Combined aerobic and strength exercise training on biological ageing in Singaporean breast cancer patients: protocol for the Breast Cancer Exercise Intervention (BREXINT) Pilot Study.

Breast cancer (BC) is the most prominent cancer amongst women, but fortunately, early diagnosis and advances in multimodality treatments have improved patient survivability. Cancer survivors, however, experience increased biological ageing which may accelerate other co-morbidities. Exercise intervention is a promising clinical adjuvant approach to improve BC patients' physiological function, recovery from treatment, and quality of life. However, the effects of combined aerobic and strength exercise training on biological ageing in BC patients have not been studied. The Breast Cancer Exercise Intervention (BREXINT) Pilot Study will evaluate the effects of a 24-week combined aerobic and strength exercise intervention against usual care in 50 BC patients' post-treatment randomised to either group. The primary outcomes include changes in cardiorespiratory fitness, muscle strength, cancer-related symptoms, and rate of biological ageing following exercise intervention period. The secondary outcomes include habitual physical activity measured with tri-axial accelerometery and supporting questionnaires, including physical activity, food diary, and quality of life questionnaires. This study will identify the effects of combined aerobic exercise strength training on biological ageing in BC patients from Singapore. Results from this study could further support the implementation of regular exercise programmes as routine care for cancer patients.

Cost-effectiveness analysis of add-on pertuzumab to trastuzumab biosimilar and chemotherapy as neoadjuvant treatment for human epidermal growth receptor 2-positive early breast cancer patients in Singapore.

OBJECTIVES: The Asian PEONY trial showed that add-on pertuzumab to trastuzumab and chemotherapy significantly improved pathological complete response in the neoadjuvant treatment of patients with human epidermal growth factor receptor 2-positive (HER2+) early breast cancer (EBC). This study evaluated the cost-effectiveness of pertuzumab as an add-on therapy to trastuzumab and chemotherapy for neoadjuvant treatment of patients with HER2+ EBC in Singapore. METHODS: A six-state Markov model was developed from the Singapore healthcare system perspective, with a lifetime time horizon. Model outputs were: costs; life-years (LYs); quality-adjusted LYs (QALYs); incremental cost-effectiveness ratios (ICERs). Sensitivity/scenario analyses explored model uncertainties. RESULTS: The base case projected the addition of pertuzumab to be associated with improved outcomes by 0.277 LYs and 0.271 QALYs, increased costs by S$1,387, and an ICER of S$5,121/QALY. The ICER was most sensitive to the pCR rate, and the probabilistic sensitivity analysis showed that add-on pertuzumab had an 81.3% probability of being cost-effective at a willingness-to-pay threshold of S$45,000/QALY gained. CONCLUSIONS: This model demonstrated that the long-term clinical impact of early pertuzumab use, particularly the avoidance of metastatic disease and thus avoidance of higher costs and mortality rates, make neoadjuvant pertuzumab a cost-effective option in the management of patients with HER2+ breast cancer in Singapore.

Alterations to DNA methylation patterns induced by chemotherapy treatment are associated with negative impacts on the olfactory pathway.

BACKGROUND: Exposure to cytotoxic chemotherapy treatment may alter DNA methylation (DNAm) in breast cancer patients. METHODS: We performed DNAm analysis in 125 breast cancer patients with blood drawn before and after chemotherapy, using the Illumina MethylationEPIC array. DNAm changes of 588,798 individual CpGs (including 41,207 promoter regions) were evaluated using linear regression models adjusted for monocyte proportion. Gene set enrichment analyses (GSEA) were conducted to identify key Gene Ontology (GO) biological processes or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with chemotherapy. Results were validated in a separate cohort of breast cancer patients who were treated (n = 1273) and not treated (n = 872) by chemotherapy (1808 blood, 337 saliva). RESULTS: A total of 141 differentially methylated CpGs and 11 promoters were significantly associated with chemotherapy after multiple testing corrections in both the paired sample and single time point analyses. GSEA of promoter regions (pre-ranked by test statistics) identified six suppressed biological processes (p < 4.67e-8) related to sensory perception and detection of chemical stimuli, including smell perception (GO:0007606, GO:0007608, GO:0009593, GO:0050906, GO:0050907, and GO:0050911). The same six biological processes were significantly suppressed in the validation dataset (p < 9.02e-14). The KEGG pathway olfactory transduction (hsa04740) was also found to be significantly suppressed (ppaired-samples = 1.72e-9, psingle-timepoint-blood = 2.03e-15 and psingle-timepoint-saliva = 7.52e-56). CONCLUSION: The enrichment of imprinted genes within biological processes and pathways suggests a biological mechanism by which chemotherapy could affect the perception of smell.

How Asian Breast Cancer Patients Experience Unequal Incidence of Chemotherapy Side Effects: A Look at Ethnic Disparities in Febrile Neutropenia Rates.

The majority of published findings on chemotherapy-induced febrile neutropenia (FN) are restricted to three ethnic groups: Asians, Caucasians, and African Americans. In this two-part study, we examined FN incidence and risk factors in Chinese, Malay, and Indian chemotherapy-treated breast cancer (BC) patients. Hospital records or ICD codes were used to identify patients with FN. In both the Singapore Breast Cancer Cohort (SGBCC) and the Joint Breast Cancer Registry (JBCR), the time of the first FN from the start of chemotherapy was estimated using Cox regression. Multinomial regression was used to evaluate differences in various characteristics across ethnicities. FN was observed in 170 of 1014 patients in SGBCC. The Cox model showed that non-Chinese were at higher risk of developing FN (HRMalay [95% CI]:2.04 [1.44-2.88], p < 0.001; HRIndian:1.88 [1.11-3.18], p = 0.018). In JBCR, FN was observed in 965 of 7449 patients. Univariable Cox models identified ethnicity, a lower baseline absolute neutrophil count, non-luminal A proxy subtypes, and anthracycline-containing regimens as risk factors. Disparities across ethnicities' risk (HRMalay:1.29 [1.07-1.54], p = 0.006; HRIndian:1.50 [1.19-1.88], p < 0.001) remained significant even after further adjustments. Finally, an age-adjusted multinomial model showed that Malays (p = 0.006) and Indians (p = 0.009) were significantly more likely to develop multiple episodes of FN during treatment. Ethnic differences in chemotherapy-induced FN among BC patients exist. Further studies can focus on investigating pharmacogenetic differences across ethnicities.

Will Absolute Risk Estimation for Time to Next Screen Work for an Asian Mammography Screening Population?

Personalized breast cancer risk profiling has the potential to promote shared decision-making and improve compliance with routine screening. We assessed the Gail model's performance in predicting the short-term (2- and 5-year) and the long-term (10- and 15-year) absolute risks in 28,234 asymptomatic Asian women. Absolute risks were calculated using different relative risk estimates and Breast cancer incidence and mortality rates (White, Asian-American, or the Singapore Asian population). Using linear models, we tested the association of absolute risk and age at breast cancer occurrence. Model discrimination was moderate (AUC range: 0.580-0.628). Calibration was better for longer-term prediction horizons (E/Olong-term ranges: 0.86-1.71; E/Oshort-term ranges:1.24-3.36). Subgroup analyses show that the model underestimates risk in women with breast cancer family history, positive recall status, and prior breast biopsy, and overestimates risk in underweight women. The Gail model absolute risk does not predict the age of breast cancer occurrence. Breast cancer risk prediction tools performed better with population-specific parameters. Two-year absolute risk estimation is attractive for breast cancer screening programs, but the models tested are not suitable for identifying Asian women at increased risk within this short interval.

Ironing out exercise on immuno-oncological outcomes.

Despite accumulating evidence that supports the beneficial effects of physical exercise in inhibiting cancer progression, whether exercise modulates its effects through systemic and cellular changes in iron metabolism and immune-tumor crosstalk is unknown. Cancer cells have greater metabolic requirements than normal cells, with their survival and proliferation depending largely on iron bioavailability. Although iron is an essential mineral for mitogenesis, it also participates in a form of iron-dependent programmed cell death termed ferroptosis. In this short hypothesis paper, we speculate that modulating iron bioavailability, transport and metabolism with regular exercise can have significant implications for tumor and stromal cells in the tumor microenvironment, by affecting multiple tumor-autonomous and stromal cell responses.

Association between Breast Cancer Polygenic Risk Score and Chemotherapy-Induced Febrile Neutropenia: Null Results.

BACKGROUND: The hypothesis that breast cancer (BC) susceptibility variants are linked to chemotherapy-induced toxicity has been previously explored. Here, we investigated the association between a validated 313-marker-based BC polygenic risk score (PRS) and chemotherapy-induced neutropenia without fever and febrile neutropenia (FNc) in Asian BC patients. METHODS: This observational case-control study of Asian BC patients treated with chemotherapy included 161 FNc patients, 219 neutropenia patients, and 936 patients who did not develop neutropenia. A continuous PRS was calculated by summing weighted risk alleles associated with overall, estrogen receptor- (ER-) positive, and ER-negative BC risk. PRS distributions neutropenia or FNc cases were compared to controls who did not develop neutropenia using two-sample t-tests. Odds ratios (OR) and corresponding 95% confidence intervals were estimated for the associations between PRS (quartiles and per standard deviation (SD) increase) and neutropenia-related outcomes compared to controls. RESULTS: PRS distributions were not significantly different in any of the comparisons. Higher PRSoverall quartiles were negatively correlated with neutropenia or FNc. However, the associations were not statistically significant (PRS per SD increase OR neutropenia: 0.91 [0.79-1.06]; FNc: 0.87 [0.73-1.03]). No dose-dependent trend was observed for the ER-positive weighted PRS (PRSER-pos) and ER-negative weighted PRS (PRSER-neg). CONCLUSION: BC PRS was not strongly associated with chemotherapy-induced neutropenia or FNc.

Overlap of high-risk individuals predicted by family history, and genetic and non-genetic breast cancer risk prediction models: implications for risk stratification.

BACKGROUND: Family history, and genetic and non-genetic risk factors can stratify women according to their individual risk of developing breast cancer. The extent of overlap between these risk predictors is not clear. METHODS: In this case-only analysis involving 7600 Asian breast cancer patients diagnosed between age 30 and 75 years, we examined identification of high-risk patients based on positive family history, the Gail model 5-year absolute risk [5yAR] above 1.3%, breast cancer predisposition genes (protein-truncating variants [PTV] in ATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, or TP53), and polygenic risk score (PRS) 5yAR above 1.3%. RESULTS: Correlation between 5yAR (at age of diagnosis) predicted by PRS and the Gail model was low (r=0.27). Fifty-three percent of breast cancer patients (n=4041) were considered high risk by one or more classification criteria. Positive family history, PTV carriership, PRS, or the Gail model identified 1247 (16%), 385 (5%), 2774 (36%), and 1592 (21%) patients who were considered at high risk, respectively. In a subset of 3227 women aged below 50 years, the four models studied identified 470 (15%), 213 (7%), 769 (24%), and 325 (10%) unique patients who were considered at high risk, respectively. For younger women, PRS and PTVs together identified 745 (59% of 1276) high-risk individuals who were not identified by the Gail model or family history. CONCLUSIONS: Family history and genetic and non-genetic risk stratification tools have the potential to complement one another to identify women at high risk.

Multi-center evaluation of artificial intelligent imaging and clinical models for predicting neoadjuvant chemotherapy response in breast cancer.

BACKGROUND: Neoadjuvant chemotherapy (NAC) plays an important role in the management of locally advanced breast cancer. It allows for downstaging of tumors, potentially allowing for breast conservation. NAC also allows for in-vivo testing of the tumors' response to chemotherapy and provides important prognostic information. There are currently no clearly defined clinical models that incorporate imaging with clinical data to predict response to NAC. Thus, the aim of this work is to develop a predictive AI model based on routine CT imaging and clinical parameters to predict response to NAC. METHODS: The CT scans of 324 patients with NAC from multiple centers in Singapore were used in this study. Four different radiomics models were built for predicting pathological complete response (pCR): first two were based on textural features extracted from peri-tumoral and tumoral regions, the third model based on novel space-resolved radiomics which extract feature maps using voxel-based radiomics and the fourth model based on deep learning (DL). Clinical parameters were included to build a final prognostic model. RESULTS: The best performing models were based on space-resolved and DL approaches. Space-resolved radiomics improves the clinical AUCs of pCR prediction from 0.743 (0.650 to 0.831) to 0.775 (0.685 to 0.860) and our DL model improved it from 0.743 (0.650 to 0.831) to 0.772 (0.685 to 0.853). The tumoral radiomics model performs the worst with no improvement of the AUC from the clinical model. The peri-tumoral combined model gives moderate performance with an AUC of 0.765 (0.671 to 0.855). CONCLUSIONS: Radiomics features extracted from diagnostic CT augment the predictive ability of pCR when combined with clinical features. The novel space-resolved radiomics and DL radiomics approaches outperformed conventional radiomics techniques.

Special Histologic Type and Rare Breast Tumors - Diagnostic Review and Clinico-Pathological Implications.

Breast cancer is the most common malignant tumor in females. While most carcinomas are categorized as invasive carcinoma, no special type (NST), a diverse group of tumors with distinct pathologic and clinical features is also recognized, ranging in incidence from relatively more common to rare. So-called "special histologic type" tumors display more than 90% of a specific, distinctive histologic pattern, while a spectrum of tumors more often encountered in the salivary gland may also arise in the breast. Metaplastic carcinomas can present diagnostic challenges. Some uncommon tumors harbor pathognomonic genetic alterations. This article provides an overview of the key diagnostic points and differential diagnoses for this group of disparate lesions, as well as the salient clinical characteristics of each entity.

The Role of the Extracellular Matrix and Tumor-Infiltrating Immune Cells in the Prognostication of High-Grade Serous Ovarian Cancer.

Ovarian cancer is the eighth global leading cause of cancer-related death among women. The most common form is the high-grade serous ovarian carcinoma (HGSOC). No further improvements in the 5-year overall survival have been seen over the last 40 years since the adoption of platinum- and taxane-based chemotherapy. Hence, a better understanding of the mechanisms governing this aggressive phenotype would help identify better therapeutic strategies. Recent research linked onset, progression, and response to treatment with dysregulated components of the tumor microenvironment (TME) in many types of cancer. In this study, using bioinformatic approaches, we identified a 19-gene TME-related HGSOC prognostic genetic panel (19 prognostic genes (PLXNB2, HMCN2, NDNF, NTN1, TGFBI, CHAD, CLEC5A, PLXNA1, CST9, LOXL4, MMP17, PI3, PRSS1, SERPINA10, TLL1, CBLN2, IL26, NRG4, and WNT9A) by assessing the RNA sequencing data of 342 tumors available in the TCGA database. Using machine learning, we found that specific patterns of infiltrating immune cells characterized each risk group. Furthermore, we demonstrated the predictive potential of our risk score across different platforms and its improved prognostic performance compared with other gene panels.

Germline breast cancer susceptibility genes, tumor characteristics, and survival.

BACKGROUND: Mutations in certain genes are known to increase breast cancer risk. We study the relevance of rare protein-truncating variants (PTVs) that may result in loss-of-function in breast cancer susceptibility genes on tumor characteristics and survival in 8852 breast cancer patients of Asian descent. METHODS: Gene panel sequencing was performed for 34 known or suspected breast cancer predisposition genes, of which nine genes (ATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, and TP53) were associated with breast cancer risk. Associations between PTV carriership in one or more genes and tumor characteristics were examined using multinomial logistic regression. Ten-year overall survival was estimated using Cox regression models in 6477 breast cancer patients after excluding older patients (≥75years) and stage 0 and IV disease. RESULTS: PTV9genes carriership (n = 690) was significantly associated (p < 0.001) with more aggressive tumor characteristics including high grade (poorly vs well-differentiated, odds ratio [95% confidence interval] 3.48 [2.35-5.17], moderately vs well-differentiated 2.33 [1.56-3.49]), as well as luminal B [HER-] and triple-negative subtypes (vs luminal A 2.15 [1.58-2.92] and 2.85 [2.17-3.73], respectively), adjusted for age at diagnosis, study, and ethnicity. Associations with grade and luminal B [HER2-] subtype remained significant after excluding BRCA1/2 carriers. PTV25genes carriership (n = 289, excluding carriers of the nine genes associated with breast cancer) was not associated with tumor characteristics. However, PTV25genes carriership, but not PTV9genes carriership, was suggested to be associated with worse 10-year overall survival (hazard ratio [CI] 1.63 [1.16-2.28]). CONCLUSIONS: PTV9genes carriership is associated with more aggressive tumors. Variants in other genes might be associated with the survival of breast cancer patients. The finding that PTV carriership is not just associated with higher breast cancer risk, but also more severe and fatal forms of the disease, suggests that genetic testing has the potential to provide additional health information and help healthy individuals make screening decisions.

Fatty acid oxidation is a druggable gateway regulating cellular plasticity for driving 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 ß-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 cancer risk stratification for mammographic screening: A nation-wide screening cohort of 24,431 women in Singapore.

BACKGROUND: Breast cancer incidence is increasing in Asia. However, few women in Singapore attend routine mammography screening. We aim to identify women at high risk of breast cancer who will benefit most from regular screening using the Gail model and information from their first screen (recall status and mammographic density). METHODS: In 24,431 Asian women (50-69 years) who attended screening between 1994 and 1997, 117 developed breast cancer within 5 years of screening. Cox proportional hazard models were used to study the associations between risk classifiers (Gail model 5-year absolute risk, recall status, mammographic density), and breast cancer occurrence. The efficacy of risk stratification was evaluated by considering sensitivity, specificity, and the proportion of cancers identified. RESULTS: Adjusting for information from first screen attenuated the hazard ratios (HR) associated with 5-year absolute risk (continuous, unadjusted HR [95% confidence interval]: 2.3 [1.8-3.1], adjusted HR: 1.9 [1.4-2.6]), but improved the discriminatory ability of the model (unadjusted AUC: 0.615 [0.559-0.670], adjusted AUC: 0.703 [0.653-0.753]). The sensitivity and specificity of the adjusted model were 0.709 and 0.622, respectively. Thirty-eight percent of all breast cancers were detected in 12% of the study population considered high risk (top five percentile of the Gail model 5-year absolute risk [absolute risk ≥1.43%], were recalled, and/or mammographic density ≥50%). CONCLUSION: The Gail model is able to stratify women based on their individual breast cancer risk in this population. Including information from the first screen can improve prediction in the 5 years after screening. Risk stratification has the potential to pick up more cancers.

Cost-effectiveness of pertuzumab and trastuzumab biosimilar combination therapy as initial treatment for HER2-positive metastatic breast cancer in Singapore.

BACKGROUND: This study evaluates the cost-effectiveness of pertuzumab with trastuzumab biosimilar and docetaxel as initial treatment for HER2-positive metastatic breast cancer (MBC) in Singapore. METHODS: A partitioned survival model with three health states was developed to evaluate the cost-effectiveness of trastuzumab biosimilar and docetaxel with or without pertuzumab from a healthcare system perspective over a 15-year time horizon for patients with HER2-positive MBC. Key clinical inputs were derived from the CLEOPATRA trial. Health state utilities were derived from the literature and direct medical costs were obtained from local public healthcare institutions. RESULTS: The base-case resulted in an incremental cost-effectiveness ratio (ICER) of SGD366,658 (USD272,244) per quality-adjusted life-year (QALY) gained. One-way sensitivity analyses showed that the ICER was sensitive to utilities in the progression-free state, price of pertuzumab and time horizon. When the price for trastuzumab reference biologic (branded) was applied, the ICER was even higher (SGD426,996 [USD317,045]/QALY). CONCLUSION: Although trastuzumab biosimilar reduced the cost of the pertuzumab combination regimen, the ICER remained high and was not cost effective in Singapore's context. As pertuzumab contributed 80% of the overall combination treatment cost, price reductions for pertuzumab will be required to improve the cost-effectiveness of combination treatment to an acceptable level.

Efficacy, patterns of use and cost of Pertuzumab in the treatment of HER2+ metastatic breast cancer in Singapore: The National Cancer Centre Singapore experience.

BACKGROUND: Pertuzumab is a humanized anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody found in a Phase III clinical trial to significantly improve median survival in HER2 positive metastatic breast cancer (MBC) when used in combination with a taxane and Trastuzumab, and its clinical efficacy has transformed the therapeutic landscape of HER2-positive breast cancer. There are currently few reports on the pattern of use and value of Pertuzumab in real world settings. Our study describes the clinical efficacy and treatment costs of Pertuzumab in HER2-positive MBC treated in a tertiary cancer centre in Singapore in a predominantly Asian population. AIM: To investigate the clinical efficacy and treatment costs of Pertuzumab in HER2-positive MBC in an Asian population in Singapore. METHODS: A retrospective study of 304 HER2-positive MBC patients seen at National Cancer Centre Singapore between 2011-2017 was conducted. Demographic and clinical data were extracted from electronic medical records. Clinical characteristics and billing data of patients who received Pertuzumab were compared with those who did not. RESULTS: Thirty-one (62.0%) of the fifty (16.4%) patients who received Pertuzumab as first-line therapy. With a median follow-up of 21.5 mo, there was a statistically significant difference in the median overall survival between Pertuzumab and non-Pertuzumab groups [51.5 (95%CI: 35.8-60.0) vs 32.9 (95%CI: 28.1-37.5) mo; P = 0.0128]. Two (4.88%) patients in the Pertuzumab group experienced grade 3 (G3) cardiotoxicity. The median treatment cost incurred for total chemotherapy for the Pertuzumab group was 130456 Singapore Dollars compared to 34523 Singapore Dollars for the non-Pertuzumab group. The median percentage of total chemotherapy costs per patient in the Pertuzumab group spent on Pertuzumab was 50.3%. CONCLUSION: This study shows that Pertuzumab use in the treatment of metastatic breast cancer is associated with a significantly better survival and a low incidence of serious cardiotoxicity. However, the proportionate cost of Pertuzumab therapy remains high and further cost-effectiveness studies should be conducted.

Detection and prognostic relevance of circulating tumour cells (CTCs) in Asian breast cancers using a label-free microfluidic platform.

OBJECTIVES: We aimed to study the prevalence of CTCs in breast cancer (BC) patients undergoing neoadjuvant or palliative therapy with a label-free microfluidic platform (ClearCell FX), and its prognostic relevance in metastatic BC (mBC). MATERIALS AND METHODS: Peripheral blood samples were collected from 108 BC patients before starting a new line of treatment ("baseline"), majority of whom had mBC (76/108; 70.4%). CTCs were retrieved by dean flow fractionation that enriched for larger cells, and enumerated using immunofluorescence-based staining. Progression-free survival (PFS) in mBC patients was analysed using Kaplan-Meier method; cox proportional hazard models were used for univariable and multivariable analyses. RESULTS: The detection rate of CTCs before starting a new line of treatment was 75.9% (n = 108; median: 8 CTCs/7.5 ml blood) at a cut off of ≥2 CTCs. PFS was inferior for mBC patients with baseline CTC count ≥5 CTCs/7.5 ml blood vs. those with < 5 CTCs/7.5 ml blood (median PFS: 4.3 vs. 7.0 months; p-value: 0.037). The prognostic relevance of CTCs was most significant in patients with HER2- mBC (median PFS: 4.1 vs. 8.3 months; p-value: 0.032), luminal (HR+HER2-) subtype (median PFS: 4.2 vs. 8.3 months; p-value: 0.048), and patients who had one or more prior treatments (median PFS: 4.2 vs. 7.0 months; p-value: 0.02). On multivariable analysis, baseline CTC level (hazard ratio (HR): 1.84, p-value: 0.02) and pre-treatment status (HR: 1.87, p-value: 0.05) were independent predictors of PFS. CONCLUSIONS: This work demonstrates the prognostic significance of CTCs in mBC detected using a label-free size-based enrichment platform.

Evaluation of phospho-histone H3 in Asian triple-negative breast cancer using multiplex immunofluorescence.

PURPOSE: We used multiplex immunofluorescence (mIF) to determine whether mitotic rate represents an independent prognostic marker in triple-negative breast cancer (TNBC). Secondary aims were to confirm the prognostic significance of immune cells in TNBC, and to investigate the relationship between immune cells and proliferating tumour cells. METHODS: A retrospective Asian cohort of 298 patients with TNBC diagnosed from 2003 to 2015 at the Singapore General Hospital was used in the present study. Formalin-fixed, paraffin-embedded breast cancer samples were analysed on tissue microarrays using mIF, which combined phospho-histone H3 (pHH3) expression with cytokeratin (CK) and leukocyte common antigen (CD45) expression to identify tumour and immune cells, respectively. RESULTS: Multivariate analysis showed that a high pHH3 index was associated with significantly improved overall survival (OS; p = 0.004), but this was not significantly associated with disease-free survival (DFS; p = 0.22). Similarly, multivariate analysis also revealed that a pHH3 positive count of > 1 cell per high-power field in the malignant epithelial compartment was an independent favourable prognostic marker for OS (p = 0.033) but not for DFS (p = 0.250). Furthermore, a high CD45 index was an independent favourable prognostic marker for DFS (p = 0.018), and there was a significant positive correlation between CD45 and pHH3 index (Spearman rank correlation coefficient, 0.250; p < 0.001). CONCLUSIONS: Mitotic rates as determined by pHH3 expression in epithelial cells are significantly associated with improved survival in TNBC. mIF analysis of pHH3 in combination with CK and CD45 could help clinicians in prognosticating patients with TNBC.

The role of Ki-67 in Asian triple negative breast cancers: a novel combinatory panel approach.

The proliferation marker Ki-67 is frequently used to assess aggressiveness in the pathological evaluation of cancer, but its role remains uncertain in triple-negative breast cancer (TNBC). We aimed to quantify and localize Ki-67 expression in both epithelial and immune compartments in TNBC and investigate its association with clinicopathological parameters and survival outcomes. A total of 406 TNBC cases diagnosed between 2003 and 2015 at Singapore General Hospital were recruited. Using state-of-the-art, 7-colour multiplex immunofluorescence (mIF) tissue microarrays (TMAs) were stained to assess the abundance, density and spatial distribution of Ki-67-positive tumour cells and immune cells co-decorated with cytokeratin (CK) and leukocyte common antigen (CD45) respectively. Furthermore, MKI67 mRNA profiles were analysed using NanoString technology. In multivariate analysis adjusted for tumour size, histologic grade, age at diagnosis, and lymph node stage, a high Ki-67 labelling index (LI) > 0.3% was associated with improved disease-free survival (DFS; HR = 0.727; p = 0.027). High Ki-67-positive immune cell count per TMA was a favourable prognostic marker for both DFS (HR = 0.379; p = 0.00153) and overall survival (OS; HR = 0.473; p = 0.0482). The combination of high Ki-67 LI and high MKI67 expression was associated with improved DFS (HR = 0.239; p = 0.00639) and OS (HR = 0.213; p = 0.034). This study is among the first to highlight that Ki-67 is associated with favourable prognosis in an adjuvant setting in TNBC, and the mIF-based evaluation of Ki-67 expression on both tumour and immune cells represents a novel prognostic approach.

Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer.

The role of programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) in triple negative breast cancer (TNBC) remains to be fully understood. In this study, we investigated the role of PD-1 as a prognostic marker for TNBC in an Asian cohort (n = 269). Samples from patients with TNBC were labeled with antibodies against PD-L1 and PD-1, and subjected to NanoString assays to measure the expression of immune-related genes. Associations between disease-free survival (DFS), overall survival (OS) and biomarker expression were investigated. Multivariate analysis showed that tumors with high PD-1+ immune infiltrates harbored significantly increased DFS, and this increase was significant even after controlling for clinicopathological parameters (HR 0.95; P = 0.030). In addition, the density of cells expressing both CD8 and PD-1, but not the density of CD8-PD-1+ immune infiltrates, was associated with improved DFS. Notably, this prognostic significance was independent of clinicopathological parameters and the densities of total CD8+ cell (HR 0.43, P = 0.011). At the transcriptional level, high expression of PDCD1 within the tumor was significantly associated with improved DFS (HR 0.38; P = 0.027). In line with these findings, high expression of IFNG (HR 0.38; P = 0.001) and IFN signaling genes (HR 0.46; p = 0.027) was also associated with favorable DFS. Inclusion of PD-1 immune infiltrates and PDCD1 gene expression added significant prognostic value for DFS (ΔLRχ2 = 6.35; P = 0.041) and OS (ΔLRχ2 = 9.53; P = 0.008), beyond that provided by classical clinicopathological variables. Thus, PD-1 mRNA and protein expression status represent a promising, independent indicator of prognosis in TNBC.