The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women.

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non-small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

Human epidermal growth factor receptor 2 (HER2) status in breast cancer: practice points and challenges.

Human epidermal growth factor receptor 2 (HER2)-enriched breast cancer benefits significantly from anti-HER2 targeted therapies. This highlights the critical need for precise HER2 immunohistochemistry (IHC) interpretation serving as a triage tool for selecting patients for anti-HER2 regimens. Recently, the emerging eligibility of patients with HER2-low breast cancers for a novel HER2-targeted antibody-drug conjugate (T-DXd) adds challenges to HER2 IHC scoring interpretation, notably in the 0-1+ range, which shows high interobserver and interlaboratory staining platform variability. In this review, we navigate evolving challenges and suggest practical recommendations for HER2 IHC interpretation.

Outcomes in Nonmetastatic Hormone Receptor-Positive HER2-Negative Pure Mucinous Breast Cancer: A Multicenter Cohort Study.

BACKGROUND: Although considered a favorable subtype, pure mucinous breast cancer (PMBC) can recur, and evidence for adjuvant therapy is limited. We aimed to compare outcomes of nonmetastatic PMBC with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) to address these uncertainties. METHODS: Individual patient-level data from 6 centers on stage I-III hormone receptor-positive and HER2-negative PMBC, IDC, and ILC were used to analyze recurrence-free interval (RFI), recurrence-free survival (RFS), and overall survival (OS), and to identify prognostic factors for PMBC. RESULTS: Data from 20,684 IDC cases, 1,475 ILC cases, and 943 PMBC cases were used. Median follow-up was 6.6 years. Five-year RFI, RFS, and OS for PMBC were 96.1%, 94.9%, and 98.1%, respectively. On multivariable Cox regression, PMBC demonstrated superior RFI (hazard ratio [HR], 0.59; 95% CI, 0.43-0.80), RFS (HR, 0.70; 95% CI, 0.56-0.89), and OS (HR, 0.71; 95% CI, 0.53-0.96) compared with IDC. ILC showed comparable outcomes to IDC. Fewer than half (48.7%) of recurrences in PMBC were distant, which was a lower rate than for IDC (67.3%) and ILC (80.6%). In contrast to RFI, RFS events were driven more by non-breast cancer deaths in older patients. Significant prognostic factors for RFI among PMBC included positive lymph node(s) (HR, 2.42; 95% CI, 1.08-5.40), radiotherapy (HR, 0.44; 95% CI, 0.23-0.85), and endocrine therapy (HR, 0.25; 95% CI, 0.09-0.70). No differential chemotherapy associations with outcomes were detected across PMBC subgroups by nodal stage, tumor size, and age. A separate SEER database analysis also did not find any association of improved survival with adjuvant chemotherapy in these subgroups. CONCLUSIONS: Compared with IDC, PMBC demonstrated superior RFI, RFS, and OS. Lymph node negativity, adjuvant radiotherapy, and endocrine therapy were associated with superior RFI. Adjuvant chemotherapy was not associated with better outcomes.

Early Triple-Negative Breast Cancers in a Singapore Cohort Exhibit High PIK3CA Mutation Rates Associated With Low PD-L1 Expression.

Mutations in the PI3K pathway, particularly PIK3CA, were reported to be intimately associated with triple-negative breast cancer (TNBC) progression and the development of treatment resistance. We profiled PIK3CA and other genes on 166 early-stage TNBC tumors from Singapore for comparison to publicly available TNBC cohorts. These tumors were profiled transcriptionally using a NanoString panel of immune genes and multiplex immunohistochemistry, then manually scored for PD-L1-positivity using 2 clinically relevant clones, SP142 and 22C3. We discovered a higher rate of PIK3CA mutations in our TNBC cohort than in non-Asian cohorts, along with TP53, BRCA1, PTPN11, and MAP3K1 alterations. PIK3CA mutations did not affect overall or recurrence-free survival, and when compared with PIK3CAWT tumors, there were no differences in immune infiltration. Using 2 clinically approved antibodies, PIK3CAmut tumors were associated with PD-L1 negativity. Analysis of comutation frequencies further revealed that PIK3CA mutations tended to be accompanied by MAP kinase pathway mutation. The mechanism and impact of PIK3CA alterations on the TNBC tumor immune microenvironment and PD-L1 positivity warrant further study.

Hypoxia-regulated carbonic anhydrase IX (CAIX) protein is an independent prognostic indicator in triple negative breast cancer.

BACKGROUND: The effect of extracellular microenvironment (hypoxia and pH) has been regarded as a key hallmark in cancer progression. The study aims to investigate the effects of carbonic anhydrase IX (CAIX), a key hypoxia-inducible marker, in triple-negative breast cancer (TNBC) in correlation with clinicopathological parameters and predicting survival outcomes. METHODS: A total of 323 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital from 2003 to 2013 were used. Immunohistochemical staining (IHC) was performed using CAIX antibody and digital mRNA quantification was performed using NanoString assays. CAIX membranous expression was correlated with clinicopathological parameters using Chi-squared test or Fisher's exact tests. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test. RESULTS: Forty percent of TNBCs were observed to express CAIX protein and demonstrated significant association with larger tumour size (P = 0.002), higher histological grade (P < 0.001), and significantly worse disease-free survival (DFS) and overall survival (OS) (after adjustment: HR = 2.99, 95% CI = 1.78-5.02, P < 0.001 and HR = 2.56, 95% CI = 1.41-4.65, P = 0.002, respectively). Gene ontology enrichment analysis revealed six significantly enriched cellular functions (secretion, cellular component disassembly, regulation of protein complex assembly, glycolytic process, cellular macromolecular complex assembly, positive regulation of cellular component biogenesis) associated with genes differentially expressed (CAIX, SETX, WAS, HK2, DDIT4, TUBA4α, ARL1). Three genes (WAS, SETX and DDIT4) were related to DNA repair, indicating that DNA stability may be influenced by hypoxia in TNBC. CONCLUSIONS: Our results demonstrate that CAIX appears to be a significant hypoxia-inducible molecular marker and increased CAIX protein levels are independently associated with poor survival in TNBC. Identification of CAIX-linked seven gene-signature and its relationship with enriched cellular functions further support the implication and influence of hypoxia-mediated CAIX expression in TNBC tumour microenvironment.

Quantitative stain-free imaging and digital profiling of collagen structure reveal diverse survival of triple negative breast cancer patients.

BACKGROUND: Stromal and collagen biology has a significant impact on tumorigenesis and metastasis. Collagen is a major structural extracellular matrix component in breast cancer, but its role in cancer progression is the subject of historical debate. Collagen may represent a protective layer that prevents cancer cell migration, while increased stromal collagen has been demonstrated to facilitate breast cancer metastasis. METHODS: Stromal remodeling is characterized by collagen fiber restructuring and realignment in stromal and tumoral areas. The patients in our study were diagnosed with triple-negative breast cancer in Singapore General Hospital from 2003 to 2015. We designed novel image processing and quantification pipelines to profile collagen structures using numerical imaging parameters. Our solution differentiated the collagen into two distinct modes: aggregated thick collagen (ATC) and dispersed thin collagen (DTC). RESULTS: Extracted parameters were significantly associated with bigger tumor size and DCIS association. Of numerical parameters, ATC collagen fiber density (CFD) and DTC collagen fiber length (CFL) were of significant prognostic value for disease-free survival and overall survival for the TNBC patient cohort. Using these two parameters, we built a predictive model to stratify the patients into four groups. CONCLUSIONS: Our study provides a novel insight for the quantitation of collagen in the tumor microenvironment and will help predict clinical outcomes for TNBC patients. The identified collagen parameters, ATC CFD and DTC CFL, represent a new direction for clinical prognosis and precision medicine. We also compared our result with benign samples and DICS samples to get novel insight about the TNBC heterogeneity. The improved understanding of collagen compartment of TNBC may provide insights into novel targets for better patient stratification and treatment.

Tertiary lymphoid structures and associated plasma cells play an important role in the biology of triple-negative breast cancers.

PURPOSE: Triple-negative breast cancers (TNBC) are aggressive tumours that exhibit abundant lymphoid infiltrates which modulate tumour behaviour. Recent findings suggest that TNBC with higher densities of plasma cells are associated with a favourable prognosis, and tertiary lymphoid structures (TLS) have prognostic significance. Here, we studied the phenotype and function of plasma cells in TNBCs by assessing their association with IgG Kappa light chain expression, B cells, and TLS. METHODS: A retrospective analysis of 269 TNBC cases was performed. Tumour-infiltrating CD38+ plasma cells, CD20+ B cells, and TLS were evaluated on conventional haematoxylin-eosin-stained and immunohistochemical-stained sections of TNBC. We then selected TNBC cases demonstrating the highest and lowest densities of plasma cells, and examined their association with TLS, B cells, as well as immunoglobulin expression using Opal-Vectra multiplex immunofluorescence (IF). RESULTS: TNBC with high density of plasma cells showed significantly higher numbers of IgG Kappa+ CD38+ cells (p = 0.0089, p < 0.0001), and higher numbers of TLS (p < 0.0001), compared to TNBC with low density of plasma cells. TNBC with high density of plasma cells also showed higher numbers of CD20+ B cells in the tumour core (p < 0.0001), invasive margin (p < 0.0001), as well as stromal (p = 0.015) compartments. CONCLUSION: TNBC with high density of plasma cells are associated with higher numbers of IgG Kappa+ CD38+ cells, CD20+ B cells, and TLS. Further studies to characterize the function of plasma cell infiltrates and how they may interact with other tumour-infiltrating lymphocytes and TLS in TNBC may help improve existing immunotherapy strategies.

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma.

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

Grit protects medical students from burnout: a longitudinal study.

BACKGROUND: Burnout is a serious issue plaguing the medical profession with potential negative consequences on patient care. Burnout symptoms are observed as early as medical school. Based on a Job Demands-Resources model, this study aims to assess associations between specific job resources measured at the beginning of the first year of medical school with burnout symptoms occurring later in the first year. METHODS: The specific job resources of grit, tolerance for ambiguity, social support and gender were measured in Duke-NUS Medical School students at the start of Year 1. Students were then surveyed for burnout symptoms at approximately quarterly intervals throughout the year. Using high ratings of cynicism and exhaustion as the definition of burnout, we investigated the associations of the occurrence of burnout with student job resources using multivariable logistic regression analysis. RESULTS: Out of 59 students, 19 (32.2%) indicated evidence of burnout at some point across the first year of medical school. Stepwise multivariable logistic regression analysis identified grit as having a significant protective effect against experiencing burnout (Odds Ratio, 0.84; 95%CI 0.74 to 0.96). Using grit as a single predictor of burnout, area under the ROC curve was 0.76 (95%CI: 0.62 to 0.89). CONCLUSIONS: Grit was identified as a protective factor against later burnout, suggesting that less gritty students are more susceptible to burnout. The results indicate that grit is a robust character trait which can prognosticate burnout in medical students. These students would potentially benefit from enhanced efforts to develop grit as a personal job resource.

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.

PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing l-asparaginase.

Natural killer (NK)/T-cell lymphomas failing L-asparaginse regimens have no known salvage and are almost invariably fatal. Seven male patients with NK/T-cell lymphoma (median age, 49 years; range, 31-68 years) for whom a median of 2 (range, 1-5) regimens (including l-asparaginase regimens and allogeneic hematopoietic stem-cell transplantation [HSCT] in 2 cases) failed were treated with the anti-programmed death 1 (PD1) antibody pembrolizumab. All patients responded, according to various clinical, radiologic (positron emission tomography), morphologic, and molecular (circulating Epstein-Barr virus [EBV] DNA) criteria. Two patients achieved complete response (CR) in all parameters. Three patients achieved clinical and radiologic CRs, with two having molecular remission (undetectable EBV DNA) but minimal EBV-encoded RNA-positive cells in lesions comprising predominantly CD3+CD4+ and CD3+CD8+ T cells (which ultimately disappeared, suggesting they represented pseudoprogression) and one having detectable EBV DNA despite morphologic CR. Two patients achieved partial response (PR). After a median of 7 (range, 2-13) cycles of pembrolizumab and a follow-up of a median of 6 (range, 2-10) months, all five CR patients were still in remission. The only adverse event was grade 2 skin graft-versus-host disease in one patient with previous allogeneic HSCT. Expression of the PD1 ligand was strong in 4 patients (3 achieving CR) and weak in 1 (achieving PR). PD1 blockade with pembrolizumab was a potent strategy for NK/T-cell lymphomas failing l-asparaginase regimens.

Higher densities of Foxp3+ regulatory T cells are associated with better prognosis in triple-negative breast cancer.

PURPOSE: The role of Forkhead Box Protein 3 (Foxp3) expressing regulatory T cells (Tregs) in breast cancer remains unclear. We examined the abundance and localisation of total T cells, B cells and Tregs within samples from triple-negative breast cancers (TNBCs) and asked whether these parameters were associated with clinicopathological features of the cancer or clinical outcomes. METHODS: Samples from TNBCs diagnosed between 2003 and 2010 in Singapore were divided into "high" and "low" intra-tumoural or stromal groups, based on whether they had higher or lower than median densities of specific tumour-infiltrating lymphocyte populations (CD3+ total T cells, Foxp3+CD3+ Tregs, or CD20+ B cells) in the intra-tumoural space or stroma. RESULTS: Of the 164 samples, patients bearing tumours with high Tregs within their intra-tumoural, but not stromal, areas experienced significantly longer overall and disease-free survival compared to individuals with low Treg densities. These "high intra-tumoural Treg" tumours were also characterised by relatively higher frequencies of CD8+ T cells and CD20+ B cells, and expressed significantly higher levels of some genes associated with inflammation, immune cell functions and trafficking, altogether consistent with a more "immune-activated" tumour microenvironment, in contrast to tumours bearing lower densities of Tregs. CONCLUSIONS: In summary, the combination of high densities of intra-tumoural Tregs, CD8+ T cells and CD20+ B cells represents a favourable prognostic panel in TNBCs. These data also indicate new avenues for further investigation on the interaction between immune cell types within the tumour microenvironment and highlight the potential of Treg density and localisation within tumours to affect clinical outcome.

A mussel-inspired double-crosslinked tissue adhesive on rat mastectomy model: seroma prevention and in vivo biocompatibility.

BACKGROUND: Seroma formation is a common postsurgical complication of breast cancer surgery. It delays wound healing and may lead to other more serious complications. Conventional methods of reducing seroma formation through suturing or placement of surgical drainage produce inconsistent clinical outcomes. Tissue adhesives are viable alternatives but most of them are unsuitable for internal use and for large-area applications because of weak tissue adhesion strength or biocompatibility issues. The aim of this study was to evaluate the efficacy and biocompatibility of a mussel-inspired double-crosslinked tissue adhesive (DCTA) in reducing seroma formation after mastectomy. MATERIALS AND METHODS: Thirty-six female Sprague-Dawley rats were randomly assigned to either the saline control group (n = 12), the TISSEEL sealant (Baxter) group (n = 12), or the DCTA group (n = 12). After performing a mastectomy and applying the corresponding treatment, the efficacy of DCTA was evaluated by measurement of seroma volume while its biocompatibility was assessed via micronuclei test and histopathologic examination. RESULTS: During the 1-wk postsurgical period, the average total seroma volume of DCTA was significantly lower than the saline control group. Importantly, the mean seroma volume in DCTA showed a decreasing trend, whereas those in TISSEEL and saline control groups showed otherwise. The application of DCTA showed no genotoxic effect on the host and no severe inflammation. CONCLUSIONS: This study demonstrates that the good tissue adhesion strength and stability of DCTA were successful in reducing seroma formation over a period of 1 wk. Furthermore, the results also showed that it is biocompatible, which makes it suitable for large-area, internal use.

Increased CD4 and CD8-positive T cell infiltrate signifies good prognosis in a subset of triple-negative breast cancer.

Tumour-infiltrating lymphocytes (TILs) signify immune response to tumour in a variety of cancers including breast cancer. However, earlier studies examining the clinical significance of TILs in breast cancers have generated mixed results. There are only a few that address the relationship between TILs and clinical outcomes in triple-negative breast cancers (TNBC). The aim of this study is to evaluate the clinical significance of TILs that express CD4 + and CD8 + , in TNBC. Immunohistochemical staining of CD4 and CD8 was performed on tissue microarrays of 164 cases of TNBC. TILs were counted separately as intratumoral when within the cancer cell nests (iTILs) and as stromal when within cancer stroma (sTILs). High CD8 + iTILs and sTILs, and CD4 + iTILs correlated with histologic grade. On Kaplan-Meier analysis, a significantly better survival rate was observed in high CD8 + iTIL (disease-free survival, DFS: P = 0.004, overall survival, OS: P = 0.02) and both high CD4 + iTILs (DFS: P = 0.025, OS: P = 0.023) and sTILs (DFS: P = 0.01, OS: P = 0.002). In multivariate analysis, CD8 + iTILs (DFS: P = 0.0095), CD4 + sTILs (DFS: P = 0.0084; OS: P = 0.0118), and CD4 (high) CD8 (high) CD8 iTILs (DFS: P = 0.0121; OS: P = 0.0329) and sTILs (DFS: P = 0.0295) showed significantly better survival outcomes. These results suggest that high levels of both CD8 + iTILs and CD4 + sTILs as well as CD4 (high) CD8 (high) iTILs and sTILs are independent prognostic factors in TNBC.

Increased ID4 expression, accompanied by mutant p53 accumulation and loss of BRCA1/2 proteins in triple-negative breast cancer, adversely affects survival.

AIMS: Breast cancer 1 (BRCA1) expression is down-regulated in a significant proportion of non-hereditary breast cancers, in the absence of any mutation. This phenomenon is more pronounced in oestrogen (ER)-negative tumours. Recent studies have suggested that inhibitor of DNA binding 4 (ID4), as well as p53, participate in the transcriptional regulation of BRCA1. METHODS: Immunohistochemical expression of ID4, BRCA1, BRCA2 and p53 in 699 women with triple-negative breast cancer was investigated using tissue microarrays. The prognostic role of these biomarkers was also evaluated. Survival outcomes were estimated with the Kaplan-Meier method and compared between groups with log-rank statistics. RESULTS: Loss of BRCA1 and BRCA2 expression and overexpression of ID4 and p53 was observed in 75%, 90%, 95% and 66% of tumours, respectively. ID4 expression was increased in higher tumour grade (P < 0.001) and was associated significantly with basal-like subtype (P < 0.001), BRCA2 down-regulation (P = 0.037) and p53 accumulation (P < 0.001). Patients with strong ID4 expression displayed worse disease-free survival in both triple-negative breast cancers (P = 0.041) and basal-like triple-negative breast cancers (P = 0.026). CONCLUSION: There is frequent ID4 expression and concomitant loss of BRCA proteins in triple-negative breast cancer. We hypothesize that strong ID4 expression could be useful as a prognostic marker in triple-negative breast cancer, predicting early tumour recurrence.

Role of epithelial-mesenchymal transition markers in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) are a heterogeneous group of breast tumours that are often associated with adverse pathological characteristics, poorer clinical outcomes and lack of targeted therapeutic options. Epithelial-mesenchymal transition, which plays a crucial role in tumour development and progression, is characterised by a transition from epithelial to mesenchymal phenotype and loss of proteins involved in maintaining cell junctions. We aimed to correlate protein expression of E-cadherin, Snail2 and transforming growth factor beta (TGF-ß) with clinicopathological parameters and survivals of a series of patients with TNBC. The study cohort comprised 767 TNBCs diagnosed at the Department of Pathology, Singapore General Hospital from 1994 to 2012. Immunohistochemistry was performed on sections cut from tissue microarrays using the polymeric method. Staining intensity and percentage of positive tumour cells were evaluated and correlated with clinicopathological findings and clinical outcomes. Loss of E-cadherin expression, Snail2 positivity, cytoplasmic and nuclear expression of TGF-ß were observed in 265 (35.2 %), 241 (32.0 %), 272 (36.2 %) and 262 (34.8 %) tumours, respectively. Histological grade significantly correlated with Snail2 positivity (P < 0.001) and loss of membranous E-cadherin expression (P = 0.003). Nuclear expression of TGF-ß was inversely correlated with histological grade (P = 0.010). Median follow-up was 73 months, with a maximum of 236 months. Despite a graphical curve for earlier recurrence in patients with tumours harbouring a combinational phenotype of loss of membranous E-cadherin and positive Snail2 expression, there was no statistical significance. Similarly for women with tumours expressing cytoplasmic TGF-ß, graphical representation showed poorer metastasis-free survival but without statistical significance. Loss of membranous E-cadherin and positive Snail2 expression are significantly associated with high-grade TNBCs. More work is needed to improve understanding of the role of TGF-ß in TNBC.

Characteristics and behaviour of screen-detected ductal carcinoma in situ of the breast: comparison with symptomatic patients.

Breast cancer is the most common malignancy in Singapore women. Ductal carcinoma in situ (DCIS) is the putative, non-obligate precursor of the majority of invasive breast cancers. The efficacy of the Singapore breast-screening pilot project in detecting early stage breast cancer led to the launch of a national breast-screening programme, BreastScreen Singapore (BSS), in January 2002. In this study, we compared clinicopathological and immunohistochemical characteristics, as well as clinical outcomes, between screen-detected and symptomatic DCIS. The study cohort comprised 1202 cases of DCIS diagnosed at Singapore General Hospital from 1994 to 2010. Comparison of clinicopathological parameters, immunohistochemical results of ER, PR, HER2, CK14, EGFR, and 34ßE12, and clinical outcomes was carried out between the 2 groups. Amongst 1202 cases, 610 (50.7%) were screen-detected and 592 (49.3%) were symptomatic DCIS. Screen-detected cases were smaller in size (P < 0.001), of lower nuclear grade (P = 0.004), and more frequently expressed ER (P < 0.001). Luminal A phenotype was more frequently observed in screen-detected DCIS, while triple-negative and HER2 phenotypes were more common in symptomatic DCIS (P < 0.001). The basal-like phenotype was also more frequent in symptomatic DCIS (P = 0.041). Mean and median follow-up was 99.7 and 97.8 months, respectively, with a maximum follow-up of 246.0 months. More symptomatic patients developed invasive recurrences compared to screen-detected patients (P = 0.001). A trend for better disease-free survival was observed in screen-detected patients (P = 0.076). Patients who were screen-detected experienced better overall survival than those with symptomatic DCIS (P = 0.007). Our data indicate a more favourable outcome of screen-detected DCIS patients confirming the role of BSS in early identification of this curable disease.

Loss of androgen receptor expression predicts early recurrence in triple-negative and basal-like breast cancer.

Treatment of triple-negative invasive breast cancers, defined by the absence of estrogen and progesterone receptors and c-erbB2 expression, remains challenging. Androgen receptor, a member of the nuclear receptor superfamily that is involved in signaling pathways regulating cell proliferation, has been implicated in breast tumorigenesis. We immunohistochemically examined the expression of androgen receptor, basal markers (CK14, 34ßE12) and EGFR in 699 triple-negative invasive breast cancers in tissue microarrays using the streptavidin-biotin method, and correlated the findings with clinical outcome. Positive androgen receptor expression was defined as staining of 1% or more of tumor cell nuclei. Survival outcomes were estimated with the Kaplan-Meier method and compared between groups with log-rank statistics. Cox proportional hazards models were used to determine the effect of androgen receptor on survival outcomes. Immunohistochemical positivity was observed in 38% of tumors, with the proportion of stained tumor cells ranging from 1 to 95% (mean 29%, median 10%). Androgen receptor expression was inversely associated with histologic grade and mitotic score. CK14, 34ßE12 and EGFR confirmed 85% of cases to be basal-like, without significant association of basal-like phenotype with androgen receptor expression. Disease-free survival was significantly better in androgen receptor-positive triple-negative breast cancer, with a trend for improved overall survival. Decreased recurrence likelihood in both triple-negative and basal-like tumors (hazard ratio, 0.704; 95% confidence intervals, 0.498-0.994; P=0.0464; and hazard ratio, 0.675; 95% confidence intervals, 0.468-0.974; P=0.0355, respectively) was noted within 5 years of diagnosis but not thereafter. Our study suggests that loss of androgen receptor in triple-negative breast cancers augurs a worse prognosis, including those with basal-like features. More work in elucidating its relationship with mechanisms of progression, as well as trials of targeted treatment for androgen receptor-expressing triple-negative tumors, needs to be performed.

Lysine-Specific Demethylase 1 (LSD1)-Mediated Epigenetic Modification of Immunogenicity and Immunomodulatory Effects in Breast Cancers.

Tumor evolution to evade immune surveillance is a hallmark of carcinogenesis, and the modulation of tumor immunogenicity has been a challenge to present therapeutic responses in immunotherapies alone for numerous cancers. By altering the cell phenotype and reshaping the tumor microenvironment, epigenetic modifications enable tumor cells to overcome immune surveillance as a mechanism of cancer progression and immunotherapy resistance. Demethylase enzymatic activity of lysine-specific demethylase 1 (LSD1), a histone demethylase first identified in 2004, plays a pivotal role in the vast cellular processes of cancer. While FDA-approved indications for epigenetic therapies are limited to hematological malignancies, it is imperative to understand how epigenetic machinery can be targeted to prime immunotherapy responses in breast cancers. In this review, we discuss the potential roles of epigenetics and demethylating agent LSD1 as a potent new cancer management strategy to combat the current challenges of breast cancers, which have presented modest efficacy to immune checkpoint inhibitors till date. Additionally, we describe the combined use of LSD1-specific inhibitors and immune checkpoint inhibitors in existing breast cancer preclinical and clinical trials that elicits a robust immune response and benefit. Overall, the promising results observed in LSD1-targeting therapies signify the central role of epigenetics as a potential novel strategy to overcome resistance commonly seen in immunotherapies.

Insulin growth factor receptor-1 expression and loss of PTEN protein predict early recurrence in triple-negative breast cancer.

AIMS: Insulin-like growth factor receptor-1 (IGFR-1) and its signalling axis promote tumorigenesis, metastasis, and resistance to existing forms of cancer therapy, and have become a major focus for the development of anticancer drugs. As oncological management options for triple-negative breast cancers (TNBCs) are limited, there is potential for the rapid development of novel selective anticancer agents specifically targeting components of the PTEN-phosphoinositide 3-kinase-AKT pathway, including the phosphorylated form of AKT (pAKT) and the tumour suppressor molecule PTEN. The aim of this study was to conduct immunohistochemical analyses to examine the levels of PTEN, IGFR-1 and pAKT expression in TNBCs, and determine whether these levels correlated with poor prognosis in this subset of aggressive breast cancers. METHODS AND RESULTS: Immunohistochemistry was performed on paraffin-embedded tumour tissues from a consecutive cohort of 144 female patients diagnosed with TNBC. Associations of IGFR-1, PTEN and pAKT expression with clinicopathological parameters, disease-free survival (DFS) and overall survival (OS) were evaluated. There were significant increases in IGFR-1 expression (99%) and pAKT expression (92%) with concomitant loss of PTEN expression in the majority of cases (63%). Increased IGFR-1 expression and loss of PTEN expression were associated with reduced OS and DFS, respectively. pAKT expression showed a strong correlation with basal-like expression. Combinatorial immunophenotypic analyses showed that loss of PTEN expression with concomitant IGFR-1 expression correlated with poor DFS. CONCLUSION: A high percentage of PTEN loss with overexpression of IGFR-1 and pAKT in TNBC indicates the potential of these molecules for predicting early recurrence and/or as targets in the formulation of effective alternative therapy regimens.