Nan Hu

Background: Human Cub and Sushi Multiple Domains 1 (CSMD1) is a novel candidate tumor-suppressor gene that codes for multiple domains, including complement regulatory and adhesion proteins, and has recently been shown to have alterations in multiple cancers. We investigated CSMD1 in esophageal squamous cell carcinoma (ESCC) by performing an integrated analysis on somatic copy number alterations (CNAs), including copy-number gain or loss, allelic imbalance (AI), loss of heterozygosity (LOH), and the expressions of mRNA and its target miRNAs on specimens from the same patients with ESCC. Results: (i) Two-thirds of ESCC patients had all three types of alterations studied—somatic DNA alterations in 70%, and abnormal expressions of CSMD1 RNA in 69% and in target miRNAs in 66%; patterns among these alterations were complex. (ii) In total, 97% of 888 CSMD1 SNPs studied showed somatic DNA alterations, with most located near exons 4–11, 24–25, 39–40, 55–56, and 69–70. (iii) In total, 68% of SNPs with a CNA were correlated with expression of CSMD1. (iv) A total of 33 correlations between non-coding SNPs and expression of CSMD1 target miRs were found. Conclusions: Our results indicate that the CSMD1 gene may play a role in ESCC through complex patterns of DNA alterations and RNA and miRNA expressions. Alterations in some somatic SNPs in non-coding regions of CSMD1 appear to influence expression of this gene and its target miRNAs.

Background Epstein–Barr virus (EBV) positivity is associated with better gastric cancer prognosis and is found in a relatively fixed 9% of tumors worldwide. Aim We aimed to examine the EBV status of gastric adenocarcinomas in a very high-incidence population and to compare prevalence between cardia and non-cardia anatomic subsites. Methods We evaluated 1035 adult gastric adenocarcinoma cases presenting during 1997–2005 to the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, China. EBV-encoded RNA was detected in alcohol-fixed paraffin-embedded tumor specimens by in situ hybridization. Associations were assessed in case–case comparisons using the Chi-squared test for categorical variables and the Mann–Whitney U test for continuous variables, with p values < 0.05 considered statistically significant. Adjusted odds ratios were calculated using logistic regression, and mortality hazard ratios (HRs) were estimated by Cox proportional hazards regression. Results Sixty-four percent of the evaluated cancers were found in the cardia. Cardia tumor localization was associated with male sex, advanced primary tumor stage, better differentiated histology, and intestinal-type Lauren classification. Four percent of the non-cardia and only 0.9% of cardia cancers were EBV-positive. EBV positivity was associated with better overall survival (adjusted HR 0.30, 95% CI 0.14–0.63). Conclusions Our study highlights unusually low EBV prevalence in gastric adenocarcinoma among a high-incidence population, particularly for cardia cancers. These findings suggest a unique risk factor profile for the high incidence of gastric cancer in this population.

Terminal duct lobular unit (TDLU) involution is a physiological process of breast tissue aging characterized by a reduction in the epithelial component. In studies of women with benign breast disease, researchers have found that age-matched women with lower levels of TDLU involution are at increased risk of developing breast cancer. We previously showed that breast cancer cases with core basal phenotype (CBP; estrogen receptor negative [ER−], progesterone receptor-negative [PR−], human epidermal growth factor receptor 2-negative [HER2−], cytokeratins (CK 5 or CK5/6)-positive [CK5/6+] and/or epidermal growth factor receptor-positive [EGFR+]) tumors had significantly reduced TDLU involution compared with cases with luminal A (ER+ and/or PR+, HER2−, CK5/6−, EGFR−) tumors from a population-based case-control study in Poland. We evaluated the association of TDLU involution with tumor subtypes in an independent population of women in China, where the breast cancer incidence rate, prevalence of known risk factors, and mammographic breast density are thought to be markedly different from those of Polish women. We performed morphometric assessment of TDLUs by using three reproducible semiquantitative measures that inversely correlate with TDLU involution (TDLU count/100 mm2, TDLU span in micrometer, and acini count/TDLU) by examining benign tissue blocks from 254 age-matched luminal A and 250 triple-negative (TN; ER−, PR−, HER2−, including 125 CBP) breast cancer cases treated in a tertiary hospital in Beijing, China. Overall, we found that TN and particularly CBP cases tended to have greater TDLU measures (less involution) than luminal A cases in logistic regression models accounting for age, body mass index, parity, and tumor grade. The strongest association was observed for tertiles of acini count among younger women (aged <50 years) (CBP vs. luminal A; ORtrend 2.11, 95% CI 1.22–3.67, P = 0.008). These data extend previous findings that TN/CBP breast cancers are associated with reduced TDLU involution in surrounding breast parenchyma compared with luminal A cases among Chinese women, providing further support for differences in the pathogenesis of these tumor subtypes.

Background: Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets. Results: As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels. Conclusions: These data reveal insight into genes and molecular pathways mediating ESCC development and provide information potentially useful in designing novel therapeutic interventions for this tumor type.

Background: Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC. Methods: Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Results: Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment. Conclusions: Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.

Background: Cancer of the esophagus is a deadly malignancy, and development of biomarkers that predict survival is an urgent need. The apoptotic pathways have been hypothesized as important in progression of esophageal squamous cell carcinoma (ESCC). We investigated a panel of proteins that regulate apoptosis as candidate of biomarkers of prognosis in ESCC. Methods: Tissue microarray (TMA) including 313 surgically-resected cases of ESCC specimens was built for immunohistochemical interrogation. We evaluated seven genes in the FasL-Fas apoptotic pathway - FasL, Fas, FAS-associated death domain protein (FADD), phosphorylated-FADD, and caspase 8 and 10, and the antiapoptotic protein bcl-2. We studied pathway integrity and relations to risk and clinical factors, and determined the prognostic significance of each marker. Results: Five markers showed strong inter-marker correlations (r ≥ 0.28, p < 0.001), including FasL, Fas, FADD, and caspases 8 and 10. FasL and FADD also showed modest correlations with one or more cancer risk factors, but none of the markers was significantly associated with either tumor stage or lymph node metastasis, the only two clinical factors that predicted survival in these ESCC cases. Multivariate-adjusted proportional hazard regression models showed no association between protein expression and risk of death for any of the seven markers examined. Conclusion: Individual biomarkers in the apoptosis pathway do not appear to predict survival of patients with ESCC.

Family history (FH) by different relative types and risk of upper gastrointestinal (UGI) cancers has been only rarely reported; the data on UGI cancer survival are sparse.

Background: The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods: Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results: In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion: Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop) from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55%) tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies.