M.J. Kerin

Abstract P2-05-09: Identification and validation of a miRNA signature associated with breast cancer progression

Introduction: MiRNAs are aberrantly expressed in both the circulation and tissue of patients with breast tumours, however little is currently known about the relationship between circulating and tissue miRNAs. The aim of this study was to quantify circulating and tumour tissue miRNA expression in a murine model of breast cancer and identify novel circulating markers of disease progression. Materials and Methods: Athymic nude mice (n = 20) received either a mammary fat pad (n = 8, MFP), or subcutaneous (n = 7, SC) injection of MDA-MB-231 cells. Controls received no tumour cells (n = 5). Tumour volume was monitored weekly and blood sampling performed at weeks 1, 3 and 6 following tumour induction (total n=60). Animals were sacrificed at week 6 and tumour tissue (n = 15), lungs (n = 20) and enlarged lymph nodes (n = 3) harvested. RNA were extracted from all samples (n = 98) and relative expression of miRNAs previously associated with tumour progression were quantified using RQ-RCR. A microRNA array was also preformed comparing animals with early (weeks 1, n=5) versus late (week 6, n=5) disease and results were analysed using RQ-PCR in all blood samples (n = 60). Reverse transcription for the relevant targets and endogenous controls was carried out and expression was quantified using RQ-PCR. Results: MiR-10b expression was significantly higher in MFP compared to SC tumours (p MiR-10b was undetectable in the circulation. MiR-221 expression was significantly increased in tumour tissue (p MiR-195 and miR-497 were significantly decreased in tumour tissue (p miR-497 and miR-195 (r = 0.61, p Conclusions: This study highlights the importance of miRNAs in breast cancer, with each displaying distinct roles in circulation and tissue. Novel circulating miRNAs which have never previously been associated with breast cancer have been identified, with quantification of circulating levels in breast cancer patients currently ongoing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-09.

Abstract P5-10-07: Luminal A breast cancer: identification of novel circulating miRNA biomarkers

Introduction: Breast cancer is a prevalent disease in need of a reliable circulating biomarker with a role in combination with mammography to facilitate early diagnosis. Mi(cro)RNAs are a group of small molecules with ideal biomarker characteristics. The knowledge that miRNAs are aberrantly expressed in cancer, and can be detected in the circulation supports their potential role in minimally invasive cancer diagnostics. Intrinsic breast cancer subtypes provide well characterized phenotypes to study the biomarker potential of miRNAs. The aim of this study was to identify systemic miRNAs differentially expressed in Luminal A (ER+PR+HER2/neu−) breast cancer, and to study their utility as oncologic biomarkers in the clinical setting. Material and Method: Whole blood samples were collected prospectively from women diagnosed with Luminal A breast cancer (n = 57) and healthy controls (n = 57). Luminal A phenotype was confirmed by immunohistochemistry and fluorescence in situ hybridization (FISH). RNA was extracted, reverse transcribed and a Taqman Low-Density Array (TLDA, microarray) conducted on a test cohort (10 Luminal A; 10 Control). Differentially expressed miRNAs were identified using Artificial Neural Networks (ANN). Expression of specific miRNAs were validated using RQ-PCR on an independent whole blood cohort (n = 47 Luminal A; n=47 Control) and tissue (n = 30). Results were analyzed using Q-Base and Minitab V16.0. Results: The microarray performed on the test cohort identified 77 differentially expressed miRNAs. Artificial Neural Networking highlighted seven miRNAs for further analysis (Table 1). RQ-PCR quantification of expression of these seven candidate miRNAs in the validation group confirmed the biomarker potential of two miRNAs. MiR-181a and miR-652 were significantly under-expressed in the cancer group compared to the control group (p = 0.005 and p = 0.001 respectively). Circulating miR-181a expression correlated with invasive tumour size (r = −0.592, p = 0.008). Furthermore, a combination of these two miRNAs provided a sensitivity and specificity for detection of Luminal A breast cancer of 70% and 65%, respectively (Area Under the Curve, AUC=0.77). MiR-181a and miR-652 were also under-expressed in Luminal A tumor tissue compared to Tumor Associated Normal (TAN, p = 0.019 and p = 0.001, respectively). Conclusion: This study provides insight into the underlying molecular portrait of the Luminal A subtype by identifying 77 miRNAs with altered systemic expression levels. Two novel miRNA oncologic biomarkers are identified, which are altered in both the tumor and circulation. These miRNAs may have a role in combination with mammography to facilitate accurate subtype-specific breast tumor detection. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-07.

Abstract P3-10-02: Circulating miRNA Signature: Potential Screening and Prognostic Tool for Breast Cancer

Introduction: Genetic profiling of breast tumors revolutionized the classification of breast cancer and unravelled the complexity of this phenotypically diverse disease. However there appears to be discordance between the various mRNA-based single sample predictors which stratify tumors into subgroups. Clinical decision making based on a tumor9s mRNA expression is therefore concerning. The potential of mi(cro) RNAs as novel tumor markers has been the focus of recent scrutiny due to their tissue specificity, stability and superiority to mRNA in tumor classification. Additionally, the ability to quantify tumor-associated miRNAs in the circulation and their correlations with clinicopathologic variables, highlights their potential to improve upon existing breast tumor classification methods. Systemic miR-195 and let-7a have been shown to hold properties as breast tumor markers. The aim of this study was to identify a larger panel of miRNAs which augment the sensitivity and specificity of circulating miRNAs as diagnostic and prognostic markers for breast cancer. Methods: The expression levels of 9 miRNAs were evaluated in 345 preoperative cancer patients including 265 breast cancers and 80 non-breast malignancies, and 63 age-matched disease-free controls using RQ-PCR. MiRNA quantification was also performed on tumor tissue from 83 age and stage matched breast cancer patients. Advanced QBase Plus software and SPSS were used for biostatistical analysis of the data and correlation with clinicopathologic variables. Results: This study confirmed significantly deranged expression levels of systemic miR-195 and let-7a in an independent validation cohort of breast cancer patients (p miR-181c and miR-342 were identified as breast cancer specific biomarkers. Elevated levels of this 4-miRNA signature in breast cancer patients, including those with in-situ carcinoma, increased the discriminatory power of this test for breast cancer (all types) to 94% (P Circulating levels of these 4 miRNAs correlated with tumor miRNA expression, and decreased to basal levels by 2 weeks following curative tumor resection. Additionally circulating miRNA levels correlated with clinicopathological variables such as tumor size and hormone receptor status. A subset of 2 systemic miRNAs was predictive of the Luminal A subtype of breast cancer (ER/PR positive, Her2/neu negative) with 91% accuracy. Conclusion: This study validates the recent novel finding of dysregulated tumor-specific miRNAs in the circulation of breast cancer patients. Considering that the sensitivity of mammography is 75-90%, this circulating miRNA signature could improve upon existing breast cancer screening methods, given that it was significantly altered even in patients with in-situ carcinoma. These results indicate that circulating miRNA analysis holds immense potential in the future individualized management of breast cancer patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-02.

Abstract P5-10-04: Amplification of Topoisomerase 2 Alpha (TOP2A) in HER2/neu Negative Breast Cancer

Background: TOP2A is of particular interest in breast cancer because of its association with response to anthracycline-based chemotherapies. Much of the work which has examined levels of TOP2A has concluded that amplification is restricted to those who are also HER2 /neu amplified, leading some commentators to conclude that HER2 /neu negative patients should not receive anthracyclines as part of their treatment. A couple of studies have identified TOP2A alterations in the absence of that of HER2/ neu however, and the relationship between these targets needs to be better defined. We aimed to investigate the relationship between HER2 /neu and TOP2A, whilst elucidating their association with clinicopathological variables thereby providing further insight into which patients are most amenable to anthracycline-based regimens. Materials and Methods: Hierarchical clustering was performed on a 20 patient cDNA microarray to identify gene clusters of clinicopathological interest. RQ-PCR was then performed (n=96) to further assess gene amplification, and levels were determined using the comparative Ct approach & Taqman assays. An IHC microarray (n=76) was then employed to check for correlation between gene amplification and protein expression levels. Results: Amplification levels of TOP2A did not differ significantly according to HER2/neu status by either cDNA microarray (p=0.623), RQ-PCR (p=0.475) or IHC microarray (p=0.736). By RQ-PCR, 14/48 (29.1%) of the HER2/neu negative patients demonstrated levels of TOP2A above the third quartile. Similarly, 11/48 (22.9%) of the HER2/neu positive patients returned values in the first quartile, thereby indicating low-level amplification. Of 60 patients characterised as HER2 /neu negative using IHC and FISH, 14 (22.9%) were classified as TOP2A positive on the IHC microarray. Of the 14 patients deemed HER2 /neu positive using IHC and FISH, meanwhile, the majority (n=10) were classified as TOP2A positive. Discussion: Our results indicate that amplification of TOP2A in breast cancer is not confined to those who are concomitantly HER2/neu positive, and suggest that a significant proportion of HER2 /neu negative patients exhibit high levels of TOP2A. These findings were not expected, are contrary to popular opinion, and have significant implications in the rationalisation of anthracycline-based therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-04.