Human papillomavirus genotyping in high-grade vaginal intraepithelial neoplasia: A multicentric Italian study.

This study aimed to analyze the human papillomavirus (HPV) genotype distribution in a large cohort of high-grade vaginal intraepithelial neoplasia (VaIN) (vaginal HSIL, VaIN2/3) patients from two Italian referral centers. We included all patients with histologically confirmed VaIN2/3 from the Department of Surgical Sciences, Sant'Anna Hospital, University of Torino, Torino, Italy, and Ospedale Maggiore della Carità, Novara, Italy, between 2003 and 2022. After the histological evaluation of formalin-fixed paraffin-embedded samples, we performed HPV genotyping with VisionArray HPV Chip 1.0. We detected HPV DNA in 94.4% of VaIN2/3 (168/178), with HPV 16 as the most prevalent genotype, accounting for 51.8% of all infections, 41.2% of VaIN2 and 77.6% of VaIN3 cases. Other frequent genotypes were HPV 58 (8.3%, 10.9% of VaIN2 and 2.0% of VaIN3), HPV 73 (5.4%, 5.0% of VaIN2 and 6.1% of VaIN3), and HPV 31 (5.4%, 6.7% of VaIN2 and 2.0% of VaIN3). 73.2% of VaIN2/3 had a single HPV genotype infection and 26.8% a multiple infection (20.8% a double infection, 4.8% a triple infection, and 1.2% a quadruple infection). Single infection was more frequently present in VaIN3 than VaIN2 (81.6% vs. 69.8%). 69.1% of single infections and 73.3% of multiple infections had one or more genotypes covered by nine-valent HPV vaccine. HPV vaccination is expected to have a large impact on reducing the incidence of vaginal intraepithelial neoplasia.

Human cytomegalovirus infection triggers a paracrine senescence loop in renal epithelial cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.

One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.