Probes for INS

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma has unique biology and better outcomes. p16 immunostaining is used as a surrogate marker for transcriptionally active HPV. Although diffuse staining is generally accepted as positive, the significance of partial staining has not been established, nor has the cutoff for extent of p16 staining that should be used to identify a tumor as HPV-related. From three other large studies utilizing p16 immunohistochemistry, we identified all cases with partial positive staining. The p16-stained slides were reviewed by three study pathologists for staining (nuclear and cytoplasmic) extent (in quartiles), and also for percentage that was confluent (ie, back-to-back cell staining). Tumors were histologically typed (keratinizing, non-keratinizing, or non-keratinizing with maturation) and tested for high-risk HPV by RNA in-situ hybridization and reverse-transcriptase PCR. For the 16 cases, there were two 4+(13%), five 3+(31%), six 2+(38%), and three 1+(19%) p16 staining tumors. Extent of staining ranged from 5 to 90% of cells positive with 25% or more confluent staining in 4/16 (25%). Of the 16 (31%) cases, 5 were HPV-related on the basis of RNA in-situ hybridization and reverse-transcriptase PCR. All of these cases had >50% p16 staining, 4/5 (80%) had more than 25% confluent staining, and 4/7 (57%) had non-keratinizing histological features. Only one of the p16 1+/2+ tumors was HPV RNA-positive (by reverse-transcriptase PCR only and low level). All 1+/2+ cases were keratinizing type or undifferentiated. By sensitive detection methods, most partial p16-positive squamous cell carcinoma cases with >50% staining harbor transcriptionally active HPV, and most HPV+ tumors have significant amounts of confluent staining. Cases with <50% p16 staining and lacking significant confluent staining rarely harbor HPV. These results support that greater than 75% p16 staining or, alternatively, >50% staining combined with >25% confluent areas, are suitable cutoffs for defining positivity.

The prognostic impact of human papillomavirus (HPV) infection or smoking on oropharyngeal high-grade neuroendocrine carcinoma (HG-NEC) is not established.Retrospective study with patients with oropharyngeal HG-NEC seen at MD Anderson Cancer Center from 1997 to 2020, and previously reported patients with oropharyngeal HG-NEC and known p16 and HPV status from the literature review. Survival was estimated with the Kaplan-Meier method, and survival differences assessed with the log-rank test and Cox proportional hazards models.Thirty patients were included; most had a heavy (≥10 pack-years) smoking history (52%), locoregional disease (stage III-IVB; 77%), and p16-positive tumor (92%). HPV was positive in 65% of tested samples (15/23). Of 24 patients treated with curative intent, the objective response rates was 90% (9/10) and 81% (17/21), respectively, for induction chemotherapy and definitive radiotherapy. During follow-up, 54% (13/24) recurred, mostly (11/13) at distant sites. Median overall survival (OS) was 46 months (95% CI, 14.3 - NA). OS was not associated with HPV status (HR 0.73, P = 0.6) or smoking (HR 1.16, P = 0.8). Among 63 patients with known HPV status after the literature review (19 HPV- and 44 HPV + ), HPV status remained unassociated with OS (P = 0.92).This is the largest retrospective cohort of oropharyngeal HG-NEC. Distant recurrence rate after curative treatment was high, suggesting that multimodality treatment including systemic therapy may benefit patients with locally advanced disease. HPV infection did not affect survival outcomes, therefore should not lead to therapy de-intensification for this histology.

There is emerging evidence that vulvar squamous cell carcinoma (VSCC) can be prognostically subclassified into 3 groups based on human papillomavirus (HPV) and p53 status: HPV-associated (HPV+), HPV-independent/p53 wild-type (HPV-/p53wt), or HPV-independent/p53 abnormal (HPV-/p53abn). Our goal was to assess the feasibility of separating VSCC and its precursors into these 3 groups using p16 and p53 immunohistochemistry (IHC). A tissue microarray (TMA) containing 225 VSCC, 43 usual vulvar intraepithelial neoplasia (uVIN/HSIL), 10 verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), and 34 differentiated VIN (dVIN), was stained for p16 and p53. Non-complementary p16 and p53 patterns were resolved by repeating p53 IHC and HPV RNA in-situ hybridization (ISH) on whole sections, and sequencing for TP53. Of 82 p16-positive VSCC, 73 (89%) had complementary p16 and p53 patterns and were classified into the HPV+ group, 4 (4.9%) had wild-type p53 staining, positive HPV ISH, and were classified into the HPV+ group, while 5 (6.1%) had p53 abnormal IHC patterns (1 null, 4 overexpression), negativity for HPV ISH and harboured TP53 mutations (1 splice-site, 4 missense); they were classified as HPV-/p53abn. Of 143 p16-negative VSCC, 142 (99.3%) had complementary p53 and p16 patterns; 115 (80.4%) HPV-/p53abn and 27 (18.9%) HPV-/p53wt. One had a basal-sparing p53 pattern, positivity for HPV ISH, and was negative for TP53 mutations; it was classified into the HPV+ category. The use of IHC also led to the following revised diagnoses: HSIL to dVIN (3/43), dVIN to vaVIN (8/34), and dVIN to HSIL (3/34). Overall, 215/225 VSCC (95.6%) could be easily classifiable into 3 groups with p16 and p53 IHC. We identified several caveats, with the major caveat being that 'double positive' p16/p53 should be classified as HPV-/p53abn, and propose an algorithm which will facilitate the application of p16 and p53 IHC to classify VSCC in pathology practice.

Sinonasal adenosquamous carcinoma is rare, and there are almost no studies detailing morphology or characterizing their genetic driver events. Further, many authors have termed sinonasal tumors with combined squamous carcinoma and glands as mucoepidermoid carcinoma but none have analyzed for the presence of MAML2 rearrangement.Cases from 2014 to 2020 were collected and diagnosed using World Health Organization criteria. They were tested for p16 expression by immunohistochemistry (70% cut-off), DEK::AFF2 fusion by fluorescence in situ hybridization (FISH) and AFF2 immunohistochemistry, MAML2 rearrangement by FISH, and low- and high-risk HPV by RNA ISH and reverse transcription PCR, respectively. Detailed morphology and clinical features were reviewed.There were 7 male (64%) and 4 female (36%) patients with a median age of 69 years, most Caucasian (10 of 11 or 91%). Most had tobacco exposure (8/11, 73%) and most presented with epistaxis, a visible nasal mass, and/or facial pain. Several had a precursor papillomas (3 of 11, 27%). The squamous component had variable keratinization, 5 of 11 (46%) of which would be described as keratinizing, 3 non-keratinizing, and 2 with mixed features. All had gland formation, by definition, and 2 of 11 (18%) had ciliated tumor cells. None of the 11 cases had MAML2 rearrangement and one had DEK::AFF2 fusion with associated positive nuclear AFF2 protein immunostaining. Most were p16 positive (7 of 11, 64%) and all 7 of these were hrHPV positive either by RNA ISH or RT-PCR. Two of the p16-negative tumors were positive for lrHPV by RNA ISH. Treatment included surgery alone (4 of 11, 36%), surgery with adjuvant radiation (5 of 11, 45%), and surgery with radiation and chemotherapy (2 of 11, 18%). Four of 11 patients (36%) suffered disease recurrence, two requiring re-operation and who were disease free at last follow-up, one receiving additional chemotherapy and who was alive with disease. The other elected to undergo palliative therapy and died of disease.Sinonasal adenosquamous carcinoma is a somewhat heterogeneous tumor not infrequently arising ex papilloma and having various drivers including high- and low-risk HPV and rarely DEK::AFF2 fusion. The prognosis appears favorable when proper treatment is possible.