Ella Evron

Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

Tissue specific DNA methylation in normal human breast epithelium and in breast cancer.

Cancer is a heterogeneous and tissue-specific disease. Thus, the tissue of origin reflects on the natural history of the disease and dictates the therapeutic approach. It is suggested that tissue differentiation, mediated mostly by epigenetic modifications, could guide tissue-specific susceptibility and protective mechanisms against cancer. Here we studied breast specific methylation in purified normal epithelium and its reflection in breast cancers. We established genome wide methylation profiles of various normal epithelial tissues and identified 110 genes that were differentially methylated in normal breast epithelium. A number of these genes also showed methylation alterations in breast cancers. We elaborated on one of them, TRIM29 (ATDC), and showed that its promoter was hypo-methylated in normal breast epithelium and heavily methylated in other normal epithelial tissues. Moreover, in breast carcinomas methylation increased and expression decreased whereas the reverse was noted for multiple other carcinomas. Interestingly, TRIM29 regulation in breast tumors clustered according to the PAM50 classification. Thus, it was repressed in the estrogen receptor positive tumors, particularly in the more proliferative luminal B subtype. This goes in line with previous reports indicating tumor suppressive activity of TRIM29 in estrogen receptor positive luminal breast cells in contrast to oncogenic function in pancreatic and lung cancers. Overall, these findings emphasize the linkage between breast specific epigenetic regulation and tissue specificity of cancer.

High efficacy of pre-operative trastuzumab combined with paclitaxel following doxorubicin & cyclophosphamide in operable breast cancer

Background. Trastuzumab in combination with adjuvant chemotherapy improves disease free survival and overall survival in HER2 over-expressing breast cancer patients. Data concerning the use of trastuzumab in the neo-adjuvant setting is limited. We aimed to compare outcome of HER2 over-expressing breast cancer patients treated with either standard chemotherapy, consisting of doxorubicin, cyclophosphamide and a taxane to outcome of patients treated with the same chemotherapy regimen with the addition of trastuzumab in concurrence with paclitaxel. Methods. We conducted a retrospective review of all consecutive HER2 over-expressing breast cancer patients treated at the participating institutions during the study period and received neo-adjuvant therapy. Allocation to trastuzumab was not based on clinical parameters and was approved only by part of the insurers. Clinical and pathological characteristics, as well as response rate and type of surgery were analyzed. Results. Thirty seven patients received chemotherapy alone and 24 patients received chemotherapy and trastuzumab. A similar distribution of age, clinical stage and histology was noted in both groups. The rate of pathological complete response (pCR) was significantly higher among the trastuzumab-treated group compared to chemotherapy-alone group (75 vs. 24% respectively, p=0.0002). pCR in the breast was noted in 18 of 24 (75%) compared to 10 of 36 (28%, p=0.0005) and pCR in the axillary lymph nodes was noted in 19 of 20 (95%) compared to 8 of 28 (29%, p=0.0001), in the trastuzumab group compared to the chemotherapy-alone group respectively. The safety profile was similar between both groups and no clinical cardiotoxicity were noted. Conclusions. The addition of trastuzumab to standard chemotherapy in the neo-adjuvant setting improves pathological complete response rates in HER2 over-expressing breast cancer patients.

A similar cell-specific pattern of HOXA methylation in normal and in cancer tissues.

HOX genes are developmental genes that determine anterior–posterior embryonic pattern and govern the process of differentiation. Inappropriate expression of HOX genes has been implicated in developmental abnormalities and hematopoietic malignancies. In addition, HOX genes silencing by DNA methylation has been reported in cancers and related to disease aggressiveness and outcome. On the other hand, accumulating evidence suggests that epigenetic changes at HOX genes are linked to normal development and differentiation. To better understand the relationship between HOXA methylation and cancer, we analyzed the methylation pattern of HOXA genes in human primary breast and colon carcinomas, normal tissues and normal white blood cells. Genome-wide methylation arrays of breast cancers and white blood cells demonstrated similar methylation patterns. Quantitative methylation analysis of seven representative HOXA genes revealed various levels of methylation in both normal tissues and cancers. Analysis of epithelial-enriched normal breast tissue and stroma indicated that the stroma was the major origin of HOXA methylation. Furthermore, in selected dense breast cancers, minimal increase in methylation of several HOXA genes did not correlate with the predominance of malignant epithelial cells in these tumors. Our results suggest that methylation of the HOXA cluster may be a normal developmental and cell type specific process rather than a cancer specific mechanism.

Paclitaxel-induced pneumonitis in patients with breast cancer: case series and review of the literature

Doxorubicin plus cyclophosphamide followed by paclitaxel is a common adjuvant treatment for high-risk breast cancer. It has been associated with pulmonary toxicity in several case reports. We describe three patients in whom interstitial pneumonitis developed immediately after the first paclitaxel exposure and worsened clinically over time. All reported dyspnoea, fever and progressive respiratory distress. Imaging revealed diffuse bilateral pulmonary infiltrates. Other causes of respiratory failure were excluded with laboratory work-up, imaging, biopsy studies and results of antibiotic treatment. The respiratory decline was reversed only after administration of high-dose steroids, an empirical treatment previously reported to be beneficial in similar cases. Although chemotherapy using concomitant or sequential drugs may make identification of the toxic agent difficult, we noted a clear temporal relationship between exposure to paclitaxel and the development of pulmonary toxicity. Furthermore, according to the available literature, it is less likely that a respiratory decline would be caused by either cyclophosphamide or trastuzumab. In conclusion, clinicians should be aware of the potentially life-threatening risk of pulmonary toxicity following paclitaxel treatment. If paclitaxel is halted early and the patient has good lung reserve, pulmonary toxicity can be reversed with high-dose steroid administration.

Clinical outcomes in patients with node-negative breast cancer treated based on the recurrence score results: evidence from a large prospectively designed registry

The 21-gene Recurrence Score® (RS) assay is a validated prognostic/predictive tool in ER + early-stage breast cancer. However, clinical outcome data from prospective studies in RS ≥ 11 patients are lacking, as are relevant real-life clinical practice data. In this retrospective analysis of a prospectively designed registry, we evaluated treatments/clinical outcomes in patients undergoing RS-testing through Clalit Health Services. The analysis included N0 ER + HER2-negative breast cancer patients who were RS-tested from 1/2006 through 12/2010. Medical records were reviewed to verify treatments/recurrences/survival. The cohort included 1801 patients (median follow-up, 6.2 years). Median age was 60 years, 50.4% were grade 2 and 81.1% had invasive ductal carcinoma; 48.9% had RS < 18, 40.7% RS 18–30, and 10.4% RS ≥ 31, with chemotherapy use of 1.4, 23.7, and 87.2%, respectively. The 5-year Kaplan–Meier estimates for distant recurrence were 0.8, 3.0, and 8.6%, for patients with RS < 18, RS 18–30 and RS ≥ 31, respectively; the corresponding 5-year Kaplan–Meier estimates for breast cancer death were 0.0, 0.9, and 6.2%. Chemotherapy-untreated patients with RS < 11 (n = 304) and 11–25 (n = 1037) (TAILORx categorization) had 5-year Kaplan–Meier estimates for distant recurrence risk/breast cancer death of 1.0%/0.0% and 1.3%/0.4%, respectively. Our results extend those of the prospective TAILORx trial: the 5-year Kaplan–Meier estimates for distant recurrence and breast cancer death rate for the RS < 18 patients were very low supporting the use of endocrine therapy alone. Furthermore, in chemotherapy-untreated patients with RS 11–25 (where TAILORx patients were randomized to chemoendocrine or endocrine therapy alone), 5-year distant recurrence rates were also very low, suggesting that chemotherapy would not have conferred clinically meaningful benefit.Genetic testing: Diagnostic shows which node-negative patients need chemoPatients with early breast cancer that hasn’t spread to lymph nodes can likely forgo chemotherapy if they score under 25 on Oncotype DX. That’s the finding of a retrospective analysis led by Salomon Stemmer from Rabin Medical Center in Petah Tikvah, Israel, that looked at 1801 women with node-negative, ER-positive, HER2-negative disease who received the diagnostic test, which measures the expression levels of 21 genes within tumor cells. Rates of disease recurrence and death were low for patients who received only anti-hormone treatment and had low-to-intermediate Oncotype DX results, suggesting no need for additional chemotherapy (which carries an appreciable risk of toxicity). Previously, a prospective US study called TAILORx established that women with scores under 11 could be spared chemotherapy. The Israeli trial validates and extends the results to include women with scores up to 25.

Expression and methylation patterns partition luminal-A breast tumors into distinct prognostic subgroups

Breast cancer is a heterogeneous disease comprising several biologically different types, exhibiting diverse responses to treatment. In the past years, gene expression profiling has led to definition of several “intrinsic subtypes” of breast cancer (basal-like, HER2-enriched, luminal-A, luminal-B and normal-like), and microarray based predictors such as PAM50 have been developed. Despite their advantage over traditional histopathological classification, precise identification of breast cancer subtypes, especially within the largest and highly variable luminal-A class, remains a challenge. In this study, we revisited the molecular classification of breast tumors using both expression and methylation data obtained from The Cancer Genome Atlas (TCGA). Unsupervised clustering was applied on 1148 and 679 breast cancer samples using RNA-Seq and DNA methylation data, respectively. Clusters were evaluated using clinical information and by comparison to PAM50 subtypes. Differentially expressed genes and differentially methylated CpGs were tested for enrichment using various annotation sets. Survival analysis was conducted on the identified clusters using the log-rank test and Cox proportional hazards model. The clusters in both expression and methylation datasets had only moderate agreement with PAM50 calls, while our partitioning of the luminal samples had better five-year prognostic value than the luminal-A/luminal-B assignment as called by PAM50. Our analysis partitioned the expression profiles of the luminal-A samples into two biologically distinct subgroups exhibiting differential expression of immune-related genes, with one subgroup carrying significantly higher risk for five-year recurrence. Analysis of the luminal-A samples using methylation data identified a cluster of patients with poorer survival, characterized by distinct hyper-methylation of developmental genes. Cox multivariate survival analysis confirmed the prognostic significance of the two partitions after adjustment for commonly used factors such as age and pathological stage. Modern genomic datasets reveal large heterogeneity among luminal breast tumors. Our analysis of these data provides two prognostic gene sets that dissect and explain tumor variability within the luminal-A subgroup, thus, contributing to the advancement of subtype-specific diagnosis and treatment.BackgroundBreast cancer is a heterogeneous disease comprising several biologically different types, exhibiting diverse responses to treatment. In the past years, gene expression profiling has led to definition of several “intrinsic subtypes” of breast cancer (basal-like, HER2-enriched, luminal-A, luminal-B and normal-like), and microarray based predictors such as PAM50 have been developed. Despite their advantage over traditional histopathological classification, precise identification of breast cancer subtypes, especially within the largest and highly variable luminal-A class, remains a challenge. In this study, we revisited the molecular classification of breast tumors using both expression and methylation data obtained from The Cancer Genome Atlas (TCGA).MethodsUnsupervised clustering was applied on 1148 and 679 breast cancer samples using RNA-Seq and DNA methylation data, respectively. Clusters were evaluated using clinical information and by comparison to PAM50 subtypes. Differentially expressed genes and differentially methylated CpGs were tested for enrichment using various annotation sets. Survival analysis was conducted on the identified clusters using the log-rank test and Cox proportional hazards model.ResultsThe clusters in both expression and methylation datasets had only moderate agreement with PAM50 calls, while our partitioning of the luminal samples had better five-year prognostic value than the luminal-A/luminal-B assignment as called by PAM50. Our analysis partitioned the expression profiles of the luminal-A samples into two biologically distinct subgroups exhibiting differential expression of immune-related genes, with one subgroup carrying significantly higher risk for five-year recurrence. Analysis of the luminal-A samples using methylation data identified a cluster of patients with poorer survival, characterized by distinct hyper-methylation of developmental genes. Cox multivariate survival analysis confirmed the prognostic significance of the two partitions after adjustment for commonly used factors such as age and pathological stage.ConclusionsModern genomic datasets reveal large heterogeneity among luminal breast tumors. Our analysis of these data provides two prognostic gene sets that dissect and explain tumor variability within the luminal-A subgroup, thus, contributing to the advancement of subtype-specific diagnosis and treatment.

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.

Everolimus Plus Letrozole for Treatment of Patients With HR+, HER2– Advanced Breast Cancer Progressing on Endocrine Therapy: An Open-label, Phase II Trial

Purpose: In the Breast cancer trials of OraL EveROlimus‐2 (BOLERO‐2) trial, everolimus plus exemestane improved progression‐free survival (PFS) in patients with hormone receptor‐positive (HR+), human epidermal growth factor receptor 2‐negative (HER2−) advanced breast cancer (ABC) recurring or progressing on/after prior endocrine therapy (ET), suggesting that dual blockade using targeted therapy and ET was an effective treatment option. Here, we investigated the clinical benefit of combining everolimus with different endocrine partner, letrozole, in a similar patient population. Methods: In this phase II, open‐label, single‐arm, multicenter trial, postmenopausal women with HR+, HER2− ABC who had recurrence/progression on/after prior ET received everolimus 10 mg daily and letrozole 2.5 mg daily. The primary end point was objective response rate; key secondary end points included disease‐control rate, PFS, overall survival, and safety. Results: A total of 72 patients were enrolled and followed‐up for a median duration of 11.4 months. Everolimus plus letrozole achieved an overall response rate of 23.3% (95% confidence interval [CI], 13.4%‐36.0%). The median PFS was 8.8 months (95% CI, 6.6‐11.0 months), and the overall survival was 22.9 months (95% CI, 18.5‐28.9 months). Disease‐control rate was achieved in 51 (85%) patients. The safety profile was consistent with previously published data: The most frequently reported any grade adverse events (AEs) were fatigue (61.1%), stomatitis (54.2%), and rash (33.4%). The most frequently reported grade 3 AEs were stomatitis and anemia (8.3% each), fatigue and diarrhea (5.6% each), and hyperglycemia (4.2%). Only 1 patient had grade 4 AE of anemia. Conclusions: Everolimus plus letrozole demonstrated clinical benefit and could be a valid treatment option for postmenopausal women recurring/progressing on prior endocrine therapy.