Chow Yin Wong

Identification of Circulating MicroRNA Signatures for Breast Cancer Detection

Purpose: There is a quest for novel noninvasive diagnostic markers for the detection of breast cancer. The goal of this study is to identify circulating microRNA (miRNA) signatures using a cohort of Asian Chinese patients with breast cancer, and to compare miRNA profiles between tumor and serum samples. Experimental Design: miRNA from paired breast cancer tumors, normal tissue, and serum samples derived from 32 patients were comprehensively profiled using microarrays or locked nucleic acid real-time PCR panels. Serum samples from healthy individuals (n = 22) were also used as normal controls. Significant serum miRNAs, identified by logistic regression, were validated in an independent set of serum samples from patients (n = 132) and healthy controls (n = 101). Results: The 20 most significant miRNAs differentially expressed in breast cancer tumors included miRNA (miR)-21, miR-10b, and miR-145, previously shown to be dysregulated in breast cancer. Only 7 miRNAs were overexpressed in both tumors and serum, suggesting that miRNAs may be released into the serum selectively. Interestingly, 16 of the 20 most significant miRNAs differentially expressed in serum samples were novel. MiR-1, miR-92a, miR-133a, and miR-133b were identified as the most important diagnostic markers, and were successfully validated; receiver operating characteristic curves derived from combinations of these miRNAs exhibited areas under the curves of 0.90 to 0.91. Conclusion: The clinical use of miRNA signatures as a noninvasive diagnostic strategy is promising, but should be further validated for different subtypes of breast cancers. Clin Cancer Res; 19(16); 4477–87. ©2013 AACR.

Genomic landscapes of breast fibroepithelial tumors

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

Proteome-Wide Profiling of the MCF10AT Breast Cancer Progression Model

Background Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer. Methodology/Principal Findings We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the “Warburg effect”). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions. Conclusion/Significance The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.

Selective Tyrosine Hyperphosphorylation of Cytoskeletal and Stress Proteins in Primary Human Breast Cancers: Implications for Adjuvant Use of Kinase-Inhibitory Drugs

Purpose: Small-molecule growth factor receptor inhibitors block cell growth in vitro and downstream signaling in vivo, but controlled trials in patients with advanced solid tumors have yielded disappointing response rates. To clarify this discrepancy, we compared the patterns of tyrosine phosphoprotein expression in human cancer cells and primary tumors. Experimental Design: Immunoaffinity chromatography, two-dimensional electrophoresis, and antiphosphotyrosine immunoblotting were combined with mass spectrometry to determine the phosphoproteomic signatures of 40 matched normal and malignant tissues from patients with breast or liver cancer. The identities and abundance of the detected tyrosine phosphoproteins were compared with those of ligand-responsive A431 cells. Results: Patterns of tyrosine-phosphorylated proteins are similar among normal tissues of the same origin but vary markedly between different tissues. Primary breast tumors exhibit a strikingly homogeneous tyrosine phosphorylation profile, whereas liver cancers display greater phosphoproteomic diversity. The main breast-tumor-specific tyrosine phosphoproteins are cytoskeletal molecules (actin, tubulin, and vimentin) and molecular chaperones (Hsp70, Hsc71, and Grp75). In contrast, control studies in ligand-stimulated A431 human cancer cells revealed an additional phosphorylated subset of promitogenic phosphoproteins (Grb2, Shc, Jnk2, phospholipase C-γ, and phosphatidylinositol 3′-kinase). Conclusions: Identification of cytoskeletal and stress proteins as the most abundant tyrosine phosphoproteins in breast tumors implicates these molecules, rather than promitogenic effectors, as the prime stoichiometric substrates for kinase-inhibitory anticancer drugs in vivo. Because phosphorylated cytoskeletal proteins and chaperones mediate cell motility and apoptotic resistance, respectively, these data raise the intriguing possibility that small-molecule tyrosine kinase inhibitors may be of greatest value either as adjuvant antimetastatic/-invasive drugs or as chemo-/radiosensitizers.

LARG at chromosome 11q23 has functional characteristics of a tumor suppressor in human breast and colorectal cancer

Deletion of 11q23–q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, using both loss of heterozygosity analysis and customized microarray comparative genomic hybridization. LARG (leukemia-associated Rho guanine-nucleotide exchange factor) (also called ARHGEF12), identified from the analysed region, is frequently underexpressed in breast and colorectal carcinomas with a reduced expression observed in all breast cancer cell lines (n=11), in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers. Underexpression of the LARG transcript was significantly associated with genomic loss (P=0.00334). Hypermethylation of the LARG promoter was not detected in either breast or colorectal cancer, and treatment of four breast and four colorectal cancer cell lines with 5-aza-2′-deoxycytidine and/or trichostatin A did not result in a reactivation of LARG. Enforced expression of LARG in breast and colorectal cancer cells by stable transfection resulted in reduced cell proliferation and colony formation, as well as in a markedly slower cell migration rate in colorectal cancer cells, providing functional evidence for LARG as a candidate tumor suppressor gene.

Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity

Regulatory enhancer elements in solid tumours remain poorly characterized. Here we apply micro-scale chromatin profiling to survey the distal enhancer landscape of primary gastric adenocarcinoma (GC), a leading cause of global cancer mortality. Integrating 110 epigenomic profiles from primary GCs, normal gastric tissues and cell lines, we highlight 36,973 predicted enhancers and 3,759 predicted super-enhancers respectively. Cell-line-defined super-enhancers can be subclassified by their somatic alteration status into somatic gain, loss and unaltered categories, each displaying distinct epigenetic, transcriptional and pathway enrichments. Somatic gain super-enhancers are associated with complex chromatin interaction profiles, expression patterns correlated with patient outcome and dense co-occupancy of the transcription factors CDX2 and HNF4α. Somatic super-enhancers are also enriched in genetic risk SNPs associated with cancer predisposition. Our results reveal a genome-wide reprogramming of the GC enhancer and super-enhancer landscape during tumorigenesis, contributing to dysregulated local and regional cancer gene expression.

Early Detection of Breast Cancer through Population-Based Mammographic Screening in Asian Women: A Comparison Study between Screen-Detected and Symptomatic Breast Cancers

Abstract:  The first nation‐wide mammographic screening program in Asia, BreastScreen Singapore (BSS), was launched in Singapore in January 2002. This study compared the presentation and results of screen‐detected breast cancers with symptomatic breast cancers in two affiliated high‐volume institutions, one of which was an assessment centre for BSS. The medical records of patients diagnosed with primary breast cancer at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Centre, Singapore, during the period January 2002 to December 2003 were reviewed. Clinical and pathological comparisons were made between screen‐detected lesions and symptomatic lesions. Of a total of 767 cases, 640 (83.4%) were invasive carcinomas and 127 (16.6%) were ductal carcinoma in‐situ (DCIS) lesions. Only 13.4% of them were screen‐detected. Compared to symptomatic cancers, screen‐detected lesions were of smaller size (median size 18 versus 23 mm), a lower stage (stages 0–2, 95 versus 83.2%) and histologic grade (grade 1–2, 71 versus 60%), with a higher incidence of DCIS (31.0 versus 14.3%) and had higher rates of breast conservation (45.6 versus 28.2%) (all p‐values <0.05). By multivariate analysis, tumor palpability, tumor size >20 mm, nodal involvement, cerbB2 overexpression, and advanced disease stage were independent poor prognostic factors for disease‐free survival, whereas nodal involvement, advanced disease, and recurrence predicted poor cancer‐specific survival. However, there was no statistically significant difference in disease‐free survival or cancer‐specific survival between the two groups at a median follow‐up of 38 months. Screening mammography has allowed the detection of smaller and hence oncologically more favorable lesions in Asian women. Although no significant survival benefit was demonstrated in our study, a longer period of follow‐up is essential before the benefit of mortality reduction, as a result of mammography screening becomes evident in our population.

Clinical Validation of a Customized Multiple Signature Microarray for Breast Cancer

Purpose: Current histopathologic systems for classifying breast tumors require evaluation of multiple variables and are often associated with significant interobserver variability. Recent studies suggest that gene expression profiles may represent a promising alternative for clinical cancer classification. Here, we investigated the use of a customized microarray as a potential tool for clinical practice. Experimental Design: We fabricated custom 188-gene microarrays containing expression signatures for three breast cancer molecular subtypes [luminal/estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2), and “basaloid”], the Nottingham prognostic index (NPI-ES), and low histologic grade (TuM1). The reliability of these multiple-signature arrays (MSA) was tested in a prospective cohort of 165 patients with primary breast cancer. Results: The MSA-ER signature exhibited a high concordance of 90% with ER immunohistochemistry reported on diagnosis (P < 0.001). This remained unchanged at 89% (P < 0.001) when the immunohistochemistry was repeated using current laboratory standards. Expression of the HER2 signature showed a good correlation of 76% with HER2 fluorescence in situ hybridization (FISH; ratio ≥2.2; P < 0.001), which further improved to 89% when the ratio cutoff was raised to ≥5. A proportion of low-level FISH-amplified samples (ratio, 2.2-5) behaved comparably to FISH-negative samples by HER2 signature expression, HER2 quantitative reverse transcription-PCR, and HER2 immunohistochemistry. Luminal/ER+ tumors with high NPI-ES expression were associated with high NPI scores (P = 0.001), and luminal/ER+ TuM1-expressing tumors were significantly correlated with low histologic grade (P = 0.002) and improved survival outcome in an interim analysis (hazard ratio, 0.2; P = 0.019). Conclusion: The consistency of the MSA platform in an independent patient population suggests that custom microarrays could potentially function as an adjunct to standard immunohistochemistry and FISH in clinical practice.

Single institution's initial experience with sentinel lymph node biopsy in breast cancer patients

Background:  The sentinel lymph node is the first draining node from a cancer‐bearing area and is therefore the first to manifest metastasis. In breast cancer it has been shown to predict the axillary status. Axillary dissection provides information determining prognosis and need for adjuvant therapy but carries a certain morbidity. Our aim was to determine the feasibility of detecting the sentinel node in a teaching hospital and whether the sentinel node accurately predicts the axillary status.

Predicting the likelihood of additional lymph node metastasis in sentinel lymph node positive breast cancer: validation of the Memorial Sloan-Kettering Cancer Centre (MSKCC) nomogram

Aim To identify important clinicopathological parameters that are most helpful in predicting additional non-sentinel lymph node (SLN) metastasis among patients with a positive SLN biopsy in the Singapore breast cancer population. Methods A total of 1409 patients who underwent SLN biopsy were reviewed over a 5 year period from July 2004 to October 2009. A Singapore General Hospital (SGH) nomogram was developed from predictors in the Memorial Sloan-Kettering Cancer Centre (MSKCC) nomogram using 266 patients with primary invasive breast cancer and a positive SLN biopsy who subsequently had an axillary lymph node dissection. The SGH nomogram was calibrated using bootstrapped data, while the MSKCC nomogram was calibrated using SGH data. The performance of these two nomograms was compared with the calculation of the area under the receiver–operator characteristics curve and adequacy indices. Results The MSKCC nomogram achieved an area under the curve (AUC) of 0.716 (range 0.653–0.779) in our study population, while the SGH nomogram, which used only three pathological parameters, lymphovascular invasion, number of positive and negative SLN biopsies, achieved an AUC of 0.750 (range 0.691–0.808). The SGH nomogram with a higher adequacy index (0.969) provided better estimates compared with the MSKCC nomogram (0.689). Conclusions The use of the MSKCC nomogram was validated in our local patient population. The SGH nomogram showed promise to be equally, if not, more predictive as a model in our own population, while using only three pathological parameters.