Clonal relationship of synchronous head and neck cancer and esophageal cancer assessed by single nucleotide polymorphism-based loss of heterozygosity analysis.

BACKGROUND: The prognoses of head and neck squamous cell carcinoma (HNSCC) and esophageal squamous cell carcinoma (ESCC) are poor, especially when both tumors occur at the same time. We examined the clonal relatedness of HNSCCs with synchronous ESCCs to confirm whether the second tumors were metastasis or separate second primary malignancies (SPMs) using loss of heterozygosity (LOH) analysis. METHODS: Twenty-one pairs of formalin-fixed paraffin-embedded tissue from HNSCC patients with synchronous esophageal cancer were analyzed by single nucleotide polymorphism (SNP) array using the Illumina HumanCytoSNP FFPE-12 BeadChip (San Diego, CA), which contains approximately 300,000 probes. LOH was identified using Nexus Copy Number software (El Segundo, CA). RESULTS: Comparing the LOH pattern between HNSCC and paired ESCC, we found that 20 out of 21 paired tissues had a high number of discordant LOHs (LOH identified solely in the primary HNSCC but not in synchronous ESCC at the same genomic location) and a low number of concordant LOHs (LOH at the same genomic location in both HNSCC and ESCC). Only one case fell into the undetermined category. Therefore, these 20 ESCCs were classified as SPMs or second field tumors (SFTs). Moreover, the HNSCC patients with molecularly confirmed esophageal SPM had significantly poorer survival than the other patients. CONCLUSIONS: We propose the use of a genome-wide SNP array as a tool to differentiate metastatic tumors from SPM/SFT. The SNP array offers genome-wide LOH information that earlier microsatellite analysis studies lack. The ability to accurately identify SPM should contribute to a better treatment plan and follow-up care of these patients.

Oncoproteomic and gene expression analyses identify prognostic biomarkers for second primary malignancy in patients with head and neck squamous cell carcinoma.

Patients with head and neck squamous cell carcinoma are at increased risk of developing a second primary malignancy, which is associated with poor prognosis and early death. To help improve clinical outcome, we aimed to identify biomarkers for second primary malignancy risk prediction using the routinely obtained formalin-fixed paraffin-embedded tissues of the index head and neck cancer. Liquid chromatography-tandem mass spectrometry was initially performed for candidate biomarker discovery in 16 pairs of primary cancer tissues and their matched normal mucosal epithelia from head and neck squamous cell carcinoma patients with or without second primary malignancy. The 32 candidate proteins differentially expressed between head and neck cancers with and without second primary malignancy were identified. Among these, 30 selected candidates and seven more from literature review were further studied using NanoString nCounter gene expression assay in an independent cohort of 49 head and neck cancer patients. Focusing on the p16-negative cases, we showed that a multivariate logistic regression model comprising the expression levels of ITPR3, KMT2D, EMILIN1, and the patient's age can accurately predict second primary malignancy occurrence with 88% sensitivity and 75% specificity. Furthermore, using Cox proportional hazards regression analysis and survival analysis, high expression levels of ITPR3 and DSG3 were found to be significantly associated with shorter time to second primary malignancy development (log-rank test P = 0.017). In summary, we identified a set of genes whose expressions may serve as the prognostic biomarkers for second primary malignancy occurrence in head and neck squamous cell carcinomas. In combination with the histopathologic examination of index tumor, these biomarkers can be used to guide the optimum frequency of second primary malignancy surveillance, which may lead to early diagnosis and better survival outcome.

Luteinizing Hormone Receptor Gene and Regulator of G-protein Signaling 2 Gene Expression Level and Association with Oocyte Maturity in In vitro Fertilization/Intracytoplasmic Sperm Injection Cycle.

AIMS: The aim is to study the relation and distribution in gene expression level of the luteinizing hormone receptor (LHR) gene and regulator of G-protein signaling 2 (RGS2) gene expression with oocyte maturation. SETTING AND DESIGN: This cross-sectional study was undertaken in an instruction-based tertiary care infertility unit, department of obstetrics and gynecology. MATERIALS AND METHODS: After controlled ovarian hyperstimulation, cumulus granulosa cells (CCs) from 59 oocytes among 18 women being treated by in vitro fertilization/intracytoplasmic sperm injection cycle technique from November 2015 to January 2016 were collected on the day of oocyte retrieval. Total RNA was extracted and converted to cDNA in individual oocytes. LHR and RGS2 gene levels were measured and analyzed using digital droplet polymerase chain reaction. STATISTICAL ANALYSIS: Gene expression level was analyzed using software STATA, version 14.0 (College Station, TX: StataCorp LP, USA). RESULTS: CCs were obtained from 59 cumulus-oocyte complexes (COC), 46 COC from metaphase II (CCMII), 13 COC from metaphase I, and GV oocyte (CCMI + GV). The RGS2 gene expression level, when compared with the housekeeping gene in CCMII and CCMI + GV, was 0.15 (0.05-0.52) and 0.08 (0.02-0.27), respectively. The LHR gene expression when compared with the housekeeping gene in CCMII and CCMI + GV did not differ and was quite in the same value that was 0.02 (0.00-0.11) and 0.02 (0.00-0.06), respectively. CONCLUSION: This study showed that LHR gene expression did not differ in between oocyte groups. Even though the median of RGS2 gene expression was more in the mature oocyte group, the result was inconclusive due to scattering and overlapping of gene expression data between oocyte groups.

The Association of Gut Microbiota with Nonalcoholic Steatohepatitis in Thais.

OBJECTIVES: Nonalcoholic steatohepatitis (NASH) can progress to advanced fibrosis; the link between intestinal bacterial overgrowth and NASH has been proposed. Gut microbiota may promote inflammation and provoke disease progression. We evaluated gut microbiota pattern in NASH and its influencing factors. METHODS: A case-controlled study with sixteen NASH and eight control subjects was done. We performed DNA extraction from stool samples and bacterial 16S rRNA sequencing using MiSeq™. The sequences were clustered into operational taxonomic units using Quantitative Insights Into Microbial Ecology software. We calculated relative abundances, determined alpha diversity, obtained beta diversity by principal coordinate analysis, and conducted the partial least-squares regression model. RESULTS: The relative abundance of Bacteroidetes tended to be higher in NASH group. The Bacteroidetes/Firmicutes (B/F) ratio was significantly elevated in NASH patients. The pattern of gut microbiota in NASH was clearly separated from that of control subjects. Factors influencing the separation of NASH from control subjects were age, diabetes, body mass index, Bacteroidetes phylum, metformin, Actinobacteria, Verrucomicrobia, Thermotogae, and Caldithrix and Bacteroidetes/Firmicutes ratio. CONCLUSIONS: Bacteroidetes phylum (Bacteroides and Prevotella genus) is abundant in NASH subjects, who exhibited an elevated B/F ratio. NASH patients showed a specific pattern of gut microbiota independent of diabetes or metformin use.

Engineered U7 snRNA mediates sustained splicing correction in erythroid cells from ß-thalassemia/HbE patients.

Repair of a splicing defect of ß-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with ß-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of ßE-globin pre-mRNA and achieves a long-term therapeutic effect. Using a two-step tiling approach, we systematically screened U7 snRNAs carrying splice-switching SSO sequences targeted to the cryptic 5' splice site created by HbE mutation. We tested this approach and identified the most responsive element for mediating splicing correction in engineered U7 snRNAs in HeLa-ßE cell model cell line. Remarkably, the U7 snRNA lentiviral vector (U7 ßE4+1) targeted to this region effectively restored the correctly-spliced ßE-globin mRNA for at least 5 months. Moreover, the effects of the U7 ßE4+1 snRNA lentiviral vector were also evident as upregulation of the correctly-spliced ßE-globin mRNA in erythroid progenitor cells from ß-thalassemia/HbE patients treated with the vector, which led to improvements of pathologies in erythroid progenitor cells from thalassemia patients. These results suggest that the splicing correction of ßE-globin pre-mRNA by the engineered U7 snRNA lentiviral vector provides a promising, long-term treatment for ß-thalassemia/HbE.