Probes for INS

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH “low” and RNA CISH “high”. Negative HPV RNA CISH cases were scored as RNA CISH “low”. This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

Abstract

Background

Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.

Methods

To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).

Results

105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%.

The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07–0.51, p = 0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08–0.66, p = 0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1–0.65, p = 0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13–0.81, p = 0.02).

Conclusions

Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Abstract

BACKGROUND:

There is increasing evidence that high-risk human papillomavirus plays significant role in oropharyngeal cancer; however, there is lack of knowledge on the interplay between the virus and its downstream related molecules and their possible prognostic values. The objectives of the study are to better understand the interplay of the HR-HPV and its associated downstream molecules and to evaluate potential biomarkers for patient outcomes.

METHODS:

We conducted a retrospective study with available formalin-fixed, paraffin-embedded tissue from 244 oropharyngeal cancer patients that received curative radiotherapy or concurrent chemoradiotherapy from 2000 to 2008. In addition to chart review, we performed HPV DNA and RNA in situ hybridization and immunohistochemistry for p53, the retinoblastoma protein, p16, and cyclin D1 analysis. Cox-proportional hazard and Kaplan-Meier survival analysis were used to determine the prognostic markers for clinical outcomes.

RESULTS:

Patients averaged 57.3±9.4 year-old and were mostly males (76.2%) and ever-smokers (76.2%). All patients received curative radiotherapy and 44.3% received concurrent chemoradiotherapy. We detected the human papillomavirus in 77.9% of study patients. Ever-smokers, more advanced tumor stage, and receiving radiotherapy only had poorer 5-year overall survival, disease-specific survival, and loco-regional recurrence. Cases with positive human papillomavirus and p53 overexpression had poorer disease-specific survival. Cases without human papillomavirus, but cyclin D1 overexpression, was associated with poorer 5-year overall survival.

CONCLUSIONS:

Our data suggests that additional p53 and cyclin D1 testing may benefit oropharyngeal cancer patients with known human papillomavirus status.

Hedgehog (Hh) pathway inhibitors such as vismodegib are highly effective for treating basal cell carcinoma (BCC); however, residual tumor cells frequently persist and regenerate the primary tumor upon drug discontinuation. Here, we show that BCCs are organized into two molecularly and functionally distinct compartments. Whereas interior Hh+/Notch+ suprabasal cells undergo apoptosis in response to vismodegib, peripheral Hh+++/Notch− basal cells survive throughout treatment. Inhibiting Notch specifically promotes tumor persistence without causing drug resistance, while activating Notch is sufficient to regress already established lesions. Altogether, these findings suggest that the three-dimensional architecture of BCCs establishes a natural hierarchy of drug response in the tumor and that this hierarchy can be overcome, for better or worse, by modulating Notch.

The aim of this study is comparing two in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, i.e. the RNAscope™ 2.0 High Definition (HD) and the upgraded RNAscope™ 2.5 HD version. The RNAscope™ 2.5 HD has recently replaced the RNAscope™ 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma (OSCC) with the final goal to apply it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify OSCC that are truly driven by high risk-HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real time reverse transcriptase-polymerase chain reaction (RT-PCR), in a fraction of cases where material for HPV E6/E7 mRNA real time RT-PCR was available. Furthermore, the algorithm that associates p16 immunohistochemistry (IHC) with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 IHC with the identification of HPV DNA by ISH.

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals are regulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it. Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis

HPV-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk human papillomavirus (HR-HPV) infection. Using HR-HPV mRNA in situ hybridization (ISH), we reported six new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma (ACC). Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified six HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium and one also had invasive squamous cell carcinoma (SCC). All HMSC were SOX10-positive whereas the overlying atypical squamous epithelium and the SCC were SOX10-negative. One atypical HMSC-like case was also identified which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals than HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium as well as the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.

Under injury conditions, dedicated stem cell populations govern tissue regeneration. However, the molecular mechanisms that induce stem cell regeneration and enable plasticity are poorly understood. Here, we investigate stem cell recovery in the context of the hair follicle to understand how two molecularly distinct stem cell populations are integrated. Utilizing diphtheria-toxin-mediated cell ablation of Lgr5+(leucine-rich repeat-containing G-protein-coupled receptor 5) stem cells, we show that killing of Lgr5+ cells in mice abrogates hair regeneration but this is reversible. During recovery, CD34+ (CD34 antigen) stem cells activate inflammatory response programs and start dividing. Pharmacological attenuation of inflammation inhibits CD34+ cell proliferation. Subsequently, the Wnt pathway controls the recovery of Lgr5+ cells and inhibition of Wnt signalling prevents Lgr5+ cell and hair germ recovery. Thus, our study uncovers a compensatory relationship between two stem cell populations and the underlying molecular mechanisms that enable hair follicle regeneration.