The calvaria (upper part of the skull) is made of plates of bone and fibrous joints (sutures and fontanelles), and the proper balance and organization of these components are crucial to normal development of the calvaria. In a mouse embryo, the calvaria develops from a layer of head mesenchyme that surrounds the brain from shortly after mid-gestation. The mesenchyme just above the eye (supra-orbital mesenchyme, SOM) generates ossification centers for the bones, which then grow toward the apex gradually. In contrast, the mesenchyme apical to SOM (early migrating mesenchyme, EMM), including the area at the vertex, does not generate an ossification center. As a result, the dorsal midline of the head is occupied by sutures and fontanelles at birth. To date, the molecular basis for this regional difference in developmental programs is unknown. The current study provides vital insights into the genetic regulation of calvarial patterning. First, we showed that osteogenic signals were active in both EMM and SOM during normal development, which suggested the presence of an anti-osteogenic factor in EMM to counter the effect of these signals. Subsequently, we identified Lmx1b as an anti-osteogenic gene that was expressed in EMM but not in SOM. Furthermore, head mesenchyme-specific deletion of Lmx1b resulted in heterotopic ossification from EMM at the vertex, and craniosynostosis affecting multiple sutures. Conversely, forced expression of Lmx1b in SOM was sufficient to inhibit osteogenic specification. Therefore, we conclude that Lmx1b plays a key role as an anti-osteogenic factor in patterning the head mesenchyme into areas with different osteogenic competence. In turn, this patterning event is crucial to generating the proper organization of the bones and soft tissue joints of the calvaria.

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Background Oral squamous cell carcinoma (OSCC) is a capricious cancer with poor survival rates, even for early-stage patients. There is a pressing need to develop more precise risk assessment methods to appropriately tailor clinical treatment. Genome-wide association studies have not produced a viable biomarker. However, these studies are limited by using heterogeneous cohorts, not focusing on methylation although OSCC is a heavily epigenetically-regulated cancer, and not combining molecular data with clinicopathologic data for risk prediction. In this study we focused on early-stage (I/II) OSCC and created a risk score called the REASON score, which combines clinicopathologic characteristics with a 12-gene methylation signature, to predict the risk of 5-year mortality. Methods We combined data from an internal cohort (n = 515) and The Cancer Genome Atlas (TCGA) cohort (n = 58). We collected clinicopathologic data from both cohorts to derive the non-molecular portion of the REASON score. We then analyzed the TCGA cohort DNA methylation data to derive the molecular portion of the risk score. Results 5-year disease specific survival was 63% for the internal cohort and 86% for the TCGA cohort. The clinicopathologic features with the highest predictive ability among the two the cohorts were age, race, sex, tobacco use, alcohol use, histologic grade, stage, perineural invasion (PNI), lymphovascular invasion (LVI), and margin status. This panel of 10 non-molecular features predicted 5-year mortality risk with a concordance (c)-index = 0.67. Our molecular panel consisted of a 12-gene methylation signature (i.e., HORMAD2, MYLK, GPR133, SOX8, TRPA1, ABCA2, HGFAC, MCPH1, WDR86, CACNA1H, RNF216, CCNJL), which had the most significant differential methylation between patients who survived vs. died by 5 years. All 12 genes have already been linked to survival in other cancers. Of the genes, only SOX8 was previously associated with OSCC; our study was the first to link the remaining 11 genes to OSCC survival. The combined molecular and non-molecular panel formed the REASON score, which predicted risk of death with a c-index = 0.915. Conclusions The REASON score is a promising biomarker to predict risk of mortality in early-stage OSCC patients. Validation of the REASON score in a larger independent cohort is warranted.