Breast cancers in monoallelic MUTYH germline mutation carriers have clinicopathological features overlapping with those in BRCA1 germline mutation carriers.

PURPOSE: Breast cancer patients referred to genetic counseling often undergo genetic testing with broad panels that include both breast cancer susceptibility genes as well as genes more specific for extramammary sites. As a result, patients are often incidentally found to have germline mutations in genes that are not necessarily related to breast cancer risk. One such gene is MUTYH. To understand the role MUTYH may play in breast cancer, the clinicopathological features of patients with monoallelic MUTYH germline mutation and breast cancer were examined. METHODS: The clinicopathological characteristics of the breast cancers from patients with monoallelic MUTYH mutation were compared to breast cancer patients with other germline mutations in known breast cancer susceptibility genes, including ATM, BRCA1/2, CHEK2, and PALB2. The breast cancer patients who received genetic counseling but tested negative for the aforementioned gene mutations were used as a control group. RESULTS: Histologic characteristics of the breast cancers arising in monoallelic MUTYH mutation carriers had significantly larger tumor size, higher tumor grade, and more high-risk biomarker profiles (i.e., Her2-positive and triple-negative) than breast cancer patients with susceptibility genes, except for BRCA1. MUTYH mutation carriers also showed a trend of more frequent intratumoral divergency in terms of tumor grade and biomarker profiles. CONCLUSION: Although germline monoallelic MUTYH mutation is not thought to confer a meaningfully increased risk of breast cancer development, it may contribute to pathological aggressiveness and diversity of breast cancers when they sporadically arise in MUTYH carriers.

Divergent Malignant Melanocytic Differentiation in Ovarian Endometrioid Adenocarcinoma With Aberrant ß-Catenin Expression: A Case Expanding the Histologic Spectrum of ß-Catenin Activated Gynecologic Neoplasia.

Divergent differentiation in gynecologic carcinomas encompasses a broad range of lineages, including mesenchymal, germ cell, high-grade neuroendocrine, neuroectodermal, and cutaneous adnexal differentiation. Here we present a case of ovarian endometrioid adenocarcinoma with divergent malignant melanocytic differentiation (MMeD). The background ovarian endometrioid adenocarcinoma showed focally aberrant ß-catenin expression and histologic patterns associated with ß-catenin activation, including spindled elements and corded and hyalinized foci. The areas with MMeD had both spindled and epithelioid morphology, diffusely aberrant ß-catenin expression, expression of melanocytic markers (an HMB45/Mart-1 cocktail, MITF, and S100), and no staining for myogenic markers (SMA and desmin) or epithelial markers (cytokeratins and E-cadherin). INI1, BRG1, PMS2, and MSH6 were retained, and p53 showed a wild-type expression pattern. No areas with definitive carcinosarcomatous differentiation were identified despite extensive sampling. While a single case of gynecologic carcinosarcoma with a serous epithelial component and a small focus on malignant melanoma has been reported in the English literature, the current case represents what is, to the best of our knowledge, the first case of MMeD arising in the context of a ß-catenin activated endometrioid adenocarcinoma. Pathogenetic and differential diagnostic considerations are discussed.

Immunohistochemistry as an adjunct for challenging histological patterns of borderline Brenner tumors: An illustrative study of 4 cases.

Borderline Brenner tumors (BBT) have a range of morphology that shows considerable overlap with that of malignant Brenner tumors (MBT). In particular, two histological patterns of BBT can be particularly challenging: 1) BBT with intraepithelial carcinoma (BBT-IEC) and 2) BBT with a small nested pattern (BBT-SNP). BBT-IEC is characterized by a tumor with the low-power non-infiltrative silhouette of a conventional BBT, but with increased cytological atypia and mitotic activity similar to that of MBT. Conversely, BBT-SNP is characterized by a complex proliferation of small tumor nests that closely resemble the infiltrative growth pattern of MBT, but without the obligate cytologic atypia and mitotic activity of MBT. We suggest that the combination of p16, p53 and Ki-67 may be helpful in distinguishing these 2 patterns of BBT from both conventional BBT and from MBT. While both conventional BBT and BBT-IEC show a null pattern of p16 expression, our case of BBT-IEC showed aberrant p53 overexpression, albeit with a maturation pattern similar to that described for TP53 mutant mucinous ovarian carcinoma and differentiated vulvar intraepithelial neoplasia (dVIN). Similarly, while BBT-SNP shows an infiltrative-like growth pattern similar to that of MBT, our case also showed a wild-type pattern of p53 expression and a Ki-67 proliferative index similar to areas with conventional BBT histology. In conclusion, in our small case series, we show that the use of immunohistochemistry for p53 and Ki-67 may help to distinguish challenging patterns of BBT from MBT. Further studies are needed to validate this finding in a larger case cohort.

Spatial transcriptomics of a giant pilomatricoma.

Pilomatricomas (PMs) are common benign adnexal tumors that show a predilection for the head and neck region and are characterized at the molecular level by activating mutations in the beta-catenin (CTNNB1) gene. Giant PMs are a rare histopathological variant, according to the World Health Organization, which are defined by a size greater than 4 cm and are reported to show upregulation of yes-associated protein compared to PMs of typical 1-3 cm size. We describe the case of a 67-year-old man with an 8 cm giant PM involving his temporal scalp, whose PM we characterized by 10X spatial gene expression analysis. This revealed five total transcriptomic clusters, including four distinct clusters within the giant PM, each with a unique transcriptional pattern of hair follicle-related factors, keratin gene expression, and beta-catenin pathway activity.

Clinicopathological characteristics of the SOX10+ subset of HER2+ breast cancer.

HER2-positive breast cancers (HER2+ BC) are a heterogeneous group of tumors with variable clinical behavior. SOX10, a biomarker that has been studied in the context of breast carcinomas, especially triple-negative breast carcinomas (TNBC), has yet to be systematically investigated in a cohort of HER2+ BC. Our aim was to investigate the clinicopathological features of the SOX10+ subset of HER2+ BC. 80 HER2+/ER- invasive breast carcinomas were stained for SOX10. All SOX10+ cases and a matched number of SOX10- cases were also stained for vimentin and androgen receptor (AR). 18 % (14/80) of our cases were SOX10+. SOX10 expression was seen in both IHC positive (3+) and equivocal (2+) but ISH-amplified cases. The SOX10+ tumors were significantly associated with both greater vimentin expression (36 % vs 0 %, p = 0.0407) and less AR expression (14 % vs 100 %, p = 0.0001) compared to SOX10- tumors. Interestingly, the vimentin+/AR- subset of our SOX10+ cases showed uniformly apocrine-like morphology, while all SOX10- cases, including those with apocrine-like morphology, were vimentin-/AR+. Our findings suggest that SOX10+/HER2+ BC are more likely to show a peculiar apocrine-like, vimentin+/AR- phenotype as compared to SOX10-/HER2+ BC.

FIGO Grade 3 Endometrioid Adenocarcinomas With Diffusely Aberrant ß-Catenin Expression: An Aggressive Subset Resembling Cutaneous Pilomatrix Carcinomas.

Uterine endometrioid adenocarcinomas are known for their morphologic plasticity. In addition to a multiplicity of metaplasias, uterine endometrioid adenocarcinomas may also undergo high-grade divergent differentiation in the form of high-grade neuroendocrine carcinoma, neuroectodermal differentiation or carcinosarcoma; others may dedifferentiate completely. Here we describe 5 cases of uterine endometrioid adenocarcinomas with high-grade divergent differentiation showing a striking morphologic and immunophenotypic resemblance to cutaneous pilomatrix carcinoma. Specifically, the high-grade component in all cases exhibited solid, basaloid morphology with conspicuous tumor cell necrosis and the presence of shadow cells, accompanied by diffusely aberrant (nuclear and cytoplasmic) ß-catenin expression as well as variably diffuse CDX2 expression. In addition, the high-grade component in all cases showed loss of ER and PAX8 expression, retained MMR expression, wild-type p53 expression, patchy p16 expression, and diffusely positive cytokeratin expression (AE1/AE3 and CK7); at least focal neuroendocrine marker expression was present in all cases. CK20 was negative in all cases, with the exception of very focal staining in a single case (2% of tumor cells). All 5 of our tumors had at least a focal conventional FIGO grade 1 component. In all 4 cases tested, the low-grade component retained both PAX8 and ER expression and had, at best, focally aberrant ß-catenin expression. Two of our cases had molecular analysis performed and both harbored mutations in exon 3 of CTNNB1 as expected; molecular analysis also revealed that both cases lacked POLE or TP53 mutations and showed no microsatellite instability. The tumors in this series were uniformly aggressive. Four of the 5 patients in our cohort had available follow-up information; of these, 3/4 died of their disease within 14 mo of diagnosis and the fourth patient had distant metastatic disease at presentation and is alive with disease 1 mo following diagnosis. The 1 patient without follow-up information also had distant metastatic disease at presentation and was lost to follow-up 17 mo later. The cases described in this series (1) represent a highly aggressive CTNNB1-mutated subset of the "no specific molecular profile" category of endometrioid adenocarcinomas; (2) illustrate a form of high-grade divergent differentiation resembling cutaneous pilomatrix carcinoma already described in carcinomas at other anatomic sites; and (3) underscore the difficulty in recognizing this phenotype at distant metastatic sites, which are frequent even at the time of presentation, given the consistent loss of ER and PAX8 expression and concurrent CDX2 expression.

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

HER2 Expression in Endometrial Cancers Diagnosed as Clear Cell Carcinoma.

There is increasing evidence that many endometrial cancers (EC) diagnosed as clear cell carcinoma (CCC) have substantial overlap with both serous carcinoma (SC) and endometrioid carcinoma (EmC), not only in terms of morphology and immunophenotype but also by molecular characterization. Now with use of HER2-based therapy in SC, a CCC diagnosis in serous-like tumors has the potential to exclude patients from receiving beneficial therapy. To assess HER2 in CCC in relation to other characteristics, a tissue microarray of archived CCC, EmC, and SC was stained for HER2 alongside a battery of immunostains used in EC. Cases with equivocal HER2 IHC were also assessed by in situ hybridization. HER2 status was assessed in 229 cases (23 CCC, 74 SC, 132 EmC). HER2 was positive in 48% of cases diagnosed as CCC, 19% of SC, and 0% of EmC. Rigorous morphologic and immunophenotypic review by 5 gynecologic pathologists revealed diagnostic disagreement in 8/11 HER2+ cases diagnosed as CCC, with SC as the other major diagnostic consideration. All HER2+ (n=25) cases were MMR-intact and most HER2+ EC had aberrant p53 staining (22/25, 88%); the 3 cases with a wild type pattern for p53 (12%) were all negative for ER. Based on these findings, patients with a diagnosis of CCC should be included in future clinical trials of HER2-targeted therapy. Moreover, given the diagnostic difficulty surrounding CCC, immunohistochemistry-based algorithms that include aberrant p53 and/or the absence of ER expression may provide a more objective means of establishing eligibility criteria than is currently possible using traditional histologic classification.

Dedifferentiated secretory breast carcinoma with fibrosarcomatous features harboring an ETV6-NTRK3 fusion in both components.

Secretory carcinomas are low-grade translocation-driven carcinomas occurring in patients over a wide age range. These tumors most frequently arise in the breast and salivary gland, but may occasionally arise at other anatomic sites, such as the skin, the thyroid gland or the upper or lower respiratory tract. In concert with their low-grade morphology, secretory carcinomas most often follow an indolent clinical course. However, rare cases have shown dedifferentiation (also known as high-grade transformation) and aggressive clinical behavior. To date, the dedifferentiated component in all molecularly confirmed cases of secretory carcinoma has taken the form of a high-grade carcinoma. Here we present a case of an ETV6-NTRK3 fusion-positive secretory carcinoma of the breast with sarcomatous dedifferentiation. The sarcomatous component showed an infantile or adult fibrosarcoma-like morphology including a herringbone fascicular pattern and a hemangiopericytic vascular pattern. By immunohistochemistry, the sarcomatous component showed focal CD34 immunoreactivity and loss of all of the markers expressed in the conventional secretory carcinoma component, including SOX10, S100, GATA-3, AE1/AE3 and E-cadherin. Fluorescence in situ hybridization analysis revealed that the sarcomatous component retained the ETV6-NTRK3 fusion, but also acquired homozygous deletion of CDKN2A. The tumor followed an aggressive clinical course and the patient eventually succumbed to her disease 14 months after diagnosis. The histomorphologic and molecular genetic features of this tumor are discussed, including its ability to mimic kinase-rearranged infantile or adult fibrosarcomas at extramammary sites and the theragnostic importance of its distinction from conventional metaplastic spindle cell carcinomas in the breast.

Genetic characterisation of adult primary pleomorphic uterine rhabdomyosarcoma and comparison with uterine carcinosarcoma.

AIMS: To characterise the genetic alterations in adult primary uterine rhabdomyosarcomas (uRMSs) and to investigate whether these tumours are genetically distinct from uterine carcinosarcomas (UCSs). METHODS AND RESULTS: Three tumours originally diagnosed as primary adult pleomorphic uRMS were subjected to massively parallel sequencing targeting 468 cancer-related genes and RNA-sequencing. Mutational profiles were compared with those of UCSs (n = 57) obtained from The Cancer Genome Atlas. Sequencing data analyses were performed using validated bioinformatic approaches. Pathogenic TP53 mutations and high levels of genomic instability were detected in the three cases. uRMS1 harboured a likely pathogenic YTHDF2-FOXR1 fusion. uRMS2 harboured a PPP2R1A hotspot mutation and amplification of multiple genes, including WHSC1L1, FGFR1, MDM2, and CCNE1, whereas uRMS3 harboured an FBXW7 hotspot mutation and an ANKRD11 homozygous deletion. Hierarchical clustering of somatic mutations and copy number alterations revealed that these tumours initially diagnosed as pleomorphic uRMSs and UCSs were similar. Subsequent comprehensive pathological re-review of the three uRMSs revealed previously unidentified minute pan-cytokeratin-positive atypical glands in one case (uRMS3), favouring its reclassification as UCS with extensive rhabdomyosarcomatous overgrowth. CONCLUSIONS: Adult pleomorphic uRMSs harbour TP53 mutations and high levels of copy number alterations. Our findings underscore the challenge in discriminating between uRMS and UCS with rhabdomyosarcomatous differentiation.

A Novel Ciliated, Mucin-producing Variant of HPV-related Cervical Adenosquamous Carcinoma In Situ: A Case Report.

Uterine cervical adenosquamous carcinoma in situ was originally defined as having either a uniform population of cells with features intermediate in appearance between glandular and squamous cells, or a mixture of distinct glandular and squamous components within a single lesion. The former type would likely be reclassified today as stratified mucin-producing intraepithelial lesion, while the latter type is vanishingly rare. Here, we report a novel case of bona fide adenosquamous carcinoma in situ, which exhibits 2 morphologically and immunophenotypically distinct components: (1) an inner glandular component composed of a single layer of p40-negative, ciliated, mucin-producing dysplastic columnar cells and (2) an outer p40-positive, stratified dysplastic squamous component otherwise identical to cervical intraepithelial neoplasia-3. Both components show block-positive staining for p16 and are positive for high-risk human papillomavirus RNA by in situ hybridization. Our finding expands the histological spectrum of human papillomavirus-associated preinvasive cervical lesions while also providing further evidence that human papillomavirus-driven processes can exhibit ciliated morphology.

PAX8-positive, Cytokeratin-positive Intra-abdominal Ewing Sarcoma Masquerading as a Mullerian Carcinoma in a Postmenopausal Female.

Extraskeletal Ewing sarcoma presenting as intra-abdominal or pelvic disease in adult female patients is very rare and may lead to diagnostic difficulty due to clinical and histologic overlap with Mullerian adenocarcinomas, which are far more common. We report a case of an intra-abdominal Ewing sarcoma in a postmenopausal female patient whose clinical and radiological presentation closely resembled that of peritoneal carcinomatosis. Biopsy of an omental nodule revealed numerous histologic features suggestive of a Mullerian carcinoma, including gland-like rosettes, strong, diffuse PAX8 immunoreactivity and cytokeratin expression. After excluding other differential diagnostic considerations, the possibility that this might represent an intra-abdominal Ewing sarcoma was entertained. Reverse transcriptase polymerase chain reaction testing demonstrated the presence of an EWSR1-ERG fusion transcript, confirming the diagnosis. The differential diagnostic considerations when dealing with this unusual clinical scenario and the uncommon yet important pitfall of PAX8 immunoreactivity in Ewing sarcoma are discussed.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Fatal adrenal crisis due to Addison's disease arising in the context of autoimmune polyglandular syndrome type 1.

The autoimmune polyglandular syndromes (APS) are rare immune-mediated endocrinopathies causing destruction of multiple endocrine and non-endocrine organs. Involvement of adrenal glands associated with any type of APS results in Addison's disease. While patients with Addison's disease often suffer from symptoms of neuroglycopenia, lethal hypotension and hypoglycemia are uncommon. Here, we report a fatal case of APS type 1 with hypotension and profound hypoglycemia in a 24-year-old man who was found unconsciousness at home and progressively evolved into pulseless electrical activity. Although his condition was initially considered to be possibly due to drug toxicity, subsequent drug screening tests failed to detect alcohol or any other substances. Emergent medical evaluation revealed severe hypotension (51/30 mm/Hg) and profound hypoglycemia (blood glucose of 20-30 mg/dl). Despite vigorous supportive care, the patient died following 3 days of respiratory dependency due to irreversible anoxic brain injury. Postmortem examination revealed severely atrophic adrenal glands with lymphocytic infiltration. Subsequent review of the patient's medical history and correlation with autopsy findings confirmed the presence of multiple organ involvement, consistent with APS type 1. This case serves as a reminder for forensic pathologists that death from an acute adrenal (Addisonian) crisis, while uncommon, should remain a differential diagnostic consideration. Furthermore, it underscores the fact that Addison's disease can occur as part of a constellation of autoimmune manifestations within the context of an underlying polyglandular syndrome, such as APS type 1.

The Evolving Role of Companion Diagnostics for Breast Cancer in an Era of Next-Generation Omics.

A companion diagnostic is a test for a specific biomarker-approved by the United States Food and Drug Administration-qualifying a patient to receive a specific, associated therapy. As interest has grown in precision medicine over the past decade, the principle of companion diagnostics has gained increasing purchase among laboratory professionals, clinicians, regulators, and even patients. The evolution of the biomarkers used to stratify and treat breast cancer illustrates the history of companion diagnostics and provides a lens through which to examine potential challenges. As new targeted therapies and corresponding biomarkers accumulate, algorithms for diagnosis and treatment necessarily become lengthier and more complex. To accommodate future needs of breast cancer patients, the companion diagnostic model will continue to adapt and evolve.

Genetic alterations of triple negative breast cancer by targeted next-generation sequencing and correlation with tumor morphology.

Triple negative breast cancer represents a heterogeneous group of breast carcinomas, both at the histologic and genetic level. Although recent molecular studies have comprehensively characterized the genetic landscape of these tumors, few have integrated a detailed histologic examination into the analysis. In this study, we defined the genetic alterations in 39 triple negative breast cancers using a high-depth targeted massively parallel sequencing assay and correlated the findings with a detailed morphologic analysis. We obtained representative frozen tissue of primary triple negative breast cancers from patients treated at our institution between 2002 and 2010. We characterized tumors according to their histologic subtype and morphologic features. DNA was extracted from paired frozen primary tumor and normal tissue samples and was subjected to a targeted massively parallel sequencing platform comprising 229 cancer-associated genes common across all experiments. The average number of non-synonymous mutations was 3 (range 0-10) per case. The most frequent somatic alterations were mutations in TP53 (74%) and PIK3CA (10%) and MYC amplifications (26%). Triple negative breast cancers with apocrine differentiation less frequently harbored TP53 mutations (25%) and MYC gains (0%), and displayed a high mutation frequency in PIK3CA and other PI3K signaling pathway-related genes (75%). Using a targeted massively parallel sequencing platform, we identified the key somatic genetic alterations previously reported in triple negative breast cancers. Furthermore, our findings show that triple negative breast cancers with apocrine differentiation constitute a distinct subset, characterized by a high frequency of PI3K pathway alterations similar to luminal subtypes of breast cancer.

Mesonephric-like adenocarcinoma of the ovary with squamoid morular metaplasia, aberrant ß-catenin expression, and concurrent FGFR2 and CTNNB1 mutations: a case report.

In general, endometrioid-defining features such as squamoid morular metaplasia are not thought to be associated with mesonephric adenocarcinoma (MA) and mesonephric-like adenocarcinoma (MLA). Here, we report a case of FGFR2-mutated ovarian MLA with squamoid morular metaplasia accompanied by aberrant nuclear and cytoplasmic ß-catenin expression and CTNNB1 mutation. Histologically, the tumor showed classical MLA histology, including well-formed glands with intraluminal eosinophilic secretions and cells with papillary thyroid carcinoma-like nuclei. Squamoid morular metaplasia was intimately associated with the tumor. Glandular epithelial elements, including those immediately associated with the squamoid morules, were negative for ER, but positive for both GATA3 and PAX8; aberrant ß-catenin expression was limited to the squamoid morules. This case illustrates the ability of mesonephric neoplasia to exhibit histological features previously thought to be restricted to an endometrioid phenotype.

Adenosquamous carcinoma in situ of the anus: a case series.

While human papillomavirus (HPV)-associated adenocarcinoma of the anus resembling endocervical adenocarcinoma has recently been described, anal adenocarcinoma in situ/AIS has not. Moreover, to the best of our knowledge, truly biphasic anal adenosquamous carcinoma in situ (ADSQ-IS) is essentially non-existent in the literature. Here, we report four cases of anal ADSQ-IS including one with associated invasive adenosquamous carcinoma. Histologically, all cases of ADSQ-IS showed a basal/peripheral population of stratified, p40-positive dysplastic squamous cells adjacent to luminal, columnar, p40-negative dysplastic glandular cells, bearing a striking resemblance to the normal anal transitional epithelium. Both lesional components were diffusely and strongly positive for p16 and positive for high-risk HPV by RNA in situ hybridization. These cases expand the morphological spectrum of high-risk HPV-associated pre-invasive lesions and underscore the plasticity of HPV-associated neoplasia.