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PDF file - 189K, Supplementary Fig. S1: Three components principal component analysis of (a) tissue and (b) serum samples. Breast cancer tumour tissues (a) and serum from breast cancer patients (b) are indicated in red. Adjacent normal tissues (a) and serum from healthy individuals (b) are indicated in blue. Supplementary Fig. S2: Correlation plots from inter-platform comparison studies. (a) same breast cancer tumour sample extracted by mirVana and miRNeasy; (b) same breast cancer tumour sample ran on microarray and RT-PCR panels.

Supplementary Table 1

Supplementary Figure 1 and 2

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.

Supplementary Data from Clinical Validation of a Customized Multiple Signature Microarray for Breast Cancer

Purpose:Trastuzumab-based chemotherapy has shown remarkable clinical benefits for patients with HER2-positive breast cancer. However, treatment regimens involving trastuzumab had little or no effect for a subset of patients. Preliminary studies revealed WW-binding protein 2 (WBP2), an oncogenic transcription coactivator, to be coamplified with HER2 in 36% of HER2-positive breast cancers. We hypothesize that WBP2 regulates and correlates with the response of HER2-positive breast cancer to trastuzumab.Experimental Design:The coexpression of WBP2 and HER2 in breast tumors was validated using IHC. The role and mechanism of WBP2 in regulating breast cancer response to trastuzumab was elucidated using in vitro, patient-derived xenograft and murine xenograft models. A multicenter retrospective study involving 143 patients given neoadjuvant trastuzumab-based chemotherapy was conducted to determine whether WBP2 expression correlates with pathologic complete response (pCR).Results:Elevated expression of WBP2 significantly enhanced breast cancer's response to trastuzumab by augmenting trastuzumab-induced HER2 downregulation and cell-cycle arrest via inhibition of cyclin D expression. High level of WBP2 correlated with better pCR (67.19%) compared with low WBP2 level (26.58%). The highest response was observed in subgroups of patients with high WBP2–expressing tumors also aged below 50 years (77.78%) or were premenopausal in status (73.33%). Retrospectively, WBP2 demonstrated sensitivity of 80% to 81% and specificity of 76.5% to 80% in discriminating between patients showing pCR and non-pCR.Conclusions:WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab-based neoadjuvant chemotherapy.

PDF file - 127K, Supplementary Table S1: Details of breast cancer patients employed for this study. IDC (NOS), Invasive Ductal Carcinoma (Not Otherwise Specified); DCIS, Ductal Carcinoma In-Situ; ILC, Invasive lobular carcinoma; LN, lymph node status; na, not available; nd, not detected. Supplementary Table S2: Complete list of significant microRNAs identified from breast cancer tumour and serum samples. Supplementary Table S3: Complete list of significant breast cancer tumour microRNAs associated with clinicopathological features. Supplementary Table S4: Complete list of significant serum microRNAs associated with clinicopathological features.

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.

file contains tables, figures and methods

file contains tables, figures and methods

The Nottingham prognostic index (NPI) is a widely used clinicopathological staging system for breast cancer prognostication. Using a step-wise classification approach where breast tumor expression profiles were first divided into general “molecular subtypes” [estrogen receptor (ER)+, ER−, ERBB2+], followed by an independent analysis of each subtype, we identified a 62-gene expression signature (NPI-ES) highly correlated to the NPI in ER+ tumors. The NPI-ES classified the majority of ER+ tumors into two distinct groups with high confidence and was significantly correlated to NPI status in two independent sets of ER+ tumors derived from different centers. The NPI-ES is comparable to the classical NPI in identifying patients likely to exhibit a poor clinical prognosis, as well as to a recently described “prognosis signature” for breast cancer. Our findings demonstrate how expression profiling can complement classical staging systems employing histopathological parameters scored over a continuous range of values.

Male breast cancer (MBC) is rare, representing1% of all breast cancers. Treatment recommendations have been extrapolated from trial data of female breast cancer patients. This study aims to report our institutional experience of MBC across a 20 year period, analyse the survival outcome and prognosis of this group against female breast cancer patients treated at the same centre.Clinical, histopathological, treatment and survival data of male and female breast cancer patients treated between Jan 1999 and July 2019 at Singapore General Hospital and National Cancer Centre Singapore were identified and analysed.Fifty-seven male patients were identified. The median age at diagnosis was 63 years. Majority had invasive ductal carcinoma (86%) and presented at an early disease stage: 70.2% presented as Tis/T1/T2 and 49.1% had no axillary nodal involvement. 84.2% had a simple mastectomy with either a sentinel lymph node biopsy or axillary clearance. The median follow up was 5.69 years for males and 5.83 years for females. The median survival was 11.86 years for males and 16.3 years for females. At 5 years, overall survival (OS) was 69.9% (52.3-82.1%) and disease free survival (DFS) was 62.9% (44.9-76.5%) for males compared with OS 83.8% (83.21-84.39%) and DFS 74.5% (73.91-75.09%) for females.MBC remains understudied. Our institutional data indicates that good long term survival in South-East Asian patients can be achieved with treatment protocols that are similar to female breast cancer. More prospective studies are required.

IntroductionStatins, HMG-CoA reductase inhibitors, are commonly used cholesterol-lowering medications which are also increasingly recognized to have anti-cancer properties for various cancers, including breast cancer. Most clinical evidence supports a protective effect of statin on reducing breast cancer recurrence, particularly in hormone-receptor positive breast cancers.This study seeks to study the impact of statin use on breast cancer recurrence in an Asian population.MethodsThis is a retrospective study of patients diagnosed with breast cancer at the National Cancer Centre and Singapore General Hospital from 2005-2015. Statin use was defined as use after surgery. Associations between statin use, breast cancer recurrence and overall survival were estimated using Cox proportional hazards regression with adjustment for age, TNM stage, grade, ER/HER2 status, and co-morbidities. Associations between statin-use and disease-specific survival were estimated using competing risks regression.ResultsA total of 7858 females with breast cancer were studied, 1353(17.2%) were statin users, 6505(82.8%) were non-statin users, with a median follow-up of 8.67 years. Distribution of cancer stage, histology, molecular subtypes and grades were similar in both groups. Estrogen receptor(ER) positive (HR 0.57,95%CI 0.43-0.76,pConclusionThis is the first known retrospective study on the effect of statin use and breast cancer recurrence in an Asian population. Similar to previous international studies, statin use is associated with a risk reduction in breast cancer recurrence. This is especially beneficial in patients who have ER+ and HER2- invasive breast cancer. Statin use is also associated with a reduced risk of breast cancer recurrence in all subtypes of breast cancer in the long term (>6 years post diagnosis).

Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous disease which is increasingly diagnosed through improved screening measures. Multiple prognostic scores have been devised to predict the risk of local recurrence (LR), and the optimal adjuvant management for DCIS is still debated. Hence, the aim of this analysis is to investigate the factors contributing to the prognosis of DCIS, in particular the role of its hormonal status. From 2005 to 2016, a total of 1221 female patients diagnosed with DCIS at the National Cancer Centre Singapore and Singapore General Hospital were studied. The mean age of diagnosis was 54 years of age (sd = 11.0), with estrogen receptor (ER)-positive DCIS tumors presenting earlier (mean age 54 vs 57 years of age; P < .001). DCIS with negative hormonal status (HS) correlates significantly with a larger size (mean 23.5mm vs 13.0 mm, P < .001) and higher grade of tumor (P < .001). Patients with positive HS were more likely to undergo breast conservation surgery over a mastectomy, in contrast to patients with negative HS (P < .001). Patients with negative HS had a poorer prognosis, with a shorter time of overall survival time (HR = 26.3, P = .020). In conclusion, our study shows that the hormonal status, age of diagnosis, and positive margins are important prognostic factors for DCIS, at least in our Asian population.

Studies that report equivalent oncologic outcomes of sentinel lymph node biopsy (SLNB) alone versus axillary lymph node dissection (ALND) for T1-2N1mi breast cancers are heavily weighted with patients who received breast-conserving surgery (BCS). The impact of omitting ALND in N1mi patients treated with mastectomy is not well studied. It is also unknown if these patients would benefit from post-mastectomy radiotherapy (PMRT). This study reports the outcomes of patients with T1-2N1mi breast cancer treated by mastectomy without axillary therapy.Patients who had T1-2N1mi breast cancer and underwent mastectomy from January 1998 to December 2018 were identified from our multi-institutional prospective database. Axillary recurrence rate (ARR), disease-free survival (DFS), and overall survival (OS) are reported.260 patients with pT1-2N1mi breast cancer who had mastectomy were identified. They had either SLNB (35.4%) or ALND (64.6%). Majority of these patients received adjuvant systemic therapy (93.8%). 77 (29.6%) patients received radiotherapy, 31 after SLNB and 46 after ALND. At median follow-up of 61 months, ARR was 1.1% (n = 1) in the SLNB only group, vs. 0.6% (n = 1) in the ALND group (p = 0.752). DFS and OS were not significantly different between patients with SLNB alone versus ALND (p = 0.40 and p = 0.27, respectively). Among 92 patients who had SLNB only, no DFS or OS difference was observed with the use of PMRT.In T1-2N1mi patients with mastectomy and SLNB, axillary recurrences were rare. No statistically significant differences were noted between patients with SLNB, ALND, or PMRT. Our findings suggest that these patients may be safely treated without axillary therapy.

Description, Fatty acid oxidation is a metabolic gateway that regulates cellular plasticity and supports metastasis in breast cancer., Cell state transitions control the functional behavior of cancer cells. Epithelial-to-mesenchymal transition (EMT) confers cancer stem cell-like properties, enhanced tumorigenicity and drug resistance to tumor cells, while mesenchymal-epithelial transition (MET) reverses these phenotypes. Using high-throughput chemical library screens, retinoids are found to be potent promoters of MET that inhibit tumorigenicity in basal-like breast cancer. Cell state transitions are defined by reprogramming of lipid metabolism. Retinoids bind cognate nuclear receptors, which target lipid metabolism genes, thereby redirecting fatty acids for β-oxidation in the mesenchymal cell state towards lipid storage in the epithelial cell state. Disruptions of key metabolic enzymes mediating this flux inhibit MET. Conversely, perturbations to fatty acid oxidation (FAO) rechannel fatty acid flux and promote a more epithelial cell phenotype, blocking EMT-driven breast cancer metastasis in animal models. FAO impinges on the epigenetic control of EMT through acetyl-CoA-dependent regulation of histone acetylation on EMT genes, thus determining cell states.

Breast fibroepithelial lesions are biphasic tumors which comprise the common benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). This study analyzed 262 (42%) conventional FAs, 45 (7%) cellular FAs, and 321 (51%) benign PTs contributed by the International Fibroepithelial Consortium, using a previously curated 16 gene panel. Benign PTs were found to possess a higher number of mutations, and higher rates of cancer driver gene alterations than both groups of FAs, in particular MED12, TERT promoter, RARA, FLNA, SETD2, RB1, and EGFR. Cases with MED12 mutations were also more likely to have TERT promoter, RARA, SETD2, and EGFR. There were no significant differences detected between conventional FAs and cellular FAs, except for PIK3CA and MAP3K1. TERT promoter alterations were most optimal in discriminating between FAs and benign PTs. Our study affirms the role of sequencing and key mutations that may assist in refining diagnoses of these lesions.

Nipple-sparing mastectomy (NSM) allows for excellent postmastectomy reconstruction aesthetics and is used for both therapeutic and risk-reducing purposes. Reservations regarding the potential for locoregional recurrence and concerns about nipple–areolar complex (NAC) necrosis remain amongst many surgeons. We review the surgical and oncological outcomes after NSM in our institution. All NSM cases at the National Cancer Centre Singapore and Singapore General Hospital between 2005 and 2015 were reviewed. Tumour characteristics, reconstruction methods, surgical and oncological outcomes are described. A total of 139 NSMs were performed for 130 patients. The median age was 46 years (range 21–66). The use of NSM increased from 2% of all breast reconstructions in 2005 to 37% in 2015. The majority (n = 119; 86%) were for cancer treatment and 20 (14%) for risk-reducing purposes. Among those performed for cancer, patients mainly had early stage breast cancer (n = 106, 89%). Autologous reconstruction (n = 111, 80%) was most common. Early complications requiring surgical intervention occurred in 24 (17%) NSMs, including 9 partial/complete flap loss and 2 complete NAC loss. Smoking, previous breast radiation and periareolar incision were all not associated with a higher re-intervention rate (p = 0.93, 0.41 and 0.91, respectively). Median follow-up was 43 months (range 5–145). Five patients (4%) developed local recurrence, including 2 NAC recurrences. The 2- and 5-year overall survival rate is 97 and 90%, respectively. NSM is an oncologically safe procedure in selected patients with acceptable low complication rates.

Background Women with unilateral breast cancer have an increased risk of developing bilateral breast cancer (BBC). Patients with metachronous BBC (mBBC) usually have an earlier age of onset, and their prognoses have been shown to be either similar or poorer than those with synchronous BBC (sBBC). Given the differing presentation and characteristics of breast cancers in the Asian population and the West, this study aims to characterize Asian patients with BBC. Methods All patients who had oncological breast surgery between 2001 and 2010 at the Singapore General Hospital and National Cancer Centre Singapore were reviewed. Patients with BBC were identified and studied. Results A total of 5520 Singaporean women had oncological breast surgery, 155 women (2.8%) had BBC. Of those with BBC, 47.1% (n = 73) were synchronous and 52.9% (n = 82) metachronous (mean interval of 39.4 months), and there was no difference in median age in both groups (54 years of age). Patients with sBBC were more likely to have a positive family history and had asymptomatic contralateral tumours. Although patients with sBBCs were more likely to have ER/PR positive and Her2 negative tumours, they had a lower 5-year overall survival than those with mBBC (P = 0.022). Conclusion Our study shows that Asian women with BBC have different characteristics to their Western counterparts. In particular, women with sBBC tended to have a lower 5-year overall survival compared to those with mBBC, despite having seemingly biologically favourable tumours, which suggest that there may be more underlying their tumour biology and genetics.

Purpose: Atypical ductal hyperplasia (ADH) is a high risk lesion with an increased risk of developing breast cancer. This study aims to identify factors predictive of malignant upgrade for ADH diagnosed on core needle biopsy (CNB) and to develop a nomogram to facilitate evidence-based decision making.Methods: Retrospective analysis of women with CNB diagnosed ADH at the National Cancer Centre Singapore between 2010 and 2015 was performed. Cox proportional hazards regression was used to identify independent clinical, radiological and histological factors associated with malignant upgrade. A nomogram was constructed and multivariable logistic regression coefficients were used to estimate the predicted probability of upgrade for each factor combination. Combinations with the lowest predicted probabilities (≤5%) were identified as low risk. Model sensitivity, specificity, positive and negative predictive values were assessed.Results: From 2010-2015, 238,122 women underwent screening under the national breast cancer screening programme. 29,564 women were recalled and 5742 CNBs were performed, of which 2686 were performed at NCCS. 88 patients (90 lesions) were diagnosed with ADH. 26 lesions were upgraded to a breast malignancy on excision biopsy. On univariate analysis, presence of a mass on either ultrasound (p= 0.018) or mammogram (p=0.026), presence of mammographic microcalcifications (p=0.047), diffuse microcalcification distribution (p=0.034), mammographic parenchymal density (p=0.008), presence of microcalcifications on biopsy (p=0.037) and three or more separate foci of ADH found on biopsy (p=0.024) were associated with malignant upgrade. Mammographic parenchymal density (Hazard ratio= 0.04, 95% CI 0.005-0.35, p=0.014), presence of a mass on ultrasound (Hazard ratio= 10.50, 95% CI 9.21-25.2, p=0.010) and number of foci of ADH (Hazard ratio = 1.877, 95% CI 1.831-1.920, p=0.002) remained significant on multivariate analysis and were included in the normogram which demonstrated good discrimination with C-statistic of 0.81 [95% CI, 0.74 to 0.88].Conclusion: Our model provides good discrimination of breast cancer risk prediction in patients with ADH on CNB. A subset of women at low risk (

// Claire Hian Tzer Chan 1, * , Prabhakaran Munusamy 1, * , Sau Yeen Loke 1 , Geok Ling Koh 1 , Audrey Zhi Yi Yang 1 , Hai Yang Law 2 , Chui Sheun Yoon 2 , Chow Yin Wong 3 , Wei Sean Yong 4 , Nan Soon Wong 5, 6 , Raymond Chee Hui Ng 5 , Kong Wee Ong 4 , Preetha Madhukumar 4 , Chung Lie Oey 4 , Gay Hui Ho 4, 7 , Puay Hoon Tan 8 , Min Han Tan 5, 9, 10 , Peter Ang 5, 6 , Yoon Sim Yap 5 and Ann Siew Gek Lee 1, 11, 12 1 Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore 2 DNA Diagnostic and Research Laboratory, KK Women’s and Children’s Hospital, Singapore 3 Department of General Surgery, Singapore General Hospital, Singapore 4 Department of Surgical Oncology, National Cancer Centre, Singapore 5 Department of Medical Oncology, National Cancer Centre, Singapore 6 Oncocare Cancer Centre, Gleneagles Medical Centre, Singapore 7 Koong and Ho Surgery Centre, Singapore 8 Department of Pathology, Singapore General Hospital, Singapore 9 Institute of Bioengineering and Nanotechnology, Singapore 10 Lucence Diagnostics Pte Ltd, Singapore 11 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 12 Office of Clinical and Academic Faculty Affairs, Duke-NUS Graduate Medical School, Singapore * These authors contributed equally to the work Correspondence to: Ann Siew Gek Lee, email: dmslsg@nccs.com.sg Keywords: breast cancer; single-nucleotide polymorphism; risk loci; genotyping; polygenic risk score Received: July 27, 2017 Accepted: January 25, 2018 Published: January 31, 2018 ABSTRACT Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. The majority of these studies are on European populations, with limited SNP association data in other populations. We genotyped 51 GWAS-identified SNPs in two independent cohorts of Singaporean Chinese. Cohort 1 comprised 1294 BC cases and 885 controls and was used to determine odds ratios (ORs); Cohort 2 had 301 BC cases and 243 controls for deriving polygenic risk scores (PRS). After age-adjustment, 11 SNPs were found to be significantly associated with BC risk. Five SNPs were present in

BackgroundKnown collectively as breast fibroepithelial lesions (FELs), the common fibroadenomas (FAs) and the rarer phyllodes tumors (PTs) are a heterogenous group of biphasic neoplasms. Owing to limited tissue availability, inter-observer variability, overlapping histological features and heterogeneity of these lesions, diagnosing them accurately on core biopsies is challenging. As the choice management option depends on the histological diagnosis; a novel 16-gene panel assay was developed to improve the accuracy of preoperative diagnosis on core biopsy specimens.MethodsUsing this 16-gene panel, targeted amplicon-based sequencing was performed on 275 formalin-fixed, paraffin-embedded (FFPE) breast FEL specimens, archived at the Singapore General Hospital, from 2008 to 2012.ResultsIn total, 167 FAs, 24 benign, 14 borderline and 6 malignant PTs, were profiled. Compared to FAs, PTs had significantly higher mutation rates in theTERTpromoter (p RARA(p FLNA,RB1andTP53(p = 0.002, 0.020 and 0.018, respectively). In addition to a higher mutational count (p TERTpromoter (p p = 0.001, A multivariate logistic regression model was built using these as variables and a predictive scoring system was developed. It classifies a FEL at low or high risk (score p = 0.945) and is significant in predicting PTs (p ConclusionThis novel study demonstrates the ability to extract DNA of sufficient quality and quantity for targeted sequencing from FFPE breast core biopsy specimens, along with their successful characterization and profiling using our customized 16-gene panel. Prospective work includes validating the utility of this promising 16-gene panel assay as an adjunctive diagnostic tool in clinical practice.

Fibroepithelial lesions (FELs) are a heterogeneous group of tumours comprising fibroadenomas (FAs) and phyllodes tumours (PTs). Here we used a 16-gene panel that was previously discovered to be implicated in pathogenesis and progression, to characterise a large international cohort of FELs via targeted sequencing. The study comprised 303 (38%) FAs and 493 (62%) PTs which were contributed by the International Fibroepithelial Consortium. There were 659 (83%) Asian and 109 (14%) non-Asian FELs, while the ethnicity of the rest was unknown. Genetic aberrations were significantly associated with increasing grade of PTs, and were detected more in PTs than FAs for MED12, TERT promoter, RARA, FLNA, SETD2, TP53, RB1, EGFR, and IGF1R. Most borderline and malignant PTs possessed ≥ 2 mutations, while there were more cases of FAs with ≤ 1 mutation compared to PTs. FELs with MED12 mutations had significantly higher rates of TERT promoter, RARA, SETD2, EGFR, ERBB4, MAP3K1, and IGF1R aberrations. However, FELs with wild-type MED12 were more likely to express TP53 and PIK3CA mutations. There were no significant differences observed between the mutational profiles of recurrent FAs, FAs with a history of subsequent ipsilateral recurrence or contralateral occurrence, and FAs without a history of subsequent events. We identified recurrent mutations which were more frequent in PTs than FAs, with borderline and malignant PTs harbouring cancer driver gene and multiple mutations. This study affirms the role of a set of genes in FELs, including its potential utility in classification based on mutational profiles. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Post-mastectomy breast reconstruction is an integral component of breast cancer treatment. It is often perceived that women in Asian countries have a lower rate of post-mastectomy reconstruction than Western populations. This study describes trends in timing and types of breast reconstruction performed in the largest healthcare provider in Singapore, over a period of 12 years. It also reports on the oncological outcomes and surgical safety. A retrospective review of all patients who underwent post-mastectomy reconstruction from January 2001 to December 2012 at the National Cancer Centre Singapore and Singapore General Hospital was performed. Six hundred and twenty post-mastectomy reconstructions were performed in 579 patients. The proportion of reconstructions increased from 4% in 2001 to 18% in 2012. Younger patients (

Purpose: Trastuzumab-based chemotherapy has shown remarkable clinical benefits for patients with HER2-positive breast cancer. However, treatment regimens involving trastuzumab had little or no effect for a subset of patients. Preliminary studies revealed WW-binding protein 2 (WBP2), an oncogenic transcription coactivator, to be coamplified with HER2 in 36% of HER2-positive breast cancers. We hypothesize that WBP2 regulates and correlates with the response of HER2-positive breast cancer to trastuzumab. Experimental Design: The coexpression of WBP2 and HER2 in breast tumors was validated using IHC. The role and mechanism of WBP2 in regulating breast cancer response to trastuzumab was elucidated using in vitro, patient-derived xenograft and murine xenograft models. A multicenter retrospective study involving 143 patients given neoadjuvant trastuzumab-based chemotherapy was conducted to determine whether WBP2 expression correlates with pathologic complete response (pCR). Results: Elevated expression of WBP2 significantly enhanced breast cancer's response to trastuzumab by augmenting trastuzumab-induced HER2 downregulation and cell-cycle arrest via inhibition of cyclin D expression. High level of WBP2 correlated with better pCR (67.19%) compared with low WBP2 level (26.58%). The highest response was observed in subgroups of patients with high WBP2–expressing tumors also aged below 50 years (77.78%) or were premenopausal in status (73.33%). Retrospectively, WBP2 demonstrated sensitivity of 80% to 81% and specificity of 76.5% to 80% in discriminating between patients showing pCR and non-pCR. Conclusions: WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab-based neoadjuvant chemotherapy.

Women with unilateral breast cancer have an increased risk of developing bilateral breast cancer (BBC). Patients with metachronous BBC (mBBC) usually have an earlier age of onset, and their prognoses have been shown to be either similar or poorer than those with synchronous BBC (sBBC). Given the differing presentation and characteristics of breast cancers in the Asian population and the West, this study aims to characterize Asian patients with BBC.All patients who had oncological breast surgery between 2001 and 2010 at the Singapore General Hospital and National Cancer Centre Singapore were reviewed. Patients with BBC were identified and studied.A total of 5520 Singaporean women had oncological breast surgery, 155 women (2.8%) had BBC. Of those with BBC, 47.1% (n = 73) were synchronous and 52.9% (n = 82) metachronous (mean interval of 39.4 months), and there was no difference in median age in both groups (54 years of age). Patients with sBBC were more likely to have a positive family history and had asymptomatic contralateral tumours. Although patients with sBBCs were more likely to have ER/PR positive and Her2 negative tumours, they had a lower 5-year overall survival than those with mBBC (P = 0.022).Our study shows that Asian women with BBC have different characteristics to their Western counterparts. In particular, women with sBBC tended to have a lower 5-year overall survival compared to those with mBBC, despite having seemingly biologically favourable tumours, which suggest that there may be more underlying their tumour biology and genetics.

Malignant transformation of the epithelial component of phyllodes tumours (PT) is rare and only reported in literature as sporadic cases of carcinoma associated with PTs. We report the clinicopathological characteristics of in situ and invasive carcinoma coexisting with PT in 10 patients treated in our institution over an 11-year period from 1992 to 2012. Ten patients with coexisting PT and in situ or invasive carcinoma were identified from our records. Six had carcinoma found within the PT. All were female with a median age of 47 (43-72) years. One patient had a history of PT in the same breast while another had a history of PT in the same breast as well as invasive ductal carcinoma in the contralateral breast. The rest did not have any risk factors of breast cancer. Five patients had a preoperative core needle biopsy performed with the report of a fibroepithelial lesion. The rest of the patients had surgery upfront for their breast masses. Two patients who had ER/PR positive invasive carcinoma received adjuvant hormonal therapy. Patients were followed up for a mean of 3.6 years (9 months-10 years) and all patients were alive and recurrence free. PT associated with carcinoma is rare, and we present a series of cases that add to the limited current literature. It is often difficult to detect the presence of the carcinomatous component preoperatively. Hence, close examination of resected PT specimens must be carried out to allow prompt detection of any associated carcinomas, however rare, such that adequate treatment can be given.

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.

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., Gene expression is regulated by enhancers and super-enhancers, which can be identified by chromatin profiling. Here, the authors surveyed gastric cancer samples and cell lines to identify enhancer elements exhibiting somatic alterations.

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P < 0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells. © 2011 Wiley-Liss, Inc.

The current need for high-throughput genotyping platforms for targeted validation of disease-associated single nucleotide polymorphisms (SNPs) motivated us to evaluate a novel nanofluidics platform for genotyping DNA extracted from peripheral blood and buccal wash samples. SNP genotyping was performed using a Fluidigm 48.48 Dynamic Array biochip on the BioMark polymerase chain reaction platform and results were compared against standard TaqMan assays and DNA sequencing. Pilot runs using these dynamic arrays on 90 samples against 20 SNP assays had an average call rate of 99.7%, with 100% call rates for 16 of the assays. Manual TaqMan genotyping of these samples against three SNPs demonstrated 100% correlation between the two platforms. To understand the influence of DNA template variability, three sources of blood samples (CH-1, n = 20; CH-2, n = 47; KK, n = 47) and buccal washes (n = 37) were genotyped for 24 SNPs. Although both CH-1 and CH-2 batches showed good base calling (≥98.8%), the KK batch and buccal wash samples exhibited lower call rates (82.1% and 94.0%). Importantly, repurification of the KK and buccal wash samples resulted in significant improvements in their call rates (to ≥97.9%). Scale-up for genotyping 1698 cases and controls for 24 SNPs had overall call rates of 97.6% for KK and 99.2% for CH samples. The Dynamic Array approach demonstrated accuracy similar to that of TaqMan genotyping, while offering significant savings in DNA, effort, time, and costs.

Triple-negative breast cancer (TNBC), as immunohistochemically defined by its estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and human epidermal growth factor receptor-2 (HER2)-negative status, is an important subtype due to its biologically aggressive behavior and limited treatment options available. TNBC is associated with an overall poorer prognosis, with higher risk of disease recurrence/progression and shorter duration of treatment response, i.e., treatment resistance. Treatment resistance may be largely attributed to cancer stem cells (CSCs), which are intrinsically treatment resistant and continually self-renew, proliferate, and differentiate into different phenotypes. Activation of the cell biologic program, epithelial-mesenchymal transition (EMT), has been demonstrated to promote the dedifferentiation of heterogeneous subpopulations of cancer cells towards CSC phenotypes. We hypothesized that induction of the mesenchymal-epithelial transition (MET) program might disrupt CSC function, drive differentiation, and render greater susceptibility to conventional chemotherapy. In this study, we utilized high-throughput chemical-genetic screens to uncover a potent class of MET mediators. With the use of in vitro and in vivo models of TNBC, we showed that changing the malignant cell state to a differentiated phenotype by inducing MET reduced mammosphere formation, increased chemosensitivity, and decreased the tumor burden in NSG mice. Delving into the mechanisms of tumor differentiation via ChIP-seq, RNA-seq, and Gene Ontology analysis revealed differences in metabolic status between cell states, which might be exploited in the treatment of TNBC. We also assessed combinations of MET mediators, in order to increase the potency and durability of differentiation. Hence, this study assessed the role of differentiation in the treatment of TNBC and the efficacy of various MET mediators, singly and in combination, in inducing differentiation. Citation Format: Ser Yue Loo, Liping Toh, Elina Pathak, Wilson Tan, Siming Ma, Ju Yuan, Giridharan Periyasamy, Federico Torta, Jack Chan, Tira Tan, Yi Rong Sim, Veronique Tan, Benita Tan, Preetha Madhukumar, Wei Sean Yong, Kong Wee Ong, Chow Yin Wong, Markus R. Wenk, Roger Foo, Yoon-Sim Yap, Elaine Lim, Wai Leong Tam. Inducing cell state transitions in triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B22.

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.

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.

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.

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. Methods: All patients with stage I and II breast cancer and non-palpable axillary nodes were eligible, including those with previous excision biopsy. We excluded pregnant women, those with previous axillary surgery and women with advanced breast cancer with enlarged axillary nodes. The sentinel node was detected with technetium-99m-labelled tin colloid and vital blue dye and removed, and axillary clearance was performed. Results: A total of 312 patients were examined from August 1996 to December 1998. The mean age was 53 years (range 28−83) and mean tumour size 2.6 cm (range 0.2−9.0). The detection rate of the sentinel node was 86%. The sentinel lymph node predicted the axillary status with a sensitivity of 83% and specificity of 100%. The false-negative rate was 16.7%. Conclusions: Detection of the sentinel lymph node is feasible and it can accurately predict the nodal status of the axilla. ­However, the high false-negative rate precludes as yet the use of sentinel lymph node biopsy in replacing axillary clearance as the standard of care for breast cancer.

Study objectives To determine the quantity and resource utilisation in management of the severely injured patients presenting at the Singapore General Hospital (SGH). Method All patients who were initially triaged to the hospital Emergency Department's (ED) resuscitation room and all trauma related mortality in the year 1998 were studied. All records were traced and the cases were followed up. Important outcomes studied were mortality and length of hospital stay. Results Three hundred and forty-seven (0.2%) severely injured patients who were initially treated at the ED's resuscitation room were studied. The median age of the patients was 32 years old. Male patients formed 82.1% of the total. The three main causes of trauma in such patients were fall from height, motor vehicle related accidents and penetrating injuries caused by sharp instruments and firearms. Of all the patients, 62.8% were admitted to the ED between 1601 hours to 0759 hours and 30.5% of all severely injured patients were treated at the ED on weekends. One hundred and twenty three (35.4%) patients had emergency surgery within 24 hours of admission to the ED and 42.4% of the patients had an ISS score of 16 or more; 22.8% of patients had ISS score of 25 or more. The mortality of patients with ISS score of 25 or more was 39.2%. Conclusion One percent of all new ED patients with trauma were classified as ‘severely’ injured and these occurred mostly outside “normal working hours”. There was significant mortality in patients with ISS score of 25 or more.

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.

AimWe aimed to assess the one step nucleic acid amplification (OSNA) assay as an intraoperative method in comparison with frozen sections (FS) for detection of metastasis in sentinel lymph nodes (SLNs) of breast cancer.Method100 SLNs from patients with breast carcinoma were enrolled within a 3-month period. Alternate 2 mm node slices were subjected to routine FS, and later to permanent histology, and the rest for automated molecular detection of CK19 mRNA using OSNA. FS and OSNA findings were compared with permanent histology results. Difference in turnaround time was also noted.ResultsWith permanent histology as gold standard, OSNA was discrepant in 8 of 98 (3 false negative, 5 false positive) included SLNs whereas FS had 2 false negative cases. FS had higher sensitivity (89%, p=ConclusionsAutomation of SLN assessment using OSNA is a potentially useful intraoperative diagnostic tool with acceptable accuracy. Discordant findings in this study may be due to sampling allocation. Since OSNA is more time-consuming, its practical advantage over routine FS requires further study in view of current technical workflow considerations.