate with KIT mutations and cannot be used for screening purposes. Recognising the molecular heterogeneity of MM helps to identify patients who require a different therapeutic approach.

Anal intraepithelial neoplasia: a single centre 19 year review.

AIM: There is debate about whether the traditional three-tiered grading of anal intraepithelial neoplasia (AIN) should be replaced by a more reproducible two-tiered system. In this study, we review our experience with AIN to determine the most suitable classification system. METHOD: We performed a retrospective review of all histological reports over a 19 year period. All specimens were graded on haemataloxin and eosin appearance and those with dysplasia had immunohistochemistry for p16 and Ki67 performed. RESULTS: Cases included 25 condyloma acuminata, 11 dysplastic cases and 24 invasive squamous cell carcinomas. On review, 18 were classified as condyloma acuminata without dysplasia. Seven had AIN I, five had AIN II and six had AIN III when using a three-tiered system. All cases classified as dysplastic (n = 18) showed an increased proliferation index as measured by Ki67. p16 positivity was seen in all AIN III, two AIN II and none of the AIN I cases. Recurrence was not observed in any of the AIN I cases. Five of eleven AIN II and AIN III cases recurred or persisted at a similar, higher or lower grade. Both of the AIN II cases which recurred or persisted were p16 positive. None of the AIN II cases that were p16 negative recurred. Three of the p16-positive AIN III cases did not recur. None of the 18 AIN cases progressed to carcinoma. CONCLUSION: The findings support the slow progression of AIN as described in the literature. In our small series, a two-tiered system with further subclassification of the traditional AIN II group using p16 appears to be clinically useful.

Molecular and immunohistochemical profile of a basaloid (cloacogenic) carcinoma of the sigmoid colon: possible predictive value for clinical outcomes.

A 61-year-old woman was hospitalized with a 5-week history of abdominal discomfort, change in bowel habits, and weight loss. Colonoscopy showed a protruded tumor of the sigmoid colon first diagnosed as undifferentiated carcinoma. Surgical resection of the sigmoid colon was performed. Histological examination of the surgical specimen showed a proliferation of basaloid cells arranged in tumor clusters with central comedonecrosis and peripheral palisading of the nuclei. The tumor invaded the subserosa and presented liver metastasis without lymph node metastases. The tumor cells were marked by keratin AE1/AE3, keratin 5/6, epithelial membrane antigen, bcl-2, vascular endothelial growth factor, CD105, neuron-specific enolase, MLH-1, MSH-2, and p53, and were negative for keratin 7/20, chromogranin, synaptophysin, carcinoembryonic antigen, p63, c-KIT, and maspin. A high p53 nuclear index was also detected. On the basis of these characteristics and molecular examinations, the final diagnosis was microsatellite stable/human papilloma virus-negative/K-ras mutated/BRAF wild-type basaloid carcinoma (BC). Only seven BCs of the colon were reported in the literature, this being the eighth one and the first case that reports new molecular findings about microsatellite instability, K-ras/BRAF mutations, angiogenesis, and maspin expression in BC, with direct involvement in targeted therapy.