Barbara Pasculli

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.

Epigenetics of breast cancer: Biology and clinical implication in the era of precision medicine

In the last years, mortality from breast cancer has declined in western countries as a consequence of a more widespread screening resulting in earlier detection, as well as an improved molecular classification and advances in adjuvant treatment. Nevertheless, approximately one third of breast cancer patients will develop distant metastases and eventually die for the disease. There is now a compelling body of evidence suggesting that epigenetic modifications comprising DNA methylation and chromatin remodeling play a pivotal role since the early stages of breast cancerogenesis. In addition, recently, increasing emphasis is being placed on the property of ncRNAs to finely control gene expression at multiple levels by interacting with a wide array of molecules such that they might be designated as epigenetic modifiers. In this review, we summarize the current knowledge about the involvement of epigenetic modifications in breast cancer, and provide an overview of the significant association of epigenetic traits with the breast cancer clinicopathological features, emphasizing the potentiality of epigenetic marks to become biomarkers in the context of precision medicine.

Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients.

BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.

Stepwise analysis of MIR9 loci identifies miR-9-5p to be involved in Oestrogen regulated pathways in breast cancer patients

miR-9 was initially identified as an epigenetically regulated miRNA in tumours, but inconsistent findings have been reported so far. We analysed the expression of miR-9-5p, miR-9-3p, pri-miRs and MIR9 promoters methylation status in 131 breast cancer cases and 12 normal breast tissues (NBTs). The expression of both mature miRs was increased in tumours as compared to NBTs (P < 0.001) and negatively correlated with ER protein expression (P = 0.005 and P = 0.003, for miR-9-3p and miR-9-5p respectively). In addition, miR-9-5p showed a significant negative correlation with PgR (P = 0.002). Consistently, miR-9-5p and miR-9 3p were differentially expressed in the breast cancer subgroups identified by ER and PgR expression and HER2 amplification. No significant correlation between promoter methylation and pri-miRNAs expressions was found either in tumours or in NBTs. In the Luminal breast cancer subtype the expression of miR-9-5p was associated with a worse prognosis in both univariable and multivariable analyses. Ingenuity Pathway Analysis exploring the putative interactions among miR-9-5p/miR-9-3p, ER and PgR upstream and downstream regulators suggested a regulatory loop by which miR-9-5p but not miR-9-3p is induced by steroid hormone receptor and acts within hormone-receptor regulated pathways.

Predictive Value of Epigenetic Signatures

The rational development of novel cancer therapeutics is strictly dependent on a more in-depth understanding of tumor biology, and predictive biomarkers represent critical tools for identifying patients who may really benefit from the treatment with both standard chemotherapy and targeted drugs. In this context, epigenetic traits of human cancers have emerged as candidate biomarkers to predict response to common regimens and sensitivity to targeted compounds. In particular, DNA methylation and noncoding RNAs (ncRNAs) likely possess features that make them appropriate for being implemented as biomarkers of clinical utility. Herein, we highlight the importance of predictive biomarkers in the scenario of modern oncology, underlying the major challenges in biomarker definition and validating procedures needed before clinical translation. Then, we review the current state of epigenetics translational research, and report evidences supporting DNA methylation and ncRNAs as the most promising predictive epigenetic biomarkers and even potential targets for molecular drug design.

Abstract 3977: Evaluation of miR10b and miR9 expression in breast cancer and correlations with distant metastases development

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We have recently demonstrated that the evaluation of miR10b expression relative to its normal counterparts (Relative Expression Ratio, RER) is able to predict patients risk to develop distant metastases. To confirm these results and evaluate whether other microRNAs could be predictive of outcome in breast cancer we evaluated the expression of miR10b and miR9 in paired tumors and apparently normal breast tissues 2 cm distant from tumour (NBT) on an extended cohort (n = 127) with a longer follow up (median 42 months). As control normal breast tissues from 10 healthy subjects (HBT) treated by reductive mammoplasty were analyzed. An increased expression was seen for the miR9 and miR10b in NBT samples as compared with HBT (P<0.01). Whereas the expression of each of the two miRNAs was significantly lower in tumour samples as compared to the NBT specimens with RER value of 0.15 for miR10b (P = 0.001) and 0.59 for miR9 (P = 0.05). However only higher miR10b RERs were associated with distant metastases development in multivariable analysis (HR = 1.12; P = 0.04). Our results confirm the role of miR10b as predictive biomarkers of metastases development in breast cancer. In addition our data suggest a deregulation of miRNAs expression in the normal tissue surrounding tumor as compared to normal breast of healthy individuals. Citation Format: Raffaela Barbano, Barbara Pasculli, Massimiliano Copetti, Andrea Fontana, Michelina Coco, Lorenzo Vitulano, Maria Luana Poeta, Francesca Picardo, Gianfranco Marangi, Roberto Murgo, Luigi Ciuffreda, Vanna Maria Valori, Vanna Maria Valori, Maria Morritti, Evaristo Maiello, Vito Michele Fazio, Paola Parrella. Evaluation of miR10b and miR9 expression in breast cancer and correlations with distant metastases development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3977. doi:10.1158/1538-7445.AM2015-3977

Abstract 2251: nrf2-keap1 axis molecular profile in small cell lung cancer cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The Keap1/Nrf2 pathway is a master regulator of antioxidants and cellular stress responses implicated in resistance of tumor cells against chemotherapeutic drugs. The link between molecular alterations of this pathway in Non Small Cell Lung Cancer (NSCLC) is well studied and appears to depend on several main factors including the existence of activating mutations in NFE2LE gene and/or loss of function mutations and methylation in the KEAP1 gene. At present, the data concerning the mechanism of alteration of Nrf2-Keap1 pathway in Small Cell Lung Cancer (SCLC) instead are almost incomplete and correlation analysis with therapeutic strategies targeting the molecular dysfunction of this pathway is ongoing. Here we present a comprehensive molecular alteration profile of the main partners of the Nrf2/keap1 axis in 12 SCLC cell lines by integrating data from SNP-Array analysis, immunofluorescence, mutation screening by direct sequencing, methylation by QMSP and expression analyses by RT-qPCR and western blotting. Our analyses confirm the global deregulation of Nrf2/Keap1 pathway in SCLC cell lines, showing an hypermethylation of the CpGs located into the P1 promoter region of the KEAP1 in 42% (5/12) of the cell lines, and a chromosomal amplification involving the NFE2LE gene locus (2q31.1) in two cell lines. Only one just described point mutation in the kelch-repeat 2 was observed in one of the cell line analysed. Our gene-alteration profile of SCLC cell lines provides new insights into the mechanism of deregulation of Nrf2-Keap1 detoxification pathway in this group of high grade neuroendocrine lung cancers, suggesting the NFE2LE gene amplification and the KEAP1 promoter hypermethylation as alternative mechanisms of dysfunctional Nrf2/Keap1 in SCLCs.Analyses on tumor tissues are ongoing to confirm these observations. Moreover, the provided full molecular data from cell lines will be useful for in vitro functional studies aimed to establish new combined therapeutic strategies in targeted cancer treatments of this aggressive lung tumor histotype. Citation Format: Lucia Anna Muscarella, Annamaria la Torre, Angelo Sparaneo, Orazio Palumbo, Clelia Tiziana Storlazzi, Teresa Balsamo, Domenico Trombetta, Fabio Pellegrini, Raffaela Barbano, Barbara Pasculli, Paolo Graziano, Montse Sanchez-cespedes, Maria Teresa Landi, Paola Parrella, Vito Michele Fazio. nrf2-keap1 axis molecular profile in small cell lung cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2251. doi:10.1158/1538-7445.AM2014-2251

Abstract 1477: Evaluation of microRNA-10b prognostic significance in a prospective cohort of breast cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Several prognostic factors are currently used to stratify breast cancer patients based on the risk of disease progression. However, even patients classified as at low risk can progress and die for the disease. MicroRNA-10b (miR-10b) has a prominent role in regulating tumor invasion and metastasis by targeting the HOXD10 transcriptional repressor and has been found up-regulated in several tumor types including breast cancer. We evaluated the expression of miR-10b in paired tumor and normal specimens obtained from a prospective cohort of breast cancer patients with at least 36 months follow-up enrolled according to the REMARK guidelines (n=150). RNA quality was measured and only samples with RNA Integrity Number (RIN) ≥7.0 were analyzed. The relative expression of miR-10b in tumor as compared to its normal counterpart (RER) was determined by RT-qPCR. miR-10b RERs were higher in the subgroup of patients with synchronous metastases (n=11, Median 0.25; IQR 0.11-1.02) as compared with patients without metastases (n=90, Median 0.09; IQR 0.04-0.29) (p=0.028). In the subgroup of patients without synchronous metastases (n=90), higher miR-10b RERs were associated with increased risk of disease progression and death in both univariable (HR 4.35, p=0.021 and HR 6.02, p=0.015 respectively) and multivariable (HR 13.37, p<0.001, and HR 15.39, p=0.003 respectively) Cox regression models. The addition of miR-10b RERs to the Nottingham Prognostic Index (NPI) provided an improvement in discrimination power and risk reclassification abilities for the clinical outcomes at 36 months. Survival C-indices significantly increased from 0.849 to 0.889 (p=0.009) for OS and from 0.735 to 0.767 (p=0.050) for DFS. Our results provide evidences that the addition of miR-10b RERs to the prognostic factors used in clinical routine could improve the prediction abilities for both overall mortality and disease progression in breast cancer patients. Citation Format: Paola Parrella, Raffaela Barbano, Barbara Pasculli, Andrea Fontana, Massimiliano Copetti, Vanna Maria Valori, Maria Luana Poeta, Giuseppe Perrone, Michelina Coco, Teresa Balsamo, Fabio Pellegrini, Andrea Onetti Muda, Evaristo Maiello, Roberto Murgo, Vito Michele Fazio. Evaluation of microRNA-10b prognostic significance in a prospective cohort of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2014-1477

Abstract 664: Aberrant KEAP1 promoter methylation is associated with disease progression in breast cancer patients treated with epirubicin/cyclophosfamide and docetaxel chemotherapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The KEAP1 (Kelch-like ECH-Associated Protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We investigated the possible contribution of KEAP1 genetic and epigenetic abnormalities to development and progression of breast cancer. Methylation status of the KEAP1 promoter was evaluated in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by Quantitative Methylation Specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple negative breast cancers (35%) (p=0.02, Mann Whitney U-test). Interactions between ER, PR, HER2 expression and KEAP1 methylation on the mortality were investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple negative breast cancer (HR=14.73, 95% CI: 3.65-59.37). Both univariable and multivariable COX regression analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR=0.096; 95%CI: 0.011-0.832 p=0.03 and HR=0.082 95%CI: 0.007-0.934 P=0.04 respectively). Our data indicate that aberrant promoter methylation of the KEAP1 gene promoter is involved in breast cancerogenesis. In addition identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients. Citation Format: Raffaela Barbano, Lucia Anna Muscarella, Barbara Pasculli, Vanna Maria Valori, Andrea Fontana, Michelina Coco, Annamaria la Torre, Teresa Balsamo, Maria Luana Poeta, Giovanni Francesco Marangi, Evaristo Maiello, Marina Castelvetere, Fabio Pellegrini, Roberto Murgo, Vito Michele Fazio, Paola Parrella. Aberrant KEAP1 promoter methylation is associated with disease progression in breast cancer patients treated with epirubicin/cyclophosfamide and docetaxel chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2013-664