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

CADM1, MAL, and miR124 Promoter Methylation as Biomarkers of Transforming Cervical Intrapithelial Lesions

Background: Squamous intraepithelial lesions/cervical intraepithelial neoplasias (SIL/CIN) are high-risk human papilloma virus (hrHPV)-related lesions which are considered as high grade (HSIL/CIN2-3) or low grade (LSIL/CIN1) lesions according to their risk of progression to cervical cancer (CC). Most HSIL/CIN2-3 are considered as transforming hrHPV infections, so truly CC precursors, although some clear spontaneously. hrHPV testing has a high sensitivity for the detection of HSIL/CIN2-3 but a relatively low specificity for identifying transforming lesions. We aimed to determine whether the combination of CADM1, MAL and miR124 promoter methylation status assessed in histological samples can be used as a biomarker in the identification of transforming HSIL/CIN lesions. Design: 131 cervical biopsies, including 8 cases with no lesion and a negative hrHPV test result (control group), 19 low-grade (L)SIL/CIN1, 30 HSIL/CIN2, 60 HSIL/CIN3, and 14 CC were prospectively collected. hrHPV was detected and genotyped using the polymerase chain reaction (PCR)-based technique SPF10 HPV LIPA. A multiplex quantitative methylation-specific PCR (qMSP) was used to identify the methylation status of the CADM1, MAL, and miR124 promoter genes. Results: Significantly higher methylation levels of CADM1, MAL and miR-124 were found in HSIL/CIN2-3 and CC compared with normal and LSIL lesions. DNA methylation of at least one gene was detected in 12.5% (1/8) of normal samples, 31.5% (6/19) of LSIL/CIN1, 83.3% (25/30) of HSIL/CIN2, 81.6% (49/60) of HSIL/CIN3 and 100% (14/14) of CC (p < 0.001). The sensitivity and specificity for HSIL/CIN2-3 and CC of having at least one methylated gene were 84.6% and 74.0%, respectively. The sensitivity and specificity of the combination of at least one methylated gene and a positive hrHPV test were 80.7% and 85.1% for HSIL/CIN2-3 and CC, respectively. Conclusions: The methylation rate of CADM1, MAL and miR124 increases with the severity of the lesion. Further research is warranted to evaluate the usefulness of these biomarkers for the identification of transforming HSIL/CIN

Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea

Background: In northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013. Methods: A validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay. Results: CQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) &gt;100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were &lt;100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC50s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005-2007, the geometric mean (95% CI) CQ IC50 was lower (87 (71-107) vs 167 (141-197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted. Conclusions: Reflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC50s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12610000913077

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

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

HPV-negative tumors of the uterine cervix

Human papillomaviruses (HPV) are the causative agents of virtually all cervical carcinomas. Nevertheless, a small proportion of cervical cancer are negative for HPV, although the significance of this finding remains unclear. We aimed to provide insight into the differential clinico-pathological characteristics of this unusual subset of HPV-negative cervical cancer. We performed HPV-DNA detection using a highly sensitive PCR test (SPF10) and p16 immunostaining in 214 cervical carcinomas specimens from women treated at the Gynecological Oncology Unit of the Hospital Clinic (Barcelona, Spain) from 2012 to 2015. The clinical and pathological characteristics, including disease-free survival and overall survival, of HPV-negative and -positive cervical carcinomas were compared. Twenty-one out of 214 tumors (10%) were negative for HPV DNA. HPV-negative tumors were more frequently of the non-squamous type (9/21, 43% vs. 37/193, 19%; p\xE2\x80\x89<\xE2\x80\x890.01) and showed negative p16 staining (9/21; 43% vs. 7/193; 4%; p\xE2\x80\x89<\xE2\x80\x890.01). HPV-negative tumors were more frequently diagnosed at advanced FIGO stage (19/21, 91% vs. 110/193, 57%; p\xE2\x80\x89<\xE2\x80\x890.01) and more frequently had lymph node metastases (14/21, 67% vs. 69/193, 36%; p\xE2\x80\x89<\xE2\x80\x890.01). Patients with HPV-negative cervical cancer had a significantly worse disease-free survival (59.8 months, 95% confidence interval 32.0-87.6 vs. 132.2 months, 95% confidence interval 118.6-145.8; p\xE2\x80\x89<\xE2\x80\x890.01) and overall survival (77.0 months, 95% confidence interval 47.2-106.8 vs. 153.8 months, 95% confidence interval 142.0-165.6; p\xE2\x80\x89=\xE2\x80\x890.01) than women with HPV-positive tumors. However, only advanced FIGO stage and lymph node metastases remained associated with a poor disease-free survival and overall survival on multivariate analysis. In conclusion, our results suggest that a low percentage of cervical cancer arise via an HPV-independent pathway. These HPV-negative tumors are diagnosed at advanced stages, show higher prevalence of lymph nodes metastases and have an impaired prognosis

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

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

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

mRNA Detection in Anal Cytology: A Feasible Approach for Anal Cancer Screening in Men Who Have Sex with Men Living With HIV

There is growing interest in anal cancer screening strategies. However, cytological/molecular evaluation of anal samples is challenging. We aimed to determine the feasibility of detecting, in anal liquid-based cytologies, the expression of biomarkers involved in the cell cycle disturbance elicited by human papillomavirus (HPV). The accuracy of this approach in the identification of high-grade squamous intraepithelial lesions/anal intraepithelial neoplasia grade2-3 (HSIL/AIN2-3) was also evaluated. 215 anal cytologies from men having sex with men living with human immunodeficiency virus were evaluated. Patients showing concordant cytological and anoscopy-directed biopsy diagnosis were selected: 70 with negative cytology and HPV test, 70 with low-grade SIL (LSIL/AIN1) cytology and biopsy, and 75 with cytology and biopsy of HSIL/AIN2-3. CDKN2A/p16, MKI67 and TOP2A mRNA expression was analyzed. HPV detection was performed with Xpert HPV Assay (Cepheid, Sunnyvale, CA, USA). HSIL/AIN2-3 showed higher expression for the biomarkers than LSIL/AIN1 or negative samples. The specificity for HSIL/AIN2-3 detection for a sensitivity established at 70% was 44.7% (95%confidence interval [CI] 36.5-53.2) for TOP2A and MKI67 and 54.5% (95%CI 46.0-62.8%) for CDKN2A/p16. mRNA detection of cell biomarkers in anal liquid-based cytology is feasible. Further studies are warranted to confirm if strategies based on mRNA detection have any role in anal cancer screening