Background: Hereditary disposition accounts for 10-15% in breast cancers and 20-25% in ovarian cancers, in which 5-10% of women have genomic alteration in breast cancer predisposition genes, BRCA1 and BRCA2, while the rest are likely due to less penetrant genes. In specific ethnicities such as Ashkenazi Jewish, three founder mutations have been identified which covers 95 % of all the BRCA mutations identified in this race. These genes are screened prior to the gold standard Sanger Sequencing in order to reduce cost. Sanger Sequencing, however, still has the limitation on the necessity of laborious processing and results interpretation. Moreover, it limits the number of genes that can be analyzed in one setting. With the use of next-generation sequencing (NGS), identification of hereditary breast and ovarian cancer (HBOC) syndrome associated genes, other than BRCA, can be sequenced at the same time but yet a faster turnover time. This allows more timely targeted risk-reducing strategies and interventions to be implemented for mutation positive carriers and their family members. Methods: In this study cohort, 948 high-risk breast/ovarian patients who met the HBOC selection criteria were recruited for mutation screening by our NGS pipeline. With the inclusion of 90 Sanger-validated known mutation cases, the performance of the NGS pipeline were proven to be comparable to Sanger sequencing. PTEN and TP53, other than BRCA1 and BRCA2, a 4 gene sequencing panel were included in the mutation screening for high-risk patients. Results: The prevalence of BRCA1/BRCA2 germline mutations was 7.28% in our Chinese cohort and 47.8% of the mutation were recurrent mutations. Based on this finding, we further adopted a new workflow by screening the recurrent mutations including founder mutations from Chinese cohort prior to NGS for those who tested negative. In a testing cohort of 343 cases, the recurrent mutation pick-up rate was 3.5%, this implicated a more cost-effective method for mutation screening in the clinical setting. Moreover, the frequencies of PTEN and TP53 were 0.21% and 0.53% respectively in our population with breast and ovarian cases. Conclusion: Taken together, our data demonstrated a strategic upfront screening for recurrent mutations in Chinese population which is highly applicable in most of the diagnostic laboratories. Multi-gene sequencing using the NGS technology will be the upcoming strategies for mutation screening for HBOC patients. Citation Format: Kwong A, Shin VY, Au CH, Law FB, Ho DN, Ip BK, Wong AT, Lau SS, To RM, Choy G, Ford JM, Ma ES, Chan TL. Evaluation on the mutation screening by next-generation sequencing in hereditary breast and ovarian cancer: Implementation of recurrent mutation panel. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-20.
Introduction: Breast cancer is the most common malignancy and 3rd leading cause of deaths among the female population in Hong Kong. Since the establishment of The Hong Kong Hereditary Breast Cancer Family Registry in 2007, 1344 patients with breast and/or ovarian cancer who met the selection criteria were recruited for genetic testing in Hong Kong. Since 2011 we started to employ next-generation DNA sequencing (NGS) to expedite the analysis workflow and expand the panel of genes for sequencing. Aim: To evaluate the workflow of NGS in mutation screening of BRCA1, BRCA2, TP53 and PTEN genes, and compared with the sequence data obtained by Sanger sequencing. Methods: We sequenced BRCA1, BRCA2, TP53 and PTEN genes in peripheral blood samples of 410 patients, 53 positive controls and 107 healthy local individuals using 454 GS Junior System. Generation of barcoded amplicon libraries was streamlined by microfluidic PCR using Fluidigm Access Array System. Sequencing data were analyzed by an in-house developed fully automatic bioinformatics pipeline, which mainly consists of GS Amplicon Variant Analyzer, SAMtools and Ensembl Variant Effect Predictor. All putative mutations identified were validated by Sanger sequencing. Furthermore, the frequency of BRCA1, BRCA2 and PTEN missense variants of unknown significance (VUS) identified in the cohort were compared among 107 healthy local individuals and 1000 Genomes project samples. The VUS were also subjected to a panel of in silico prediction methods including PolyPhen and SIFT. Results: Among 410 patients, there were 7 in BRCA1, 6 in BRCA2 and 1 in TP53 mutations found, including 1 novel recurrent BRCA2 (c.7007G>T) and 1 novel founder BRCA2 (c.5164_5165delAG) mutations. Based on multiple criteria, 12 in BRCA1, 12 in BRCA2 and 1 in PTEN VUS could be prioritized for further investigation. The bioinformatics pipeline was extensively evaluated with Sanger-validated controls. The evaluation determined minimum sequencing coverage needed in this sequencing platform for accurate analysis. The pipeline accuracy was demonstrated by successful detecting mutations from 53 positive controls, including single nucleotide variants, insertions and deletions in different sequence context. Conclusion: BRCA1, BRCA2, TP53 and PTEN mutation screening of 410 patients were expedited by high-throughput DNA sequencing. This method could detect 14 positive cases, including recurrent mutations, in a shorter period of time when compared with Sanger full gene sequencing. High-risk patients who are negative for the gene panel may need further investigation other than screening for BRCA1/2. The in-house developed bioinformatics pipeline was validated to detect various types of mutations and potentially become a conventional platform for genetic screening. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-03.
Background: Triple-negative breast cancer is associated with higher metastatic rate and poor prognosis than other subtypes of breast cancer due to lack of targeted therapy. Epithelial-mesenchymal transition (EMT) is linked with metastasis with phenotypic conversion of epithelial cells. However, the regulation of EMT in breast cancer metastasis remains largely unstudied. Recent attention has focused on targeting the downstream of COX-2 pathway, understanding the role of prostanoid receptors in breast cancer metastasis may help the development of effective therapeutic interventions for patients with metastasis. Methods: A stable EP2-expression cell line (MB-231-EP2) was used to study tumorigenesis and distant metastasis in human breast cancer metastatic model. Localization of EP2 and EMT markers were examined by immunostaining and immunofluorescence. Profiles of drug transporters genes were compared between siEP2 and siControl cells. Functional role of EP2 on cell proliferation, invasion and apoptosis were assessed. Alteration of EMT markers were examined by real-time PCR and Western blot analysis. Results: Expression of EP2 receptor were higher in human primary tumors than non-tumor tissues. EP2 receptor was predominantly expressed in metastatic tumors than primary tumors in human breast cancer metastatic mice model. The metastatic tumors showed a higher Ki67 (cell proliferation) and CD31 (angiogenesis) than primary tumors in the xenograft tissues. Larger tumors and poor survival were seen in MD-231-EP2 bearing mice when compared with control. Silencing of EP2 by siRNA markedly reduced cell proliferation and invasion, but increased apoptosis and expression of solute carrier family 19 member A3 (SLC19A3) gene. Interestingly, SLC19A3 had a lower expression in primary tumors and was inversely correlated with EP2 expression. Ectopic expression of SLC19A3 suppressed cell proliferation and invasion through the restoration of E-cadherin and other EMT markers (Twist, Zeb1 and Snai2). Immunofluorescence staining showed that the localization of Twist and E-cadherin were altered in siEP2 cells. Conclusion: Our results showed that EP2 promoted EMT and breast cancer metastasis through the downregulation of SLC19A3 expression. Taken together, targeting EP2/SLC19A3 signaling pathway maybe a potential treatment for metastasis and adjuvant chemotherapy to reduce the metastatic risk. Citation Format: Kwong A, Siu MT, Cheuk I, Ho JC, Chen J, Shin VY. A novel mechanism of epithelial-mesenchymal transition in breast cancer metastasis: Involvement of prostanoid receptor. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-05-04.
Background: Accumulating evidence showed that long non-coding RNAs (lncRNAs) dysregulation is the hallmark of cancer. Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to overexpress in many cancers, and promote cell growth and disease progression. However, the role of NEAT1 on drug sensitivity and stem-cell like property in triple-negative breast cancer is largely unknown. Methods: LncRNA expression profile were compared between breast cancer patients and healthy individuals using lncRNA array. Large scale validation of NEAT1 expression in blood samples were performed by real-time PCR. Triple-negative breast cancer (TNBC) cells, MDA-MB-231 and its cisplatin resistance subline (MDA-MB-231/cis) were used. Stable transfection of cells with NEAT1 knockdown by shRNA, and evaluated single cell clonogenic assay, aldehyde dehydrogenase (ALDH) activity, CD44+/CD24- and other cancer stem cell (CSC) markers. Drug sensitivity assay, flow cytometry analysis, immunofluorescence staining and xenograft model were used to assess the functional role of NEAT1. Results: Array data showed that NEAT1 was the top upregulated lncRNAs in the plasma of breast cancer patients. Consistent with the array data, validation of larger cohort of patients and healthy individuals (n=369) also demonstrated a higher expression of NEAT1 in breast cancer patients, in particular TNBC subtype. Knockdown of NEAT1 by shRNA sensitized breast cancer cells to cisplatin and taxol treatment. Cell proliferation and colony formation abilities were reduced with S-phase cell cycle arrest in shNEAT1 cells. Flow cytometry analysis revealed an induction of apoptosis with increased cleaved caspase-3. Cells expressing shNEAT1 abrogated ALDH activity, CD44+/CD24- subpopulation and expression of CSC markers (SOX2, NANOG and OCT4). More importantly, shNEAT1 cells retarded tumor growth in xenograft mice model and reduced CSC markers. Conclusion: Taken together, NEAT1 expression is differentially expressed in breast cancer patients, and particularly higher in patients with TNBC. These findings suggest a potent therapeutic target to improve drug sensitivity in patients with TNBC. Citation Format: Kwong A, Chen J, Siu J, Chuek I, Shin V. NEAT1 enhances drug sensitivity by inhibiting cancer stem-like cells in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-04.
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