Shouman Wang

Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing.

Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients’ plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.

IL-32 promotes breast cancer cell growth and invasiveness.

Interleukin (IL)-32 is a newly identified cytokine in humans and primates. It has been established that IL-32 may antagonize cancer growth. However, to the best of our knowledge, the direct effect of IL-32 on breast cancer cell growth has not yet been investigated. In addition, rodents lack the expression of IL-32; hence, the effects of IL-32 on breast cancer xenografts in nude mice have not been studied. The present study aimed to examine the potential regulatory effects of IL-32 on breast cancer cells in nude mice. The effects of IL-32 on tumor cell growth in cell cuture and a tumor xenograft model were investigated, as well as the effects of IL-32 on apoptosis. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assay and TUNEL staining, respectively. The results revealed that IL-32 increases the proliferation rate of cancer cells and decreases the rate of apoptosis, In addition, IL-32 was found to enhance the growth of tumor xenografts in vivo. In summary, IL-32 may represent a useful therapeutic target for human breast cancer.

Reversal of Multidrug Resistance by Gefitinib Via RAF1/ERK Pathway in Pancreatic Cancer Cell Line

Pancreatic cancer is a devastating malignancy, characterized by intrinsic or acquired resistance to conventional chemotherapies. Recent evidences suggest an involvement of tyrosine kinase pathway in the regulation of multidrug resistance (MDR) protein gene expression. The aim of this study was to test whether gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor could regulate the MDR protein gene expression and sensitize the resistant cancer cells to chemotherapy. The gene expression of MDR proteins (MRP1, MRP2, MRP3, and PGP) were evaluated by quantitative RT‐PCR, and expression levels of various tyrosine kinases were investigated by quantitative RT‐PCR and Western Blot in pancreatic cancer cell line. MTT assay was used for evaluating the effect of chemotherapeutic agents. Chemotherapeutics induced drug resistance by regulating the gene expression of MDR proteins (MRP1, MRP2, and MRP3), and increased the gene expression of RAF1/ERK and the phosphorylation of ERK in pancreatic cancer Bxpc‐3 cells. Gefitinib caused an inhibition of p‐ERK tyrosine kinase activation in a dose‐dependent manner, and reversed gemcitabine‐induced RAF1/ERK gene expression and p‐ERK activation. In addition, a reversal of MDR proteins gene expression was achieved by gefitinib, which sensitized resistant cells to gemcitabine. This study demonstrated that MDR of Bxpc‐3 cell is involved in the RAF1/ERK tyrosine kinase pathway. Gefitinib reverses the MDR protein gene expression and restores sensitivity of resistant cells to gemcitabine via RAF1/ERK signaling pathway. Combination of gefitinib with conventional chemotherapeutic agents may offer a new approach for the treatment of patients with pancreatic cancer. Anat Rec, 2012.

Associations between body mass index and molecular subtypes as well as other clinical characteristics of breast cancer in Chinese women.

Background: Several studies have shown a positive association between body mass index (BMI) and the development of hormone receptor-positive breast cancer in postmenopausal women; however, the associations between BMI groups and molecular subtypes have yet to be well defined in premenopausal breast cancer patients. Methods: A total of 2465 female breast cancer patients diagnosed at our institution were recruited for this study. Clinicopathologic information (including age, body height and weight, as well as tumor subtypes and stages) was collected; analyses of these characteristics and the associations between them were performed. Results: A total of 1951 cases were included in the study. The mean age was 47.3 years, the majority of patients were of normal weight, premenopausal, had stage 2 cancer, and did not present with positive nodes. The prevalence of the luminal A, luminal B, human epidermal growth factor receptor 2+, and triple-negative subtypes were 57.8%, 11.6%, 6.1%, and 24.5%, respectively. There were significant differences in the clinicopathologic features among BMI groups in premenopausal patients. The case-only odds ratio (OR) analysis revealed that normal weight patients tended to have luminal B cancer (OR = 1.4, P = 0.206), and overweight and obese patients tended to have triple-negative cancer in premenopausal patients (OR = 2.8, OR = 3.7, respectively; P < 0.001). Conclusion: In Chinese women, breast cancer came with these characteristics: young mean age (premenopause), luminal A subtype, and the majority of them were within a normal weight range. In premenopausal patients, underweight patients tended to have luminal A, lower human epidermal growth factor receptor 2+ expression, stage 1 and no positive node cancer. However, overweight and obese patients tended to have a triple-negative, stage 3, and lymph node metastatic cancer.

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients

PurposeGene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management.MethodsUsing high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters.ResultsBreast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient.ConclusionsOur results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Haplotype analysis of eight genes of the monoubiquitinated FANCD2–DNA damage–repair pathway in breast cancer patients

BACKGROUND Ten genes are associated with increased susceptibility to inherited breast cancer have also been associated with population breast cancer risk, and all are involved directly or indirectly in the monoubiquitinated FANCD2-DNA damage repair pathway. We analyzed 13 haplotype blocks in eight of these genes to estimate the breast cancer risk conferred by individual haplotypes. METHODS Haplotype blocks were constructed with 48 tag single-nucleotide polymorphisms (tSNPs) identified in eight breast cancer susceptibility genes, TP53, PTEN, CHEK2, ATM, NBS1, RAD50, BRIP1, and PALB2. Genotyping was performed by SNPscan on 734 female patients and 672 female age-matched controls. RESULTS Forty-five tSNPs were successfully genotyped by SNPscan, and call rates for each tSNP were above 98.9%. Thirteen haplotype blocks of eight genes were constructed with 41 successfully genotyped tSNPs. We found that seven haplotypes from four haplotype blocks located within three genes (NBS1, PTEN, and BRIP1) were significantly associated with breast cancer risk. Among these, four haplotypes (ATC in block 1 of NBS1, GCCCC and GCCCT in block 2 of NBS1, and GCT in block 2 of BRIP1) were correlated with breast cancer risk in sporadic cases (OR (95% CI) 1.350(1.124-1.623), 0.752(0.584-0.969), 0.803(0.649-0.993), and 0.776(0.604-0.997), respectively), and only one haplotype (GGCCT in block 2 of NBS1) was significantly associated with breast cancer risk in familial and early-onset cases (OR(95% CI) 1.902(1.134-3.191)). CONCLUSIONS Four haplotypes within two genes (NBS1 and BRIP1) involved in the monoubiquitinated FANCD2-DNA damage-repair pathway are significantly associated with increased sporadic breast cancer risk, while one haplotype within NBS1 is correlated with an increased risk of familial or early-onset breast cancer, indicating that specific haplotypes may be distinct predictors of breast cancer.

Integrated analysis of promoter mutation, methylation and expression of AKT1 gene in Chinese breast cancer patients

Background As downstream mediators of PI3K /PTEN /AKT /mTORC1 pathway, the AKT isoforms play critical roles in tumorgenesis. Although the pleiotropic effects of AKT1 in breast cancer have been reported, the genetic and epigenetic characteristics of AKT1 promoter region in breast cancer remains to be identified. In this study we aimed to investigate the promoter mutation spectrum, methylation and gene expression pattern of AKT1 and their relationship with breast cancer. Methods By using PCR target sequence enrichment and next-generation sequencing technology, we sequenced AKT1 promoter region in pairs of breast tumor and normal tissues from 95 unselected Chinese breast cancer patients. The methylation of the promoter region and the expression profile of AKT1 in the same cohort were detected with bisulfite next-generation sequencing and qPCR, respectively. Results We identified 28 somatic mutations in 23 of the 95 (24.2%) breast cancer samples. And 19 of the 28 mutations were located in transcription factor (TF) binding sites. In the 23 patients with somatic mutations, no significant change of methylation or expression was found comparing with other patients. AKT1 promoter region was significantly hypo-methylated in tumor compared with matched normal tissue (P = 0.0014) in the 95 patients. The expression of AKT1 was significantly suppressed in tumor tissue (P = 0.0375). In clinicopathological factor analysis, AKT1 showed significant hypo-methylation (P = 0.0249) and suppressed expression (P = 0.0375) in HER2 negative subtype. And a trend of decrease in expression level (P = 0.0624) of AKT1 in the ER negative subtype was observed, which is significantly decreased in basal-like breast tumor (P = 0.0328). Conclusions Hypo-methylation and suppressed expression of AKT1 was observed to be associated with breast cancer in our cohort. The methylation and expression of AKT1 were both significantly associated with HER2 status. The promoter mutation of AKT1 did not show significant association with its methylation and expression status. These results suggested that the promoter mutation, methylation and gene expression of AKT1 may play distinct roles in tumorgenesis of breast cancer and the integrated analysis of methylation and expression of AKT1 might serve as potential biomarkers for diagnosis and classification of breast cancer.

Mutation screening of 10 cancer susceptibility genes in unselected breast cancer patients.

Variants of cancer susceptibility genes other than BRCA1/2 have been proved to be associated with increased risks of breast cancer. This study was performed to investigate the spectrum and prevalence of mutations in 10 cancer susceptibility genes in paired tumor/normal tissues of 292 unselected Chinese breast cancer patients. We performed an analysis of germline and somatic variants in ATM, CDH1, CHEK2, ESR1, GATA3, MAP3K1, MSH2, PALB2, RB1 and STK11 genes by integrating microfluidic PCR‐based target enrichment and next‐generation sequencing technologies. In total, 3 germline and 25 somatic deleterious mutations were found among 27 patients (9.25%), and 17 of them were novel mutations. Most deleterious mutations were prevalent in luminal A invasive breast cancer (P = .014). We also observed 83 variants of uncertain significance (VUS) in 100 patients (34.25%), 23 of which were predicted to be deleterious by in silico prediction programs (MetaSVM and MetaLR). VUS carriers had higher positive rate of lymph node metastasis than non‐carriers (P = .008) and were predominantly present in ER+ tumors (P = .018). Our findings would enhance the understanding of the molecular mechanisms of breast cancer in Chinese population.

Integrated analysis of promoter methylation and expression of telomere related genes in breast cancer

Telomeres at the ends of eukaryotic chromosomes play a critical role in tumorgenesis. Using microfluidic PCR and next-generation bisulfite sequencing technology, we investigated the promoter methylation of 29 telomere related genes in paired tumor and normal tissues from 184 breast cancer patients. The expression of significantly differentially methylated genes was quantified using qPCR method. We observed that the average methylation level of the 29 telomere related genes was significant higher in tumor than that in normal tissues (P = 4.30E-21). A total of 4 genes (RAD50, RTEL, TERC and TRF1) showed significant hyper-methylation in breast tumor tissues. RAD51D showed significant methylation difference among the four breast cancer subtypes. The methylation of TERC showed significant association with ER status of breast cancer. The expression profiles of the 4 hyper-methylated genes showed significantly reduced expression in tumor tissues. The integration analysis of methylation and expression of these 4 genes showed a good performance in breast cancer prediction (AUC = 0.947). Our results revealed the methylation pattern of telomere related genes in breast cancer and suggested a novel 4-gene panel might be a valuable biomarker for breast cancer diagnosis.

Abstract 4893: High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries

Breast cancer is one of the most common cancers and also the principal cause of cancer-related death among women in the world. BRCA1 and BRCA2 genes contribute to DNA repair and transcriptional regulation in response to DNA damage. Mutations of BRCA1/2 genes greatly increase lifetime risk to develop breast cancer and these mutations are frequently observed in hereditary breast cancer. In addition, recent studies suggested that breast cancer patients who carry a BRCA1 or BRCA2 germline mutation benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, it would be of great interest to develop a high-throughput approach for mutation screening of the full exon regions in BRCA1 and BRCA2 gene. We have developed a targeted gene exon-sequencing approach using a nanofluidic PCR platform, the Access Array™ system, which enables simultaneous amplification of 48 genetic regions (amplicons) from 48 samples in parallel. We have applied this system to generate the libraries of BRCA1 and BRCA2 coding regions. Total 120 target-specific primer pairs were designed covering all 22 exons of BRCA1 and 26 exons of BRCA2. A primer design strategy was applied to incorporate sample-specific barcodes and Illumina sequencing adaptors into each amplicon, allowing direct sequencing without further library preparation. The exon regions of these 2 genes have been amplified on Access Arrays using 48 multiplex mixes of the 120 primer sets in paired tumor/normal tissues from 144 breast cancer patients. We will present the next-generation sequencing data of BRCA1 and BRCA2 mutation screening collected from amplicon libraries generated using the Access Array system. Our results indicate the excellent utilization of this approach in the mutation screening of BRCA1 and BRCA2 in clinical samples. Note: This abstract was not presented at the meeting. Citation Format: Jun Wang, Guoli Li, Ming Chen, Jianfu Heng, Xinwu Guo, Limin Peng, Fan Zhang, Zibo Li, Shouman Wang, Zhi Xiao, Lizhong Dai, Wenjun Yi, Lili Tang. High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2015-4893