Germline HLA landscape does not predict efficacy of pembrolizumab monotherapy across solid tumor types.

We evaluated the impact of class I and class II human leukocyte antigen (HLA) genotypes, heterozygosity, and diversity on the efficacy of pembrolizumab. Seventeen pembrolizumab clinical trials across eight tumor types and one basket trial in patients with advanced solid tumors were included (n > 3,500 analyzed). Germline DNA was genotyped using a custom genotyping array. HLA diversity (measured by heterozygosity and evolutionary divergence) across class I loci was not associated with improved response to pembrolizumab, either within each tumor type evaluated or across all patients. Similarly, HLA heterozygosity at each class I and class II gene was not associated with response to pembrolizumab after accounting for the number of tests conducted. No conclusive association between HLA genotype and response to pembrolizumab was identified in this dataset. Germline HLA genotype or diversity alone is not an important independent determinant of response to pembrolizumab and should not be used for clinical decision-making in patients treated with pembrolizumab.

Hepatocellular carcinoma patients with high circulating cytotoxic T cells and intra-tumoral immune signature benefit from pembrolizumab: results from a single-arm phase 2 trial.

BACKGROUND: A limited number of studies have characterized genomic properties of hepatocellular carcinoma (HCC) patients in response to anti-PD-1 immunotherapy. METHODS: Herein, we performed comprehensive molecular characterization of immediate (D-42 to D-1) pre-treatment tumor biopsy specimens from 60 patients with sorafenib-failed HCC in a single-arm prospective phase II trial of pembrolizumab. Objective response rate was the primary efficacy endpoint. We used whole-exome sequencing, RNA sequencing, and correlative analysis. In addition, we performed single-cell RNA sequencing using peripheral blood mononuclear cells. RESULTS: The overall response rate of pembrolizumab in sorafenib-failed HCC patients was 10% ([6/60] 95% CI, 2.4-17.6). In a univariate analysis using clinicopathological features, female gender, PD-L1 positivity, and low neutrophil-to-lymphocyte ratio (NLR) were identified as contributing factors to pembrolizumab response. Somatic mutations in CTNNB1 and genomic amplifications in MET were found only in non-responders. Transcriptional profiles through RNA sequencing identified that pembrolizumab responders demonstrated T cell receptor (TCR) signaling activation with expressions of MHC genes, indicating increased levels of T cell cytotoxicity. In single-cell sequencing from 10 pre- and post-treatment peripheral blood mononuclear cells (PBMCs), patients who achieved a partial response or stable disease exhibited immunological shifts toward cytotoxic CD8+ T cells. Conversely, patients with progressive disease showed an increased number of both CD14+ and CD16+ monocytes and activation of neutrophil-associated pathways. CONCLUSIONS: Taken together, HCC patients with infiltration of cytotoxic T cells, along with increased active circulating CD8+ T cells during pembrolizumab treatment and down-regulation of neutrophil-associated markers, significantly benefited from pembrolizumab treatment. TRIAL REGISTRATION: NCT#03163992 (first posted: May 23, 2017).

High PD-L1 expression in gastric cancer (GC) patients and correlation with molecular features.

BACKGROUND: The programmed death receptor ligand 1 (PD-L1) immunohistochemistry (IHC) 22C3 pharmDx assay is a widely used selection method for pembrolizumab treatment in gastric cancer (GC) patients, especially in the U.S. The present study investigated the relationship between PD-L1 expression and the clinical features, molecular markers, and molecular subtypes of GC. METHODS: PD-L1 expression was assessed based on combined positive score (CPS) using PD-L1 IHC 22C3 pharmDx in the Asian Cancer Research Group (ACRG) GC cohort (N = 300), which has been previously genomically profiled. PD-L1 positivity was defined as PD-L1 CPS ≥ 1. The association between PD-L1 expression and clinical features, tumor burden, and molecular subtypes (ACRG and The Cancer Genome Atlas [TCGA]) was analyzed. RESULTS: Of the 300 tumors, 178 (59.3 %) had PD-L1 CPS ≥ 1 and 122 (40.7 %) had PD-L1 CPS < 1. PD-L1 CPS ≥ 1 was significantly associated with stage I tumor (P = 0.022), high microsatellite instability (MSI-H) (P < 0.001), Epstein-Barr virus (EBV) positivity (P = 0.008), and positive Helicobacter pylori status (P = 0.001). PD-L1 CPS ≥ 1 was observed in 96/193 (49.7 %) EBV-negative/microsatellite stable (MSS) tumors. In gene expression profiling, PD-L1 CPS was highly correlated with mutational load (P < 0.001) as well as EBV (P < 0.001) and MSI subtypes (P < 0.001); 27/300 (9%) GC patients had a very high PD-L1 (≥ 20) score (MSI-H, n = 10; EBV, n = 6; and non-EBV/MSS, n = 11). OS was longer in patients with PD-L1 CPS ≥ 1 tumors than in those with PD-L1 CPS < 1 tumors (median OS not reached vs. 40 months; P = 0.008; log-rank test). CONCLUSIONS: PD-L1 is expressed in 59.3 % of GC patients and is associated with MSI and EBV positivity. These results provide a basis for identifying GC patients who may benefit from anti-PD-1/PD-L1 therapy.

Correlation between the expression of Drp1 in vascular endothelial cells and inflammatory factors in hypertension rats.

The objective of this study was to investigate the expression level of dynamin-related protein 1 (Drp1) in vascular endothelium of hypertension rats and its correlation with expression of inflammatory factors. Twenty spontaneous hypertension rats (SHR) were randomly divided into SHR group (n=10) and inhibition group (MD group, n=10), and the Sprague Dawley rats were enrolled as the control group (C group, n=10). For rats in the MD group, Mdivi-1, a mitochondrial division inhibitor, was given in dosage of 25 mg/kg. After 4 weeks of administration, blood pressure was measured via tail-artery blood pressure measurement. The blood samples collected from the abdominal aorta of rats were used to assay the C-reaction protein (CRP) concentration in serum through radioimmunoassay. Hematoxylin and eosin (H&E) staining was performed for sections of thoracic aorta for morphological observation and measurement of medial thickness. Enzyme-linked immunosorbant assay (ELISA), semi-quantitative real-time polymerase chain reaction (RT-PCR) and western blotting was carried out for detecting the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Drp1 and monocyte chemotactic protein 1 (MCP-1). After 4 weeks of drug administration, the blood pressure in the MD group was significantly higher (P<0.01). The medial thickness of the thoracic aorta in the MD group was significantly decreased in comparison with the SHR group (P<0.01). The results of ELISA showed that compared with the SHR group, the expression levels of IL-6 and TNF-α in the MD group were remarkably decreased (P<0.01). Semi-quantitative RT-PCR results indicated that the mRNA expression levels of Drp1 and MCP-1 in the MD group were significantly lower than those in the SHR group (P<0.05). In the SHR rats, after administration of Mdivi-1, the expression of Drp1 is decreased, which contributes to the alleviation in inflammatory reactions and protects the vessels in SHR rats.

Systematic identification of cancer-related long noncoding RNAs and aberrant alternative splicing of quintuple-negative lung adenocarcinoma through RNA-Seq.

OBJECTIVES: Lung adenocarcinoma (LUAD) is a common subtype of non-small cell lung cancer prevalent in Asia. There is a dearth of understanding regarding the transcriptome landscape of LUAD without primary known driver mutations. In this study, LUAD samples without well-known driver mutations occurring in EGFR, KRAS, ALK, ROS1 or RET (quintuple-negative) were used for transcriptome study with a focus on long noncoding RNAs (lncRNAs), alternative splicing and gene fusions. MATERIALS AND METHODS: 24 pairs of LUAD and adjacent normal samples and 13 tumor-only samples derived from 37 quintuple-negative patients were used. Differentially expressed lncRNA transcripts were detected by paired t-test and were validated by qPCR. Functions of lncRNAs were predicted by co-expressed mRNAs. Aberrant splicing events in LUAD were identified using MISO. In addition, gene fusions were screened by SOAPfuse. RESULTS AND CONCLUSION: In total, 90 and 153 up- or down-regulated lncRNA transcripts were detected in LUAD samples in comparison with the adjacent normal samples. The most significantly differentially expressed lncRNA transcript was ENST00000598996.1 (FENDRR) down-regulated in LUAD. By lncRNA-mRNA co-expression analysis, functions of 14 lncRNAs were predicted. The predicted functions included vasculature development, immune response, cell cycle and respiratory gaseous exchange. Furthermore, six co-expressed pairs of lncRNAs and their nearby protein coding genes were identified as associated with lung development. This study also identified two highly recurrent (22 in 24) differential exon skipping events occurring in MYH14 and ESYT2 with exon including isoforms of both genes up-regulated in isoform percentage in LUAD samples. On the other hand, two out of 24 LUAD samples possessed the driver mutation exon 14 skipping of MET. The transcriptional alterations of LUAD samples without well-known driver mutations identified in the study can be used as references for future research. The translational values of these transcriptional changes are also worthy of further investigation.

Comprehensive Characterization of Oncogenic Drivers in Asian Lung Adenocarcinoma.

INTRODUCTION: The incidence rate of lung adenocarcinoma (LUAD), the predominant histological subtype of lung cancer, is elevated in Asians, particularly in female nonsmokers. The mutation patterns in LUAD in Asians might be distinct from those in LUAD in whites. METHODS: We profiled 271 resected LUAD tumors (mainly stage I) to characterize the genomic landscape of LUAD in Asians with a focus on female nonsmokers. RESULTS: Mutations in EGFR, KRAS, erb-b2 receptor tyrosine kinase 2 gene (ERBB2), and BRAF; gene fusions involving anaplastic lymphoma receptor tyrosine kinase gene (ALK), ROS1, and ret proto-oncogene (RET); and Met Proto-Oncogene Tyrosine Kinase (MET) exon 14 skipping were the major drivers in LUAD in Asians, exhibiting mutually exclusive and differing prevalence from those reported in studies of LUAD in non-Asians. In addition, we identified a novel mutational signature of XNX (the mutated base N in the middle flanked by two identical bases at the 5' and 3' positions) that was overrepresented in LUAD tumors in nonsmokers and negatively correlated with the overall mutational frequency. CONCLUSIONS: In this cohort, approximately 85% of individuals have known driver mutations (EGFR 59.4%, KRAS 7.4%, ALK 7.4%, ERBB2 2.6%, ROS1 2.2%, RET 2.2%, MET 1.8%, BRAF 1.1%, and NRAS 0.4%). Seventy percent of smokers and 90% of nonsmokers had defined oncogenic drivers matching the U.S. Food and Drug Administration-approved targeted therapies.

Identification of important long non-coding RNAs and highly recurrent aberrant alternative splicing events in hepatocellular carcinoma through integrative analysis of multiple RNA-Seq datasets.

Hepatocellular carcinoma (HCC) is an aggressive and deadly cancer. The molecular pathogenesis of the disease remains poorly understood. To better understand HCC biology and explore potential biomarkers and therapeutic targets, we investigated the whole transcriptome of HCC. Considering the genetic heterogeneity of HCC, four datasets from four studies consisting of 15 pairs of HCC and adjacent normal samples were analyzed. We observed that the number of lncRNAs expressed in each HCC sample was consistently greater than the adjacent normal sample. Moreover, 15 lncRNAs were identified expressed in five to seven HCC tissues but were not detected in any adjacent normal tissue. Differential expression analysis detected 35 up- and 80 down-regulated lncRNAs in HCC samples compared with adjacent normal samples. In addition, five differentially expressed lncRNAs were predicted to play a role in oxidation and reduction process. With regard to splicing alterations, we identified nine highly recurrent differential splicing events belonging to eight genes USO1, RPS24, CCDC50, THNSL2, NUMB, FN1 (two events), SLC39A14 and NR1I3. Of them, splicing alterations of SLC39A14 and NR1I3 were reported for the association with HCC for the first time. The splicing dysregulation in HCC may be influenced by three splicing factors ESRP2, CELF2 and SRSF5 which were significantly down-regulated in HCC samples. This study revealed uncharacterized aspects of HCC transcriptome and identified important lncRNAs and splicing isoforms with the potential to serve as biomarkers and therapeutic targets for the disease.

Genome-wide identification of RNA editing in hepatocellular carcinoma.

We did whole-transcriptome sequencing and whole-genome sequencing on nine pairs of Hepatocellular carcinoma (HCC) tumors and matched adjacent tissues to identify RNA editing events. We identified mean 26,982 editing sites with mean 89.5% canonical A→G edits in each sample using an improved bioinformatics pipeline. The editing rate was significantly higher in tumors than adjacent normal tissues. Comparing the difference between tumor and normal tissues of each patient, we found 7 non-synonymous tissue specific editing events including 4 tumor-specific edits and 3 normal-specific edits in the coding region, as well as 292 edits varying in editing degree. The significant expression changes of 150 genes associated with RNA editing were found in tumors, with 3 of the 4 most significant genes being cancer related. Our results show that editing might be related to higher gene expression. These findings indicate that RNA editing modification may play an important role in the development of HCC.

Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing.

Gastric cancer (GC) is the second most common cause of cancer-related deaths. It is known to be a heterogeneous disease with several molecular and histological subtypes. Here we perform whole-genome sequencing of 49 GCs with diffuse (N=31) and intestinal (N=18) histological subtypes and identify three mutational signatures, impacting TpT, CpG and TpCp[A/T] nucleotides. The diffuse-type GCs show significantly lower clonality and smaller numbers of somatic and structural variants compared with intestinal subtype. We further divide the diffuse subtype into one with infrequent genetic changes/low clonality and another with relatively higher clonality and mutations impacting TpT dinucleotide. Notably, we discover frequent and exclusive mutations in Ephrins and SLIT/ROBO signalling pathway genes. Overall, this study delivers new insights into the mutational heterogeneity underlying distinct histologic subtypes of GC that could have important implications for future research in the diagnosis and treatment of GC.