Probes for HPV

High-risk human papillomavirus (HPV) is a risk factor for the development of several head and neck squamous cell carcinomas (SCCs). However, there have been few reports of high-risk HPV infection in temporal bone squamous cell carcinomas (TBSCCs), and thus the prevalence and clinicopathologic significance of high-risk HPV in TBSCCs are still unclear. We retrospectively collected 131 TBSCCs and analyzed them for transcriptionally active high-risk HPV infection using messenger RNA in situ hybridization; we also assessed the utility of p16-immunohistochemistry (IHC) and Rb-IHC to predict HPV infection. Eighteen (13.7%) of the 131 TBSCCs were positive for p16-IHC, and five of them were positive for high-risk HPV infection (the estimated high-risk HPV positivity rate was 3.8% [5/131]). Interestingly, all five HPV-positive patients were male and had TBSCC on the right side. In the p16-IHC+/HPV+ cases (n = 5), the Rb-IHC showed a partial loss pattern (n = 4) or complete loss pattern (n = 1). In contrast, all p16-IHC-negative cases (n = 113) showed an Rb-IHC preserved pattern. The positive predictive value (PPV) of p16-IHC positivity for high-risk HPV infection was low at 27.8%, while the combination of p16-IHC+/Rb-IHC partial loss pattern showed excellent reliability with a PPV of 100%. The prognostic significance of high-risk HPV infection remained unclear. High-risk HPV-related TBSCC is an extremely rare but noteworthy subtype.

Liprin-α1 is a scaffold protein involved in cell adhesion, motility, and invasion in malignancies. Liprin-α1 inhibits the expression of metastatic suppressor CD82 in cancers such as oral carcinoma, and the expression of these proteins has been known to correlate negatively. The role of these proteins has not been previously studied in human papillomavirus (HPV)-related head and neck cancers. Our aim was to assess the clinical and prognostic role of liprin-α1 and CD82 in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) in comparison to HPV-negative OPSCC.The data included 139 OPSCC patients treated at the Helsinki University Hospital (HUS) during 2012-2016. Immunohistochemistry was utilized in HPV determination and in biomarker assays. Overall survival (OS) was used in the survival analysis.Stronger expression of liprin-α1 in tumor-infiltrating lymphocytes (TILs) was linked to lower cancer stage (p < 0.001) and HPV positivity (p < 0.001). Additionally, we found an association between elevated expression of liprin-α1 and weak expression of CD82 in tumor cells (p = 0.029). In survival analysis, we found significant correlation between favorable OS and stronger expression of liprin-α1 in TILs among the whole patient cohort (p < 0.001) and among HPV-positive patients (p = 0.042).Increased liprin-α1 expression in the TILs is associated with favorable prognosis in OPSCC, especially among HPV-positive patients.

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.

The diagnosis of oral epithelial dysplasia is based on the degree of architectural and cytologic atypia in the squamous epithelium. The conventional grading system of mild, moderate, and severe dysplasia is considered by many the gold standard in predicting the risk of malignant transformation. Unfortunately, some low-grade lesions, with or without dysplasia, progress to squamous cell carcinoma (SCC) in short periods. As a result, we are proposing a new approach to characterize oral dysplastic lesions that will help identify lesions at high risk for malignant transformation. We included a total of 203 cases of oral epithelial dysplasia, proliferative verrucous leukoplakia, lichenoid, and commonly observed mucosal reactive lesions to evaluate their p53 immunohistochemical (IHC) staining patterns. We identified 4 wild-type patterns, including scattered basal, patchy basal/parabasal, null-like/basal sparing, mid-epithelial/basal sparing, and 3 abnormal p53 patterns, including overexpression basal/parabasal only, overexpression basal/parabasal to diffuse, and null. All cases of lichenoid and reactive lesions exhibited scattered basal or patchy basal/parabasal patterns, whereas human papillomavirus-associated oral epithelial dysplasia demonstrated null-like/basal sparing or mid-epithelial/basal sparing patterns. Of the oral epithelial dysplasia cases, 42.5% (51/120) demonstrated an abnormal p53 IHC pattern. p53 abnormal oral epithelial dysplasia was significantly more likely to progress to invasive SCC when compared with p53 wild-type oral epithelial dysplasia (21.6% vs 0%, P < .0001). Furthermore, p53 abnormal oral epithelial dysplasia was more likely to have dyskeratosis and/or acantholysis (98.0% vs 43.5%, P < .0001). We propose the term p53 abnormal oral epithelial dysplasia to highlight the importance of utilizing p53 IHC stain to recognize lesions that are at high risk of progression to invasive disease, irrespective of the histologic grade, and propose that these lesions should not be graded using the conventional grading system to avoid delayed management.

In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.

As the most common type of HPV-independent (HPVI) endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs Although anti-HER2 therapy has been proven effective in many cancers, it has not been utilized in ECAs including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in tumor types selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using IHC and FISH and to investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (17.2%, 10/58) and amplification (15.5%, 9/58), as well as high IHC-FISH concordance rate (90%, 9/10) was found in GEAs. A lateral/basolateral staining pattern ('U-shaped') was observed, at least focally, in the majority of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (PFS) (p=0.047 and p=0.032, respectively). PD-L1 expression was associated with worse PFS (p=0.032), while mutant type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER testing guideline for GEAs.