Purpose: Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors. While most PPGL are benign, up to 20% may become metastatic with SDHB- and FH-mutated tumors showing the higher risk. We aimed at determining the contribution of immortalization mechanisms to metastatic progression. Experimental design: Immortalization mechanisms were investigated in 200 tumors. To identify telomerase (+) tumors we analyzed genomic alterations leading to transcriptional activation of TERT comprising promoter mutations, hypermethylation and gain copy number. To identify tumors that activated the alternative lengthening of telomere (ALT) mechanism, we combined analyses of telomere length by slot blot, telomere heterogeneity by telomere FISH, and ATRX mutations by next-generation sequencing. Univariate/multivariate and metastasis-free survival (MFS) and overall survival (OS) analyses were carried out for assessment of risk factors and clinical outcomes. Results: Only 37/200 (18.5%) tumors achieved immortalization. Telomerase activation occurred in 12 metastatic tumors, and was prevalent in SDHB-mutated paragangliomas (P = 2.42e-09). ALT features were present in 25 tumors, mostly pheochromocytomas, regardless of metastatic status or molecular group (P = .169), yet ATRX mutations were found preferentially in SDHB/FH-mutated metastatic (P = .0014). Telomerase activation and ATRX mutations were independent factors of poor prognosis: MFS (hazard ratio, 48.2 and 33.1; P = 6.50E-07 and 1.90E-07, respectively); OS (hazard ratio, 97.4 and 44.1; P = 4.30E-03 and 2.00E-03, respectively) and were associated with worse MFS and OS (logrank tests P < .0001). Conclusion: Assessment of telomerase activation and ATRX mutations could be used to identify metastatic PPGL, particularly in tumors at high-risk of progression.

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is a condition related to HPV 6 and 11 infection which is characterized by the repeated growth of benign exophytic papilloma in the respiratory tract. Disease progression is unpredictable: some children experience minor symptoms, while others require multiple interventions due to florid growth. The aim of this study was to explore the biomarkers of JoRRP severity on a bicentric cohort of forty-eight children. We performed a CISH on the most recent sample of papilloma with a probe targeting the mRNA of the E6 and E7 genes of HPV 6 and 11 and an immunostaining with p16^INK4a antibody. For each patient HPV RNA CISH staining was assessed semi-quantitatively to define two scores: 1+, defined as a low staining extent, and 2+, defined as a high staining extent. This series contained 19 patients with a score of 1+ and 29 with a score of 2+. Patients with a score of 2+ had a median of surgical excision (SE) per year that was twice that of patients with a score of 1+ (respectively 6.1 versus 2.8, p = 0.036). We found similar results with the median number of SE the first year. Regarding p16^INK4a, all patients were negative. To conclude, HPV RNA CISH might be a biomarker which is predictive of disease aggressiveness in JoRRP, and might help in patient care management.

The integration of Artificial Intelligence (AI) algorithms into pathology practice presents both opportunities and challenges. Although it can improve accuracy and inter-rater reliability, it is not infallible and can produce erroneous diagnoses, hence the need for pathologists to always check predictions. This critical judgment is particularly important when algorithm errors could lead to high-impact negative clinical outcomes, such as missing an invasive carcinoma. However, the influence of AI tools on pathologists’ decision-making is not well explored. This study aims to evaluate the impact of a previously developed AI tool on the diagnostic accuracy and inter-rater reliability among pathologists, while assessing whether pathologists maintain independent judgment of AI predictions. Eight pathologists from different hospitals and with varying levels of experience, participated in the study. Each of them reviewed 115 slides of laryngeal biopsies, including benign epithelium, low-grade and high-grade dysplasia, and invasive squamous carcinomas. The study compared diagnostic outcomes with and without AI assistance. The reference labels were established by an expert’s double-blind review. Results show that assisted pathologists had a higher accuracy for high-grade dysplasia, invasive carcinoma and improved inter-rater reliability. However, cases of over-reliance on AI have been observed, resulting in the omission of correctly diagnosed invasive carcinomas during the unassisted examination. The false predictions on these carcinoma slides were labeled with a low confidence score, which was not considered by the less experienced pathologists, showing the risk that they would follow the AI prediction without enough critical judgment or expertise. Our study emphasizes the potential over-reliance of pathologists on AI models and the potential harmful consequences, even with the advancement of powerful algorithms. The integration of confidence scores and the education of pathologists to use this tool could help to optimize the safe integration of AI into pathology practice.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since "low" or "high" HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.