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INTRODUCTION: Immunotherapy targeting the programmed cell death protein–1 (PD-1) axis elicits durable antitumor responses in multiple cancer types. However, clinical responses vary, and biomarkers predictive of response may help to identify patients who will derive the greatest therapeutic benefit. Clinically validated biomarkers predictive of response to the anti–PD-1 monoclonal antibody pembrolizumab include PD-1 ligand 1 (PD-L1) expression in specific cancers and high microsatellite instability (MSI-H) regardless of tumor type. Tumor mutational burden (TMB) and T cell–inflamed gene expression profile (GEP) are emerging predictive biomarkers for pembrolizumab. Both PD-L1 and GEP are inflammatory biomarkers indicative of a T cell–inflamed tumor microenvironment (TME), whereas TMB and MSI-H are indirect measures of tumor antigenicity generated by somatic tumor mutations. However, the relationship between these two categories of biomarkers is not well characterized. RATIONALE: This study assessed the potential for TMB and a T cell–inflamed GEP to jointly predict clinical response to pembrolizumab in >300 patient samples with advanced solid tumors and melanoma across 22 tumor types from four KEYNOTE clinical trials. To assess the individual and joint clinical utility of TMB and GEP, patients were stratified in four biomarker–defined clinical response groups [GEP low and TMB low (GEPlo TMBlo), GEP low and TMB high (GEPlo TMBhi), GEPhi TMBlo, and GEPhi TMBhi] based on predefined cutoffs for TMB and GEP. These patient–defined biomarker groups were further used to guide transcriptome and exome analyses of tumors in a large molecular database [The Cancer Genome Atlas (TCGA)] (n = 6384 tumors) to identify targetable patterns of biology that may modulate response and resistance. RESULTS: TMB and GEP exhibited only modest correlation and were independently predictive of response across the KEYNOTE clinical datasets. We found that objective response rates were strongest in patients with GEPhi TMBhi (37 to 57%), moderate in those with GEPhi TMBlo (12 to 35%) and GEPlo TMBhi (11 to 42%), and reduced or absent in those with GEPlo TMBlo (0 to 9%) (see the figure). Additionally, longer progression–free survival times were seen in patients with higher levels of both TMB and GEP. Findings were comparable when TMB and PD-L1 expression were jointly assessed. Within TCGA database, GEP and TMB again had a low correlation, demonstrating the potential to jointly stratify transcriptomic and genomic features across cancer types. Specific gene expression patterns reflective of TME biology showed significant associations with TMB, GEP, or both. In particular, gene set enrichment analysis identified proliferative and stromal, myeloid, and vascular biology corresponding to specific TMB-defined subgroups within GEPhi tumors. In TMBhi tumors, indication-dependent somatic DNA alterations in key cancer driver genes showed a strong negative association with GEP. CONCLUSION: This analysis shows that TMB and inflammatory biomarkers (T cell–in...

The expression of the H19 gene is governed by parental imprinting in mammals. H19, an unusual gene encoding an RNA with no known function, is exclusively expressed from the maternal chromosome. In mouse, it lies 90 kb downstream from the lgf2 gene, which encodes a fetal-specific growth factor, insulin-like growth factor II, and is expressed primarily from the paternally inherited chromosome. In this report we have utilized interspecific hybrid mice to identify male-specific DNA methylation of a 7-to 9-kb domain surrounding the 1-119 gene and its promoter. This allele-specific methylation could function as a mark to suppress transcription of the H19 paternal allele. Consistent with this proposal, the H19 promoter displayed an open chromatin conformation only on the relatively unmethylated active maternal allele. In contrast, a cell type-specific enhancer that lies outside the methylation domain is hypersensitive to restriction enzyme digestion in nuclei on both maternal and paternal chromosomes. That the allele-specific methylation domain, coupled to the two H19 enhancers, contains all the information necessary for its imprinting was tested by examining two transgenic lines containing an internally deleted H19 transgene. Both displayed paternal-specific methylation of the transgene and maternal-specific expression. Although neither line has been tested in an inbred genetic background, and therefore the action of complex modifiers cannot be formally excluded, the result suggests that the sequences necessary for the imprinting of H19 have been identified.

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to de®ne precisely the role of 5-HT 2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this de®ciency, we developed a series of naphthylpyrimidines as potentially useful 5-HT 2B receptor antagonists. 2 RS-127445 (2-amino-4-(4-¯uoronaphth-1-yl)-6-isopropylpyrimidine) was found to have nanomolar a nity for the 5-HT 2B receptor (pK i =9.5+0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. 3 In cells expressing human recombinant 5-HT 2B receptors, RS-127445 potently antagonized 5-HTevoked formation of inositol phosphates (pK B =9.5+0.1) and 5-HT-evoked increases in intracellular calcium (pIC 50 =10.4+0.1). RS-127445 also blocked 5-HT-evoked contraction of rat isolated stomach fundus (pA 2 =9.5+1.1) and (+)a-methyl-5-HT-mediated relaxation of the rat jugular vein (pA 2 =9.9+0.3). RS-127445 had no detectable intrinsic activity in these assays. 4 In rats, the fraction of RS-127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg kg 71 ) produced plasma concentrations predicted to fully saturate accessible 5-HT 2B receptors for at least 4 h. 5 In conclusion, RS-127445 is a selective, high a nity 5-HT 2B receptor antagonist suitable for use in vivo. The therapeutic potential of this molecule is being further evaluated.