Clarification of glycosylphosphatidylinositol anchorage of OTOANCORIN and human OTOA variants associated with deafness.

Otoancorin (OTOA), encoded by OTOA, is required for the development of the tectorial membrane in the inner ear. Mutations in this gene cause nonsyndromic hearing loss (DFNB22). The molecular mechanisms underlying most DFNB22 remain poorly understood. Disruption of glycosylphosphatidylinositol (GPI) anchorage has been assumed to be the pathophysiology mandating experimental validation. From a Korean deaf family, we identified two trans OTOA variants (c.1320 + 5 G > C and p.Gln589ArgfsX55 [NM_144672.3]) . The pathogenic potential of c.1320 + 5 G > C was confirmed by a minigene splicing assay. To experimentally determine the GPI anchorage, wild-type (WT) and mutant OTOA harboring p.Gln589ArgfsX55 were expressed in HEK293T cells. The mutant OTOA with p.Gln589ArgfsX55 resulted in an uncontrolled release of OTOA into the medium in contrast with phosphatidylinositol-specific phospholipase C-induced controlled release of WT OTOA from the cell surface. Together, the results of this reverse translational study confirmed GPI-anchorage of OTOA and showed that downstream sequences from the 589th amino acid are critical for GPI-anchorage.

Bridging genomics and phenomics of gastric carcinoma.

Genetic alterations are the starting point leading to numerous changes in clinical and pathologic features (phenotypes) of individual cancers; however, their inter-relationships in gastric cancers (GC) are unclear. We performed massive parallel sequencing of 381 cancer-related genes and compared the results with clinical and pathologic findings in 330 GC. High tumor mutation burden (TMB) accounted for 11% of GC (n = 37) and all 19 MSI-H GCs were high TMB. High TMB was significantly more frequent in intestinal-type by Lauren, tumor with higher host cellular immune response, earlier AJCC stage and favorable prognosis. The most significantly mutated genes were TP53 (54%), ARID1A (23%), CDH1 (22%), PIK3CA (12%), RNF43 (10%) and KRAS (9%). For receptor tyrosine kinases, amplifications detected by immunohistochemistry were higher than sequencing (HER2, 9.1% vs. 5.8%; EGFR, 11.2% vs. 6.1%; FGFR2, 4.6% vs. 3.9%, c-MET, 3.4% vs. 0.9%). PTEN protein loss (22%) correlated well with underlying PTEN alterations while ATM loss (27%) was not significantly correlated with genetic alterations of ATM. p53 protein expression predicted alterations of TP53 with high sensitivity (97.8%) and low (15.9%) specificity. The poorly cohesive histology/CDH1-mutant GC subgroup showed the worst survival (p < 0.001). PD-L1 expression was significantly associated with MSI-H, MLH1 loss, ATM loss, MET positivity, higher host immune response, and genetic alterations of ARID1A, BRD3, PIK3CA, KRAS, MAP3K13, CDH2, PTEN and ESR1. The merged clinical, pathology and genomics of GC provide a better understanding of GC and new insights into the treatment of GC.

Comprehensive somatic genome alterations of urachal carcinoma.

BACKGROUND: Urachal cancer is a rare cancer that develops in the urachus. Because of its rarity, standard treatment therapies for urachal cancer are not established, and chemotherapeutic regimens for bladder cancer have been unsuccessful for patients with urachal cancer. Hence, we aim to understand a systematic molecular characterisation of urachal cancer. METHODS: We identified somatic single-nucleotide variations (SNVs)/indels and somatic copy number aberrations (SCNAs) in the 17 patients by using whole-exome sequencing (WES) and OncoScan platform (Affymetrix) as follows: tumour-normal paired sequencing (WES, n=10), tumour-only sequencing (WES, n=1; targeted deep sequencing, n=16), and OncoScan (n=17). RESULTS: Our analyses identified 27 genes with somatic SNVs and indels, as well as six genes (APC, COL5A1, KIF26B, LRP1B, SMAD4 and TP53) that were recurrent in at least two patients. By analysing the SCNAs, we found that the extent of chromosomal amplification was highly associated with the patient's cancer stage. Interestingly, 35% (6/17) of the patients had focal DNA amplifications in fibroblast growth factor receptor family genes. The integration of somatic SNVs, indels and SCNAs revealed significant alterations in the mitogen-activated protein kinase signalling pathways. CONCLUSIONS: Our genome-wide analysis of urachal cancer suggests that molecular characteristics may be important for the treatment of urachal cancer.

Dysregulated Wnt signalling and recurrent mutations of the tumour suppressor RNF43 in early gastric carcinogenesis.

Several recurrent mutations and epigenetic changes have been identified in advanced gastric cancer, but the genetic alterations associated with early gastric carcinogenesis and malignant transformation remain unclear. We investigated the genomic and transcriptomic landscape of adenomas with low-grade dysplasia (LGD) and high-grade dysplasia (HGD), and intestinal-type early gastric cancer (EGC). The results were validated in an independent cohort that included EGCs directly adjacent to adenoma (EGC-adenomas) that were in the process of malignant transformation, and de novo EGCs that do not seem to have been derived from adenoma. The expression patterns clearly divided into normal, LGD, and EGC, whereas those of HGD overlapped with LGD or EGC. These results suggest that HGD is the critical stage determining malignant transformation. We found that genes related to focal adhesion and extracellular matrix receptor interaction pathways were upregulated as LGD progressed to EGC, whereas canonical Wnt signalling and peroxisome proliferator-activated receptor (PPAR) signalling pathway genes were downregulated in EGC. Genomic alterations such as somatic mutation, gene fusion and copy number variation increased gradually from LGD to EGC. APC mutations were present in 67% of LGDs, 58% of HGDs, and 18% of EGCs. RNF43 mutations were present only in HGD and EGC, and TP53 mutations were present only in EGC. In a validation cohort, RNF43 mutations were present in 35.2% of EGC-adenomas, but in only 8.6% of de novo EGCs. This is the first study to investigate the genomic and transcriptomic landscape of multistep gastric carcinogenesis. We investigated important alterations and their related pathways in each step as tumours progressed from LGD to HGD and eventually to EGC. We suggest that mutations and downregulation of RNF43 may play a critical role in the transition from adenoma to carcinoma. Given these findings and Wnt dependency in tumours with RNF43 mutation, intestinal-type gastric cancer or adenoma with RNF43 mutation might represent a promising indication for Wnt-targeted agents. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Four distinct immune microenvironment subtypes in gastric adenocarcinoma with special reference to microsatellite instability.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) can be overexpressed in tumours other than Epstein-Barr virus (EBV)-positive (EBV+) or microsatellite instability-high (MSI-H) gastric cancer (GC) subtypes. We aimed to determine the tumour immune microenvironment (TME) classification of GC to better understand tumour-immune interactions and help patient selection for future immunotherapy with special reference to MSI-H. METHODS: Immunohistochemistry (IHC) for PD-L1 and CD8+ T cells in three distinct subtypes of GC (43 EBV+, 79 MSI-H and 125 EBV-/MSS) were performed and analysed. In 66 MSI-H GC, mutation counts were compared with PD-L1 expression and survival of the patients. RESULTS: GC TME divided by PD-L1 IHC and tumour-infiltrating lymphocytes (TIL) measured by intratumoural CD8 density showed: (1) about 40% of GC are type I (PD-L1+/TIL+) consisting ~70% of MSI-H or EBV+ GC, and ~15% of EBV-/microsatellite stable (MSS) GC patients show the best survival in both disease-free (HR 2.044) and overall survival (HR 1.993); this type would respond to a checkpoint blockade therapy; (2) almost 30% of GC are type II (PD-L1-/TIL-) with the worst survival; (3) approximately 10% of GC are type III (PD-L1+/TIL-); and (4) up to 20% are type IV (PD-L1-/TIL+) and, unexpectedly, ~25% of EBV+ or MSI-H GC are within this subtype. In MSI-H GC, frequent frameshift mutations were observed in ARID1A, RNF43, NF1, MSH6, BRD3, NCOA3, BCORL1, TNKS2 and NPM1 and the numbers of frameshift mutation correlated significantly with PD-L1 expression (P<0.05). DISCUSSION: GC can be classified into four TME types based on PD-L1 and TIL, and numbers of frameshift mutation correlate well with PD-L1 expression in MSI-H GC.