SUMMARY This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches.

BACKGROUND: Parathyroid glands are difficult to identify during total thyroidectomies, and accidental resection can lead to problematic postoperative hypocalcemia. Our main goals were to evaluate the effectiveness of using near-infrared light (NIRL) autofluorescence intraoperatively for parathyroid gland identification and to measure its impact on postoperative hypocalcemia incidence. STUDY DESIGN: Total thyroidectomies were performed on 170 patients with different thyroid pathologies, block-randomized (1:1) into 2 equal groups. Among controls, traditional overhead white light (WL) was used throughout. In the experimental group, NIRL was used to enhance parathyroid gland recognition before thyroid dissection. The number of parathyroid glands identified was compared after thyroid dissection in controls using WL vs pre-dissection in the experimental using NIRL and with WL vs NIRL before thyroid dissection in the experimental group. Postoperative serum calcium levels and hypocalcemia rates were compared. RESULTS: The mean number of parathyroid glands identified pre-dissection with NIRL was the same identified post-dissection with WL (3.5 vs 3.6). In the experimental group, converting from WL to NIRL increased the number of glands detected from 2.6 to 3.5 (p < 0.001), and revealed at least 1 previously missed gland in 67.1% of patients. Calcium levels 7.5 mg/dL were one-tenth as common in the NIRL group (p ¼ 0.005). The adjusted odds of hypocalcemia developing increased by 15% for every 5-g increase in thyroid gland weight (odds ratio 1.15; 95% CI 1.06 to 1.25). All hypocalcemia resolved within 6 months. CONCLUSIONS: Using NIRL during thyroidectomy increases intraoperative identification of parathyroid glands, enhances their detection before thyroid dissection, and decreases the incidence of postoperative hypocalcemia.

SummaryDNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping.Availability and implementationMethylMix 2.0 was implemented as an R package and is available in bioconductor. https://www.bioconductor.org/packages/release/bioc/html/MethylMix.html

The use of NIRL for PG visualization significantly increased the number of PGs identified during thyroid and parathyroid surgery, and the differences in fluorescent intensity among PGs, thyroid glands, and background were not affected by age, sex, and histopathological diagnosis.

Chromatin modifying enzymes are frequently mutated in cancer, resulting in widespread epigenetic deregulation. Recent reports indicate that inactivating mutations in the histone methyltransferase NSD1 define an intrinsic subtype of head and neck squamous cell carcinoma (HNSC) that features pronounced DNA hypomethylation. Here, we describe a similar hypomethylated subtype of lung squamous cell carcinoma (LUSC) that is enriched for both inactivating mutations and deletions in NSD1. The ‘NSD1 subtypes’ of HNSC and LUSC are highly correlated at the DNA methylation and gene expression levels, featuring ectopic expression of developmental transcription factors and genes that are also hypomethylated in Sotos syndrome, a congenital disorder caused by germline NSD1 mutations. Further, the NSD1 subtype of HNSC displays an ‘immune cold’ phenotype characterized by low infiltration of tumor-associated leukocytes, particularly macrophages and CD8+ T cells, as well as low expression of genes encoding the immunotherapy target PD-1 immune checkpoint receptor and its ligands. Using an in vivo model, we demonstrate that NSD1 inactivation results in reduced T cell infiltration into the tumor microenvironment, implicating NSD1 as a tumor cell-intrinsic driver of an immune cold phenotype. NSD1 inactivation therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy.