bromochloroacetic-acid and Triple-Negative-Breast-Neoplasms

The tumour-stroma ratio (TSR) is identified as a promising prognostic parameter for breast cancer, but the cutoff TSR value is mostly assessed by visual assessment, which lacks objective measurement. The aims of this study were to optimize the cutoff TSR value, and evaluate its prognosis value in patients with breast cancer both as continuous and categorical variables.. Major clinicopathological and follow-up data were collected for a series of patients with breast cancer. Tissue microarray images stained with cytokeratin immunohistochemistry were evaluated by automated quantitative image analysis algorithms to assess TSR. The potential cutoff point for TSR was optimized using maximally selected rank statistics. The association between TSR and 5-year disease-free survival (5-DFS) was assessed by Cox regression analysis. Kaplan-Meier analysis and log-rank test were used to assess the significance in survival analysis.. The optimal cut-off TSR value was 33.5%. Using this cut-off point, categorical variable analysis found that low TSR (i.e., high stroma, TSR ≤ 33.5%) predicts poor outcomes for 5-DFS (hazard ratio [HR] = 2.82, 95% confidence interval [CI] = 1.81-4.40, P = 0.000). When TSR was considered as a continuous parameter, results showed that increased stroma content was associated with worse 5-DFS (HR = 1.71, 95% CI = 1.34-2.18, P = 0.000). Similar results were also obtained in three molecular subtypes in continuous and categorical variable analyses. Moreover, in the Kaplan-Meier analysis, log-rank test showed that low TSR displayed a worse 5-DFS than high TSR (P = 0.000). Similar results were also obtained in patients with triple-negative breast cancer, human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and luminal-HER2-negative breast cancer.. TSR is an independent predictor for 5-DFS in breast cancer with worse survival outcomes in low TSR. The prognostic value of TSR was also observed in other three molecular subtypes.

Topics: Disease-Free Survival; Humans; Keratins; Prognosis; Stromal Cells; Triple Negative Breast Neoplasms

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.

Topics: Adult; Aged; Aged, 80 and over; Asia; Biomarkers, Tumor; Disease-Free Survival; Female; Humans; Keratins; Ki-67 Antigen; Lymph Nodes; Middle Aged; Prognosis; Receptors, Progesterone; Triple Negative Breast Neoplasms

Circulating tumor cells (CTCs) represent a temporal "snapshot" of a patient's cancer and changes that occur during disease evolution. There is an extensive literature studying CTCs in breast cancer patients, and particularly in those with metastatic disease. In parallel, there is an increasing use of patient-derived models in preclinical investigations of human cancers. Yet studies are still limited demonstrating CTC shedding and metastasis formation in patient-derived models of breast cancer.. We used seven patient-derived orthotopic xenograft (PDOX) models generated from triple-negative breast cancer (TNBC) patients to study CTCs and distant metastases. Tumor fragments from PDOX tissue from each of the seven models were implanted into 57 NOD scid gamma (NSG) mice, and tumor growth and volume were monitored. Human CTC capture from mouse blood was first optimized on the marker-agnostic Vortex CTC isolation platform, and whole blood was processed from 37 PDOX tumor-bearing mice.. Staining and imaging revealed the presence of CTCs in 32/37 (86%). The total number of CTCs varied between different PDOX tumor models and between individual mice bearing the same PDOX tumors. CTCs were heterogeneous and showed cytokeratin (CK) positive, vimentin (VIM) positive, and mixed CK/VIM phenotypes. Metastases were detected in the lung (20/57, 35%), liver (7/57, 12%), and brain (1/57, less than 2%). The seven different PDOX tumor models displayed varying degrees of metastatic potential, including one TNBC PDOX tumor model that failed to generate any detectable metastases (0/8 mice) despite having CTCs present in the blood of 5/5 tested, suggesting that CTCs from this particular PDOX tumor model may typify metastatic inefficiency.. PDOX tumor models that shed CTCs and develop distant metastases represent an important tool for investigating TNBC.

Topics: Animals; Biomarkers, Tumor; Brain; Cell Count; Cell Line, Tumor; Female; Humans; Keratins; Liver; Lung; Mammary Neoplasms, Experimental; Mice; Mice, Mutant Strains; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Cells, Circulating; Triple Negative Breast Neoplasms; Vimentin

Cancer cells can exhibit altered dependency on specific metabolic pathways and targeting these dependencies is a promising therapeutic strategy. Triple-negative breast cancer (TNBC) is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify metabolic pathway dependencies in TNBC, we first conducted mass spectrometry-based metabolomics of TNBC and control cells. Relative levels of intracellular metabolites distinguished TNBC from nontransformed breast epithelia and revealed two metabolic subtypes within TNBC that correlate with markers of basal-like versus non-basal-like status. Among the distinguishing metabolites, levels of the cellular redox buffer glutathione were lower in TNBC cell lines compared to controls and markedly lower in non-basal-like TNBC. Significantly, these cell lines showed enhanced sensitivity to pharmacologic inhibition of glutathione biosynthesis that was rescued by N-acetylcysteine, demonstrating a dependence on glutathione production to suppress ROS and support tumor cell survival. Consistent with this, patients whose tumors express elevated levels of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, had significantly poorer survival. We find, further, that agents that limit the availability of glutathione precursors enhance both glutathione depletion and TNBC cell killing by γ-glutamylcysteine ligase inhibitors

Topics: Animals; Biosynthetic Pathways; Cell Line, Tumor; Female; Glutathione; Heterografts; Humans; Keratins; Mice; Mice, Inbred NOD; Molecular Targeted Therapy; Reactive Oxygen Species; RNA, Small Interfering; Transfection; Triple Negative Breast Neoplasms

Cytokeratin 5/6 and Cytokeratin 8/18 are basal and luminal markers of breast cancer and they have pathological and prognostic significance in breast cancer. We performed Cytokeratin 5/6 and CK8/18 immunohistochemistry on 150 cases of triple negative breast cancers and association with various clinicopathological features was evaluated.. Positive CK5/6 expression was noted in 8% (12 cases) of TNBC while 2.4% (4 cases) showed focal positive (< 10%) and 89.3% (134) were negative with CK5/6. Complete loss of CK8/18 expression was seen in 4.7% (7 cases) while 32.7% (49 cases) revealed focal loss of CK8/18 and 62.7% (94 cases) showed intact normal expression of CK8/18. No significant association of CK5/6 and CK8/18 with various clinicopathological parameters was observed. We found a low expression of basal cytokeratin (CK5/6) in TNBC our studied population, while loss/altered expression of CK8/18 in approximately 38% of TNBC. Although no prognostic relevance of these finding was noted in our study, however these findings are different from those reported in literature in other parts of the world. Therefore we suggest a more through immunohistochemical and genomic profiling of TNBC in our population for better understanding of this disease in this part of the world.

Topics: Adult; Asia; Female; Humans; Keratins; Middle Aged; Triple Negative Breast Neoplasms

Differentiation events contribute to phenotypic cellular heterogeneity within tumors and influence disease progression and response to therapy. Here, we dissect mechanisms controlling intratumoral heterogeneity within triple-negative basal-like breast cancers. Tumor cells expressing the cytokeratin K14 possess a differentiation state that is associated with that of normal luminal progenitors, and K14-negative cells are in a state closer to that of mature luminal cells. We show that cells can transition between these states through asymmetric divisions, which produce one K14

Topics: Animals; Asymmetric Cell Division; Cell Line, Tumor; Cells, Cultured; Enhancer of Zeste Homolog 2 Protein; Female; Hepatocyte Nuclear Factor 3-alpha; Humans; Keratins; Mice; Receptors, Notch; Triple Negative Breast Neoplasms

Triple negative breast cancers (TNBCs) are known to express low PGR, ESR1, and ERBB2, and high KRT5, KRT14, and KRT17. However, the reasons behind the increased expressions of KRT5, KRT14, KRT17 and decreased expressions of PGR, ESR1, and ERBB2 in TNBCs are not fully understood. Here we show that, expression of chromosome 19 miRNA cluster (C19MC) specifically marks human TNBCs. Low REST and high CEBPB correlate with expression of C19MC, KRT5, KRT14, and KRT17 and enhancers of these genes/cluster are regulated by CEBPB and REST binding sites. The C19MC miRNAs in turn can potentially target REST to offer a positive feedback loop, and might target PGR, ESR1, ERBB2, GATA3, SCUBE2, TFF3 mRNAs to contribute towards TNBC phenotype. Thus our study demonstrates that C19MC miRNA expression marks TNBCs and that C19MC miRNAs and CEBPB might together determine the TNBC marker expression pattern.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Chromosomes, Human, Pair 19; CpG Islands; DNA Methylation; Down-Regulation; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Humans; Keratins; MicroRNAs; Multigene Family; Repressor Proteins; RNA, Messenger; Triple Negative Breast Neoplasms

Topics: Breast Neoplasms; Carcinoma; Carcinoma, Ductal, Breast; Diagnosis, Differential; Female; Fibrocystic Breast Disease; Humans; Immunohistochemistry; Keratins; Neoplasm Proteins; Neoplasm Recurrence, Local; S100 Proteins; Triple Negative Breast Neoplasms

Triple-negative breast carcinomas (TNBCs) correspond to a molecular heterogeneous disease defined by lack of estrogen and progesterone receptor expression, and the absence of overexpression and/or amplification of HER2. Recent data indicate that clinical outcome in TNBC is affected by tumor-infiltrating lymphocytes, suggesting that they can benefit from immunotherapies. We selected 116 consecutive premenopausal patients with TNBC to compare the immunohistochemical profile of the group rich in tumor-infiltrating lymphocytes with those without this characteristic.. We reviewed all the original histological sections to assess pathological features, and to select a representative area for tissue microarrays and immunohistochemical study. Estrogen and progesterone receptors, HER2 and Ki-67 were evaluated in whole histological sections. The following markers were analyzed in tissue microarrays sections: androgen receptor, cytokeratin 5/6, cytokeratin 14, epidermal growth factor receptor (EGFR), vimentin, p16, claudin-3, -4, and -7, p63, and aldehyde dehydrogenase isoform 1 (ALDH1). Lymphocyte-predominant breast cancer (LPBC) was defined by the presence of more than 50% of lymphocytes in the intratumoral stroma.. Twenty-six (22.4%) patients present tumors classified as LPBC and 90 (77.6%) as non-LPBC. The two groups were similar regarding age of patients, tumor grade and Ki-67 positive cells. LPBC cases presented lower frequency of expression of the basal cytokeratins, EGFR, and basal-like immunoprofile. There was a trend to higher expression of ALDH1 by stromal intratumoral cells. The expression of all other markers were similar in the two groups.. Lymphocyte-predominant TNBC in premenopausal patients are mostly of non-basal phenotype.

Topics: Adult; Aldehyde Dehydrogenase 1 Family; Biomarkers, Tumor; Brazil; Carcinoma; Claudins; Cyclin-Dependent Kinase Inhibitor p16; ErbB Receptors; Female; Humans; Immunohistochemistry; Isoenzymes; Keratins; Lymphocytes, Tumor-Infiltrating; Membrane Proteins; Middle Aged; Premenopause; Receptors, Androgen; Retinal Dehydrogenase; Retrospective Studies; Triple Negative Breast Neoplasms; Vimentin

Patients with triple negative breast cancer (TNBC), are considered as a poor prognosis group for whom no targeted therapies are currently available. The aim of the present study was to phenotypically characterize their CTCs in order to explore potential therapeutic targets.. PBMC's cytospins were prepared from 45 early (before and after adjuvant chemotherapy), 10 metastatic TNBC and 21 hormone receptor (HR) -positive patients. The expression of Cytokeratins (CK), ER, PR, EGFR and HER2 on CTCs was assessed using immunofluoresence staining and ARIOL analysis.. In early stage TNBC, ER, PR, HER2 and EGFR expressing-CTCs were detected in 24.4%, 24.4%, 20% and 40% of patients before the initiation of adjuvant chemotherapy, and in 17.8%, 13.3% 6.7% and 51.1% respectively after the completion of adjuvant treatment. Triple staining experiments revealed distinct subpopulations of CTC expressed HR, and ErbB family receptors. In patients with metastatic disease, the frequency of HER2+ CTCs was significantly increased compared to adjuvant setting (60% vs 20%, p=0.014). The presence of CK+PR- CTCs, before adjuvant treatment was associated with reduced OS (p=0.032) and DFI (p=0.04). Furthermore, the frequency of ER-, PR- and HER2+ CTCs was higher in HR(+) than in TNBC tumors (57.1%, p=0.006; 52.4%, p=0.021 and 52.38%, p=0.009, respectively).. The CTCs in patients with early TNBC are phenotypically heterogeneous based on the expression of HR, EGFR and HER2 both before and after the completion of adjuvant chemotherapy whereas the presence of HER2+ CTCs prevails during disease evolution. These findings could be of clinical relevance in terms of CTC targeting.

Topics: Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Chemotherapy, Adjuvant; ErbB Receptors; Female; Humans; Keratins; MCF-7 Cells; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Phenotype; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Triple Negative Breast Neoplasms

Little is known about the biology, molecular profile and hence optimal treatment of African Nigerian breast cancer. The aim of this work, therefore, was to characterize the histology and molecular profile of Nigerian breast cancer.. Breast carcinomas from women at 6 centres of similar tribal origin in Nigeria were reviewed and assembled into tissue microarrays (TMAs), and sections were stained for hormone receptors, i.e. estrogen receptor (ER)α, ERβ1, ERβ progesterone receptor (PR) and androgen receptor, cyclin D, HER2, Ki67 and cytokeratins (CKs), i.e. CK5/6 and CK14 (basal) and CK18 and 19 (luminal).. A total of 835 tumours were analysed. The mean age at diagnosis was 48.62 ± 12.41 years. The most common histological subtype was ductal NST (no-special-type) carcinoma (87.3%). Over 90% of the tumours were grade 2 or 3. The predominant molecular phenotype was the non-basal, triple-negative type (47.65%) followed by the HER2-positive group (19.6%). The percentage of ER-, PR- and HER2-positive tumours was 22.4, 18.9 and 18.8%, respectively.. Nigerian breast cancer predominantly has a high-grade, triple-negative profile. It occurs at a younger age and bears similarities at the molecular level to pre-menopausal breast cancer in white women, with remarkably lower levels of ERβ expression. The early presentation and histological and molecular phenotype may explain the poor prognosis, and tailoring treatment strategies to target this unique profile are required.

Topics: Adult; Age Factors; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Middle Aged; Nigeria; Phenotype; Prognosis; Receptor, ErbB-2; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Tissue Array Analysis; Triple Negative Breast Neoplasms

Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.

Topics: Antigens, CD; Cadherins; Carcinoma; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; CRISPR-Cas Systems; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Humans; Keratins; Neoplasm Proteins; Phenotype; Protein Isoforms; Transcription Factors; Triple Negative Breast Neoplasms; Tumor Suppressor Proteins

Routine Progesterone and Estrogen hormone receptor proteins and human epidermal growth factor receptor 2 (HER-2) analysis on invasive breast carcinomas provide therapeutic and prognostic values, revealing significant subgroups: luminal A, luminal B, HER-2 and the "triple negative" tumors. The aim of this study was to determine the expression of basal cytokeratins and Epidermal Growth Factor Receptor in "triple negative" invasive breast carcinomas in Puerto Rico women.. All invasive breast carcinoma cases received from 2008 to 2010 were included. Assessment of tumoral expression of Estrogen Receptor, Progesterone Receptor and HER-2 was performed. The cases were divided into groups based on their molecular categories and analyzed according to the age. "Triple negative" tumors were further analyzed according to their expression of Epidermal Growth Factor Receptor and cytokeratins 5/6 and 14.. From 717 cases reviewed, 487 cases of invasive breast carcinoma were included. The molecular categories were 66%, 10%, 9% and 15% for the luminal A, luminal B, Her-2 and "triple negative" groups, respectively. No significant difference (p= 0.64) was observed between the molecular categories and the age of the patients. Assessment of basal cytokeratins and Epidermal Growth Factor Receptor expression was performed on 41 "triple negative" tumors; 71% expressed at least one basal cytokeratin or Epidermal Growth Factor Receptor and 29% were negative to all markers.. Prevalence and relation between the molecular categories and the expression of basal cytokeratins in "triple negative" tumors in our population is comparable to other published data.

Topics: Adult; Aged; Biomarkers, Tumor; ErbB Receptors; Female; Humans; Keratins; Middle Aged; Neoplasm Invasiveness; Puerto Rico; Retrospective Studies; Triple Negative Breast Neoplasms

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor(+) breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.

Topics: Aged; Animals; Autoantibodies; Autoimmunity; Blotting, Western; BRCA1 Protein; Cell Line, Tumor; Disease Models, Animal; Female; Glycolysis; Humans; Immunoglobulin G; Keratins; Mass Spectrometry; Mice; Middle Aged; Proteome; Proteomics; Spliceosomes; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53

To investigate the association between PTEN loss and IGFBP2 expression in a series of triple-negative breast cancers and to relate this expression to basal cytokeratin expression and clinicopathologic features.. One hundred and one formalin-fixed and paraffin-processed triple-negative breast cancer cases from the University of Malaya Medical Centre were tested immunohistochemically for cytokeratins 5/6 and 14, PTEN, and IGFBP2. The resulting slides were scored for proportion and intensity of staining.. Loss of tumor nuclear and cytoplasmic staining for PTEN occurred in 48.3% of cases and was significantly associated with younger age at diagnosis (47 years compared with 57 years in those without PTEN loss; P = .005). Independent predictors of PTEN loss were late stage at presentation (P = .026), cytokeratin 5/6 positivity (P = .028), and IGFBP2 expression (P = .042). High levels of IGFBP2 expression were seen in 32% of cases; an independent predictor of high levels was cytokeratin 14 negativity (P = .005). PTEN loss and high levels of IGFBP2 expression were associated with poorer survival, but neither of these trends was significant.. PTEN loss is a frequent event in triple-negative breast cancers and is significantly associated with younger age at onset of breast cancer, late stage, and IGFBP2 expression.

Topics: Adult; Age Factors; Aged; Disease Progression; Female; Humans; Insulin-Like Growth Factor Binding Protein 2; Keratins; Middle Aged; Neoplasm Staging; Prognosis; PTEN Phosphohydrolase; Survival Rate; Triple Negative Breast Neoplasms