We previously reported a novel association between CDKN2A nonpromoter methylation and transcription (ARF/INK4a) in human papillomavirus associated oropharyngeal tumors. In this study we assessed whether nonpromoter CDKN2A methylation in laryngeal squamous cell carcinomas (LXSCC) conferred a similar association with transcription that predicted patient outcome. We compared DNA methylation and ARF/INK4a RNA expression levels for the CDKN2A locus using the Illumina HumanMethylation27 beadchip and RT-PCR in 43 LXSCC tumor samples collected from a prospective study of head and neck cancer patients treated at Montefiore Medical Center (MMC). Validation was performed using RNAseq data on 111 LXSCC tumor samples from the Cancer Genome Atlas (TCGA). The clinical relevance of combined nonpromoter CDKN2A methylation and transcription was assessed by multivariate Cox regression for locoregional recurrence on a subset of 69 LXSCC patients with complete clinicopathologic data from the MMC and TCGA cohorts. We found evidence of CDKN2A nonpromoter hypermethylation in a third of LXSCC from our MMC cohort, which was significantly associated with increased ARF and INK4a RNA expression (Wilcoxon rank-sum, P = 0.007 and 0.003, respectively). A similar association was confirmed in TCGA samples (Wilcoxon rank-sum test P < 0.0001 for ARF and INK4a). Patients with CDKN2A hypermethylation or high ARF/INK4a expression were significantly less likely to develop a locoregional recurrence compared to those with neither of the features, independent of other clinicopatholgic risk factors (adjusted hazard ratio=0.21, 95% confidence interval:0.05-0.81). These results support the conclusion that CDKN2A nonpromoter methylation is associated with increased ARF and INK4a RNA expression, and improved locoregional control in LXSCC.

Bacterial vaginosis (BV) is a highly prevalent condition that is associated with adverse health outcomes. It has been proposed that BVs role as a pathogenic condition is mediated via bacteria-induced inflammation. However, the complex interplay between vaginal microbes and host immune factors has yet to be clearly elucidated. Here, we develop molBV, a 16 S rRNA gene amplicon-based classification pipeline that generates a molecular score and diagnoses BV with the same accuracy as the current gold standard method (i.e., Nugent score). Using 3 confirmatory cohorts we show that molBV is independent of the 16 S rRNA region and generalizable across populations. We use the score in a cohort without clinical BV states, but with measures of HPV infection history and immune markers, to reveal that BV-associated increases in the IL-1β/IP-10 cytokine ratio directly predicts clearance of incident high-risk HPV infection (HR = 1.86, 95% CI: 1.19-2.9). Furthermore, we identify an alternate inflammatory BV signature characterized by elevated TNF-α/MIP-1β ratio that is prospectively associated with progression of incident infections to CIN2 + (OR = 2.81, 95% CI: 1.62-5.42). Thus, BV is a heterogeneous condition that activates different arms of the immune response, which in turn are independent risk factors for HR-HPV clearance and progression. Clinical Trial registration number: The CVT trial has been registered under: NCT00128661.