Probes for INS

Objective The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS- or bleomycin-induced acute lung injury, sheddases (membrane-bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV-induced shedding of the glycocalyx. Hypothesis We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases. Methods We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP-7, -2, -9 and their inhibitors TIMP-1 and -2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT-qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE-12 mouse lung epithelial cells were infected with viable or heat-inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT-qPCR. Results Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP-7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP-7 is only expressed in cells that are directly infected by IAV. Expression of the MMP-7 inhibitor TIMP-1 is similar to uninfected lungs on day 1, but increases 50-fold on day 3. In contrast, MMP-2 and MMP-9, as well as their inhibitor TIMP-2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat-inactivated IAV fails to upregulate MMP-7. Conclusions Together, these data suggest that localized IAV infection increases MMP-7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP-7 may modulate IAV-induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV-ARDS pathogenesis.

Although both the 2014 and 2020 World Health Organization (WHO) criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma (ASC), in practice, ASC diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphologic, and clinical features and outcomes associated with ASCs, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed ASCs (including glassy cell carcinoma and related lesions) to confirm an ASC diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as ASCs, 34 retained their ASC diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or ASCs), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy adenocarcinomas were reclassified as poorly differentiated HPV-associated carcinomas based on morphology and immunophenotype. There were no significant immunophenotypic differences between ASCs and pure invasive stratified mucin-producing carcinomas with regard to HPV and other markers including p16 expression. Although limited by a small sample size, survival outcomes seemed to be similar between all groups. ASCs should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The 2 putative glassy cell carcinomas studied did not meet our criteria for ASC and categorizing them as such should be reconsidered.

Epidermodysplasia verruciformis (EDV) is a rare genodermatosis that predisposes individuals to persistent infection with β-human papillomavirus (HPV) genotypes. The term EDV acanthoma may be applied to lesions with incidental findings of EDV-defining histopathological features without clinical signs of EDV. We report a case of HPV-14- and -21-positive EDV acanthoma arising in association with condyloma in a female patient with a history of low-grade squamous intraepithelial lesion of the cervix positive for high-risk HPV (non-16/18), chronic kidney disease, and systemic lupus erythematosus. The patient had no family or personal history of EDV, but the patient was on immunosuppressive therapy with mycophenolate mofetil and prednisone. A biopsy specimen from one of the perianal lesions revealed histopathologic changes consistent with EDV in the setting of condyloma. Molecular testing showed HPV-14 and -21, which supported the coexistence of condyloma with EDV acanthoma.

Oropharyngeal squamous cell carcinoma is frequently associated with high-risk HPV infection, which confers a good prognosis. Immunohistochemistry for p16 is used as a surrogate for HPV status, but discrepant results are occasionally seen. Here, we report a case with a unique pattern of partial loss of p16.A 63 year old male presented with a base of tongue nonkeratinizing squamous cell carcinoma and a large metastatic neck mass. The primary lesion and multiple regions of the metastatic mass were assessed with p16 immunohistochemistry, RNA in situ hybridization for high-risk HPV, and HPV16 genome sequencing.The primary lesion was p16 negative, and the metastatic neck mass had large, confluent regions that were either strongly p16 positive or entirely p16 negative. All of these regions were positive for high-risk HPV with identical HPV16 genomes.This unusual case illustrates a potential diagnostic pitfall, and it raises important questions regarding molecular mechanisms and prognostic implications of p16 staining in oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-independent primary endometrial squamous cell carcinoma (PESCC) is a rare but aggressive subtype of endometrial carcinoma for which little is known about the genomic characteristics. Traditional criteria have restricted the diagnosis of PESCC to cases without any cervical involvement. However, given that modern ancillary techniques can detect HPV and characteristic genetic alterations that should identify the more common mimics in the differential diagnosis, including endometrial endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma, those criteria may benefit from revision. To further characterize PESCC, we identified 5 cases of pure squamous cell carcinoma dominantly involving the endometrium that had the potential to be PESCC: 1 case involving only the endometrium and 4 cases with some involvement of the cervix. Clinicopathologic features were assessed and immunohistochemical analysis (p16, estrogen receptor, progesterone receptor, and p53), HPV RNA in situ hybridization (high-risk and low-risk cocktails and targeted probes for 16 and 18), and molecular studies were performed. All tumors showed aberrant/mutation-type p53 expression, were negative for estrogen receptor, progesterone receptor, and p16, and had no detectable HPV. Per whole-exome sequencing, 4 of the 5 tumors demonstrated comutations in TP53 and CDKN2A (p16). Four patients died of disease within 20 months (range, 1 to 20 mo; mean, 9 mo), and 1 patient had no evidence of disease at 38 months. PESCC represents a unique, clinically aggressive subtype of endometrial cancer with TP53 and CDKN2A comutations. This characteristic profile, which is similar to HPV-independent squamous cell carcinoma of the vulva, is distinct from endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma and can be used to distinguish PESCC from those mimics even when cervical involvement is present. Diagnostic criteria for PESCC should be relaxed to allow for cervical involvement when other pathologic features are consistent with, and ancillary techniques are supportive of classification as such.