Effector mechanisms of cd8+ hla-dr+ t cells in breast cancer patients who respond to neoadjuvant chemotherapy

Cytotoxic T lymphocyte (CTLs) activation is an independent predictor of response to neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. Here, we go deeper into the function of CD8+ HLA-DR+T cells from NACT treated HER2 negative BC patients. Flow cytometry analysis revealed that CD8+ HLA-DR+ T cell percentage was increased in NACT responder (R) compared to non-responder (NR) patients. R patients with ER-/PR- hormone receptors had the highest CD8+HLA-DR+T cell frequencies, while no differences were found when patients were classified according to cancer stage or menopause status. Interestingly, the cytotoxicity and production of anti-tumor cytokines were enhanced when CD8+ HLA-DR+ T cells from healthy donors were cultured with plasma from R, but not from NR patients. The induced anti-tumor profile of CD8+ HLA-DR+ T cells was associated with plasmatic IL-12 and IFN-γ levels, increased cytokines in R patients. IL-12 or IFN-γ neutralization decreased cytotoxic activity and TNF-α production by cultured CD8+ HLA-DR+ T cells in R plasma presence. All these data suggest that an effective response to NACT in BC patients is associated with increased IL-12 or IFN-γ levels involved in the induction of cytotoxic and pro-inflammatory mechanisms in CD8+ HLA-DR+ T cells

Detoxifying antitumoral drugs via nanoconjugation: the case of gold nanoparticles and cisplatin

Nanoparticles (NPs) have emerged as a potential tool to improve cancer treatment. Among the proposed uses in imaging and therapy, their use as a drug delivery scaffold has been extensively highlighted. However, there are still some controversial points which need a deeper understanding before clinical application can occur. Here the use of gold nanoparticles (AuNPs) to detoxify the antitumoral agent cisplatin, linked to a nanoparticle via a pH-sensitive coordination bond for endosomal release, is presented. The NP conjugate design has important effects on pharmacokinetics, conjugate evolution and biodistribution and results in an absence of observed toxicity. Besides, AuNPs present unique opportunities as drug delivery scaffolds due to their size and surface tunability. Here we show that cisplatin-induced toxicity is clearly reduced without affecting the therapeutic benefits in mice models. The NPs not only act as carriers, but also protect the drug from deactivation by plasma proteins until conjugates are internalized in cells and cisplatin is released. Additionally, the possibility to track the drug (Pt) and vehicle (Au) separately as a function of organ and time enables a better understanding of how nanocarriers are processed by the organism.The authors acknowledge financial support from the grants “Plan Nacional” (MAT2009-14734-C02-01 and MAT2009-14734-C02-02) and NANOBIOMED-CONSOLIDER (CSD2006-00012) from the Spanish Government. Also grants VALTEC09-2-0085, VALTEC09-2-0089, and 2009-SGR-776 from the Catalan GovernmentS

Epigenetic inactivation of the putative DNA/RNA helicase SLFN11 in human cancer confers resistance to platinum drugs

Platinum-derived drugs such as cisplatin and carboplatin are among the most commonly used cancer chemotherapy drugs, but very few specific molecular and cellular markers predicting differential sensitivity to these agents in a given tumor type have been clearly identified. Epigenetic gene silencing is increasingly being recognized as a factor conferring distinct tumoral drug sensitivity, so we have used a comprehensive DNA methylation microarray platform to interrogate the widely characterized NCI60 panel of human cancer cell lines with respect to CpG methylation status and cisplatin/carboplatin sensitivity. Using this approach, we have found promoter CpG island hypermethylation-associated silencing of the putative DNA/RNA helicase Schlafen-11 (SLFN11) to be associated with increased resistance to platinum compounds. We have also experimentally validated these findings in vitro. In this setting, we also identified the BRCA1 interacting DHX9 RNA helicase (also known as RHA) as a protein partner for SLFN11, suggesting a mechanistic pathway for the observed chemoresistance effect. Most importantly, we have been able to extend these findings clinically, following the observation that those patients with ovarian and non-small cell lung cancer carrying SLFN11 hypermethylation had a poor response to both cisplatin and carboplatin treatments. Overall, these results identify SLFN11 epigenetic inactivation as a predictor of resistance to platinum drugs in human cancer

Circulating microRNAs in Early Breast Cancer Patients and Its Association With Lymph Node Metastases

MicroRNAs have emerged as important regulators of the metastatic process. In addition, circulating miRNAs appear to be surprisingly stable in peripheral blood making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to investigate the expression profile of circulating miRNAs and their association with the metastatic lymph node status in early breast cancer patients. Sentinel lymph node status was detected by one-step nucleic acid (OSNA) analysis. We performed RNA-sequencing in 16 plasma samples and validated the results by qPCR. Gene Ontology term enrichment and KEGG pathway analyses were carried out using DAVID tools. We found16 differentially expressed miRNAs (q < 0.01) in patients with positive SLNs. Fourteen miRNAs were down-regulated (miR-339-5p, miR-133a-3p, miR-326, miR-331-3p, miR-369-3p, miR-328-3p, miR-26a-3p, miR-139-3p, miR-493-3p, miR-664a-5p, miR-146a-5p, miR-323b-3p, miR-1307-3p and miR-423-3p) and 2 were up-regulated (miR-101-3pand miR-144-3p). Hierarchical clustering using differentially expressed miRNAs clearly distinguished patients according to their lymph node status. Gene ontology analysis showed a significant enrichment of biological processes associated with the regulation of the epithelial mesenchymal transition, cell proliferation and transcriptional regulation. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further validation in a larger cohort of patients will be necessary to confirm our results

Role of cooperative groups and funding source in clinical trials supporting guidelines for systemic therapy of breast cancer

Clinical research is conducted by academia, cooperative groups (CGs) or pharmaceutical industry. Here, we evaluate the role of CGs and funding sources in the development of guidelines for breast cancer therapies. We identified 94 studies. CGs were involved in 28 (30%) studies while industry either partially or fully sponsored 64 (68%) studies. The number of industry funded studies increased over time (from 0% in 1976 to 100% in 2014; p for trend = 0.048). Only 10 (11%) government or academic studies were identified. Studies conducted by GCs included a greater number of subjects (median 448 vs. 284; p = 0.015), were more common in the neo/adjuvant setting (p < 0.0001), and were more often randomized (p = 0.018) phase III (p < 0.0001) trials. Phase III trial remained significant predictor for CG-sponsored trials (OR 7.1 p = 0.004) in a multivariable analysis. Industry funding was associated with higher likelihood of positive outcomes favoring the sponsored experimental arm (p = 0.013) but this relationship was not seen for CG-sponsored trials (p = 0.53). ASCO, ESMO, and NCCN guidelines were searched to identify systemic anti-cancer therapies for early-stage and metastatic breast cancer. Trial characteristics and outcomes were collected. We identified sponsors and/or the funding source(s) and determined whether CGs, industry, or government or academic institutions were involved. Chi-square tests were used for comparison between studies. Industry funding is present in the majority of studies providing the basis for which recommendations about treatment of breast cancer are made. Industry funding, but not CG-based funding, was associated with higher likelihood of positive outcomes in clinical studies supporting guidelines for systemic therapy

Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential : a molecular and clinicopathological study

Background: The metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas. Methods: We classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis. Results: Group-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported. Conclusions: Intratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions

Prescription refill, patient self-report and physician report in assessing adherence to oral endocrine therapy in early breast cancer patients: a restrospective cohort study in Catalonia, Spain.

AIMS: To compare different methods in order to assess adherence and persistence with oral endocrine therapy in women diagnosed with breast cancer (BC) in Catalonia. MATERIALS AND METHODS: This study covered all women newly diagnosed with stage I, II or IIIa BC and positive hormone receptors at six hospitals in Catalonia (Spain) in 2004. Adherence was assessed on the basis of physician report and patient self-report using a telephone questionnaire. Persistence was measured by refill prescriptions. We used the Kappa index to compare adherence measures and logistic regression to evaluate adherence-related risk factors. RESULTS: The study covered a total of 692 women. Adherence ranged from 92% (self-report) to 94.7% (physician report), depending on the measure used; persistence was 74.7% at 5 years of follow-up. Low concordance between measures was observed (Kappa range: 0.018-0.267). Patients aged 50-74 years showed higher adherence than those aged <50 years. Adherence was also associated with: adjuvant chemotherapy and sequential hormonal therapy. CONCLUSIONS: Concordance between the different measures was remarkably low, indicating the need for further research. Adherence is an issue in the management of BC patients taking oral drugs, and should be assessed in clinical practice

ASPN and GJB2 are implicated in the mechanisms of invasion of ductal breast carcinomas

The mechanism of progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) remains largely unknown. We compared gene expression in tumors with simultaneous DCIS and IDC to decipher how diverse proteins participate in the local invasive process. Twenty frozen tumor specimens with concurrent, but separated, DCIS and IDC were mi-crodissected and evaluated. Total RNA was extracted and microarray analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Microarray data were validated by quantitative real time reverse transcription-PCR (qRT-PCR) and immunohistochemistry. Controls included seven pure in situ carcinomas, eight fragments from normal breast tissue, and a series of mouse breast carcinomas (MMTV-PyMT). Fifty-six genes were differentially expressed between DCIS and IDC samples. The genes upregulated in IDC samples, and probably associated with invasion, were related to the ep-ithelial-mesenchymal transition (ASPN, THBS2, FN1, SPARC, and COL11A1), cellular adhesion (GJB2), cell motility and progression (PLAUR, PLAU, BGN, ADAMTS16, and ENPP2), extracellular matrix degradation (MMP11, MMP13, and MMP14), and growth/proliferation (ST6GAL2). qRT-PCR confirmed the expression patterns of ASPN, GJB2, ENPP2, ST6GAL2, and TMBS10. Expression of the ASPN and GJB2 gene products was detected by immunohistochemistry in invasive carcinoma foci. The association of GJB2 protein expression with invasion was con-firmed by qRT-PCR in mouse tumors (P < 0.05). Conclusions: The upregulation of ASPN and GJB2 may play important roles in local invasion of breast ductal carcinomas

Epigenetic inactivation of the putative DNA/RNA helicase SLFN11 in human cancer confers resistance to platinum drugs

Platinum-derived drugs such as cisplatin and carboplatin are among the most commonly used cancer chemotherapy drugs, but very few specific molecular and cellular markers predicting differential sensitivity to these agents in a given tumor type have been clearly identified. Epigenetic gene silencing is increasingly being recognized as a factor conferring distinct tumoral drug sensitivity, so we have used a comprehensive DNA methylation microarray platform to interrogate the widely characterized NCI60 panel of human cancer cell lines with respect to CpG methylation status and cisplatin/carboplatin sensitivity. Using this approach, we have found promoter CpG island hypermethylation-associated silencing of the putative DNA/RNA helicase Schlafen-11 (SLFN11) to be associated with increased resistance to platinum compounds. We have also experimentally validated these findings in vitro. In this setting, we also identified the BRCA1 interacting DHX9 RNA helicase (also known as RHA) as a protein partner for SLFN11, suggesting a mechanistic pathway for the observed chemoresistance effect. Most importantly, we have been able to extend these findings clinically, following the observation that those patients with ovarian and non-small cell lung cancer carrying SLFN11 hypermethylation had a poor response to both cisplatin and carboplatin treatments. Overall, these results identify SLFN11 epigenetic inactivation as a predictor of resistance to platinum drugs in human cancer

Chromosome 17 Centromere Duplication and Responsiveness to Anthracycline-Based Neoadjuvant Chemotherapy in Breast Cancer

Human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) genes have been proposed as predictive biomarkers of sensitivity to anthracycline chemotherapy. Recently, chromosome 17 centromere enumeration probe (CEP17) duplication has also been associated with increased responsiveness to anthracyclines. However, reports are conflicting and none of these tumor markers can yet be considered a clinically reliable predictor of response to anthracyclines. We studied the association of TOP2A gene alterations, HER2 gene amplification, and CEP17 duplication with response to anthracycline-based neoadjuvant chemotherapy in 140 patients with operable or locally advanced breast cancer. HER2 was tested by fluorescence in situ hybridization and TOP2A and CEP17 by chromogenic in situ hybridization. Thirteen patients (9.3%) achieved pathologic complete response (pCR). HER2 amplification was present in 24 (17.5%) of the tumors. TOP2A amplification occurred in seven tumors (5.1%). CEP17 duplication was detected in 13 patients (9.5%). CEP17 duplication correlated with a higher rate of pCR [odds ratio (OR) 6.55, 95% confidence interval (95% CI) 1.25-34.29, P =.026], and analysis of TOP2A amplification showed a trend bordering on statistical significance (OR 6.97, 95% CI 0.96-50.12, P =.054). TOP2A amplification and CEP17 duplication combined were strongly associated with pCR (OR 6.71, 95% CI 1.66-27.01, P =.007). HER2 amplification did not correlate with pCR. Our results suggest that CEP17 duplication predicts pCR to primary anthracycline-based chemotherapy. CEP17 duplication, TOP2A amplifications, and HER2 amplifications were not associated with prognosis