Melanie Spears

In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein-protein complexes in paraffin sections from breast cancer tissues.

Background:Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2.Method and results:In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6–HER2 protein–protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012).Conclusion:Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2–PTK6 complexes are of prognostic relevance.

Proximity ligation assays for isoform-specific Akt activation in breast cancer identify activated Akt1 as a driver of progression

The PI3K/Akt signal transduction pathway plays an important role in cancer progression and cell survival. Akt activation is associated with poor outcome in endocrine‐treated breast cancer, whereas high levels of cytoplasmic Akt2 are associated with an improved overall survival. Proximity ligation assays (PLAs) were used to determine quantitative expression levels of isoform‐specific activation (phosphorylation) of Akt1 and Akt2 in formalin‐fixed, paraffin‐embedded cell lines and breast cancer tumour tissues in situ. PLAs demonstrated a range of expression in breast cancer samples for total pAkt1 and pAkt2. High levels of pAkt1 were associated with reduced DRFS (HR: 1.45, 95% CI 1.14–1.83, p = 0.002) and OS (HR: 1.42, 95% CI 1.10–1.83, p = 0.007). When PLA results were combined, patients that had high levels of pAkt1 only had a significantly decreased DRFS (HR: 1.92, 95% CI 1.34–2.76, p = 0.005) and OS (HR: 1.94, 95% CI 1.32–2.86, p = 0.008) compared to other patients. Using PLAs to discriminate activation of Akt1 versus Akt2 suggests that Akt1 drives progression in early breast cancers. In cases where both Akt1/Akt2 are activated, Akt2 may act to reverse this effect. Using PLAs, we have measured activation of Akt1 and Akt2 proteins separately in situ in FFPE breast cancer samples. Copyright

The potential role of estrogen receptors and the SRC family as targets for the treatment of breast cancer

Background: Breast cancer has a number of subtypes, the main ones are estrogen-receptor (ER)-positive, luminal type A and B. Treatment selection, with respect to hormonal therapy, is based upon ER expression. Whilst for ER-positive cancers, endocrine therapy is highly successful in the adjuvant setting, a significant proportion of cancers exhibit hormone resistance, often associated with altered growth factor receptor or ER signalling. Modulation of steroid receptor function by receptor co-activators or repressors is a potential mechanism of resistance. The p160 or SRC proto-oncogene family of co-activators are important in breast cancer response to endocrine therapy and can act as a paradigm of co-activator function. Objective/methods: This review focuses on the role of ER and ER co-activators in breast cancer and current approaches to targeting SRC co-factors for treatment of hormone-receptor-positive breast cancer. Results/conclusions: There is a drive to selectively apply aromatase inhibitors on the basis of either risk or biological evidence of resistance to tamoxifen treatment. Both strategies may yield improved treatment to benefit ratios.

The p160 ER co-regulators predict outcome in ER negative breast cancer

The SRC family of ER co-regulators are frequently overexpressed in breast cancer. Overexpression of AIB1 appears to be linked to hormone resistance in HER2 positive breast cancer. However, the role of these co-regulators in ER negative disease is poorly understood. SRC1, SRC2 and AIB1 expression was determined by immunohistochemical analysis of tissue microarrays constructed from tumours within the Edinburgh Breast Conservation Series (BCS). The BCS represents a fully documented consecutive cohort of 1,812 patients treated by breast conservation surgery in a single institution. Our results demonstrate tumours that overexpress both HER2 and AIB1 were associated with markedly reduced relapse free, distant relapse free and overall survival compared to HER2 and AIB1 only overexpressing tumours irrespective of ER status. In ER negative disease both SRC1 and AIB1 were linked to early relapse and death. The SRC family of ER co-regulators is involved in early relapse and resistance in both ER negative and ER positive breast cancer challenging the conventional concept that this effect is mediated solely via the ER.

Molecular characterisation of isogenic taxane resistant cell lines identify novel drivers of drug resistance.

BackgroundTaxanes such as paclitaxel and docetaxel are used successfully to treat breast cancer, usually in combination with other agents. They interfere with microtubules causing cell cycle arrest; however, the mechanisms underlying the clinical effects of taxanes are yet to be fully elucidated.MethodsIsogenic paclitaxel resistant (PACR) MDA‒MB‒231, paclitaxel resistant ZR75‒1 and docetaxel resistant (DOCR) ZR75‒1 cell lines were generated by incrementally increasing taxane dose in native cell lines in vitro. We used aCGH analysis to identify mechanisms driving taxane resistance.ResultsTaxane resistant cell lines exhibited an 18-170 fold increased resistance to taxanes, with the ZR75-1 resistant cell lines also demonstrating cross resistance to anthracyclines. Paclitaxel treatment of native cells resulted in a G2/M block and a decrease in the G1 phase of the cell cycle. However, in the resistant cell lines, minimal changes were present. Functional network analysis revealed that the mitotic prometaphase was lost in the resistant cell lines.ConclusionThis study established a model system for examining taxane resistance and demonstrated that both MDR and mitosis represent common mechanism of taxane resistance.

A four gene signature predicts benefit from anthracyclines: evidence from the BR9601 and MA.5 clinical trials

Chromosome instability (CIN) in solid tumours results in multiple numerical and structural chromosomal aberrations and is associated with poor prognosis in multiple tumour types. Recent evidence demonstrated CEP17 duplication, a CIN marker, is a predictive marker of anthracycline benefit. An analysis of the BR9601 and MA.5 clinical trials was performed to test the role of existing CIN gene expression signatures as predictive markers of anthracycline sensitivity in breast cancer. Univariate analysis demonstrated, high CIN25 expression score was associated with improved distant relapse free survival (DRFS) (HR: 0.74, 95% CI 0.54-0.99, p = 0.046). High tumour CIN70 and CIN25 scores were associated with aggressive clinicopathological phenotype and increased sensitivity to anthracycline therapy compared to low CIN scores. However, in a prospectively planned multivariate analysis only pathological grade, nodal status and tumour size were significant predictors of outcome for CIN25/CIN70. A limited gene signature was generated, patients with low tumour CIN4 scores benefited from anthracycline treatment significantly more than those with high CIN4 scores (HR 0.37, 95% CI 0.20-0.56, p = 0.001). In multivariate analyses the treatment by marker interaction for CIN4/anthracyclines demonstrated hazard ratio of 0.35 (95% CI 0.15-0.80, p = 0.012) for DRFS. This data shows CIN4 is independent predictor of anthracycline benefit for DRFS in breast cancer.

Human Epidermal Growth Factor Receptor Dimerization Analysis in Breast Cancer Diagnosis

Our understanding of the human epidermal growth factor receptor (HER) family of proteins has increased over the last few decades. It is clear from the vast assortment of research that has been and is currently being carried out that there is still a lot to be learned. HER dimerization is known to activate various downstream pathways that have an effect on treatment and therapy for breast cancer. HER dimerization acts as a mechanism not only for amplifying the signal pathway but also for signal diversification. There is clear evidence that molecular subtypes of cancer respond differently to different therapeutic options, which challenges the ‘one size fits all’ approach to chemotherapy in cancer. Here, we review the methods by which HER dimerization can be explored and the potential that this has in the treatment of breast cancer.

A four gene signature of chromosome instability (CIN4) predicts for benefit from taxanes in the NCIC-CTG MA21 clinical trial

Recent evidence demonstrated CIN4 as a predictive marker of anthracycline benefit in early breast cancer. An analysis of the NCIC CTG MA.21 clinical trial was performed to test the role of existing CIN gene expression signatures as prognostic and predictive markers in the context of taxane based chemotherapy. RNA was extracted from patients in cyclophosphamide, epirubicin and fluorouracil (CEF) and epirubicin, cyclophosphamide and paclitaxel (EC/T) arms of the NCIC CTG MA.21 trial and analysed using NanoString technology. After multivariate analysis both high CIN25 and CIN70 score was significantly associated with an increased in RFS (HR 1.76, 95%CI 1.07-2.86, p=0.0018 and HR 1.59, 95%CI 1.12-2.25, p=0.0096 respectively). Patients whose tumours had low CIN4 gene expression scores were associated with an increase in RFS (HR: 0.64, 95% CI 0.39-1.03, p=0.06) when treated with EC/T compared to patients treated with CEF. In conclusion we have demonstrated CIN25 and CIN70 as prognostic markers in breast cancer and that CIN4 is a potential predictive maker of benefit from taxane treatment.

Abstract P2-03-17: Assessing reproducibility of copy number arrays to assist breast cancer biomarker discovery

Introduction: Large-scale interrogation of the genome has emerged as an attractive method for identifying useful characteristics of cancer biology; in particular, the study of copy number aberrations (CNA) has recently received tremendous attention. A number of different technologies have been developed to assess the copy-number landscape, allowing us to better understand the role of CNA in cancer cells. The OncoScan CNA platform (Affymetrix Inc.) has been particularly appealing for oncology due of its ability to work well with formalin-fixed, paraffin-embedded (FFPE) materials, which is the primary form for storage of clinical samples. In addition, its high resolution, rapid analysis time and ability to interrogate different genomic characteristics (CNA, loss of heterozygosity or mutation) make the OncoScan platform highly popular: it has been widely cited in the literature for use in biomarker discovery, clonal evolution and sub-clonal detection, as well as population-based analyses. While CNAs identified by the OncoScan platform have shown good concordance with fluorescence in-situ hybridization (FISH) results, to date, no studies have been conducted to thoroughly assess the reproducibility of the assay. In this study, we have assessed the reproducibility of the OncoScan platform using identical samples performed in replicates across multiple chip batches. Moreover, we have assessed the effect on reproducibility of DNA treatment, including elution in water or TE buffer, as well as in the use of varying amounts of DNA. Methods: Affymetrix OncoScan FFPE Express 3.0 SNP Arrays were performed using the optimal input DNA as recommended by the manufacturer as well as fewer input amounts for comparison. CNAs were called using BioDiscovery Nexus Copy Number™ software (http://www.biodiscovery.com/software/nexus-copy-number/) using the SNP-FASST2 algorithm with modified parameters (significance threshold of 1 x 10 -9 and minimum number of probes per segment of 10). Results: Initial reproducibility analysis involving 12 samples repeated either 2, 4 or 6 times both within a single batch and across different batches has revealed that CNA calls were concordant between replicates for the majority of the genome (ranges between 81% to 100%), suggesting high precision of the assay. In addition, we are in the process of assessing and comparing mutation calls across replicates to gain a more in-depth understanding of the platform. Conclusion: This is the first study examining the reproducibility of OncoScan FFPE assays; initial results have suggested that the assay is precise and has the potential for robust biomarker discovery. Additional characterizations would be interesting for evaluating its use as a clinical tool in the long term. Citation Format: Cindy Q Yao, Cheryl Crozier, Mary Anne Quintayo, Jane Bayani, Melanie Spears, Julie Livingstone, Esther Jung, Clement Fung, Victoria Sabine, Paul C Boutros, John MS Bartlett. Assessing reproducibility of copy number arrays to assist breast cancer biomarker discovery [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-17.

Abstract P2-05-05: A four gene signature predicts anthracycline benefit: Evidence from the BR9601 and MA5 breast cancer trials

Background: Chromosome instability (CIN) in solid tumours is associated with poor prognosis and results in numerical and structural chromosomal aberrations. Recent evidence from both the BR9601 and MA.5 trials has demonstrated CEP17 duplication as a predictive marker of anthracycline benefit. CIN25 and CIN70 gene expression profiles have previously been published and predict survival response. An analysis of the BR9601 and MA5 clinical trials was performed to test the role of CIN gene expression signatures as a marker of anthracycline sensitivity. Methods: RNA was extracted from patients in both the BR9601 and MA5 studies and analysed through Nanostring technology. Log-rank analyses explored the prognostic values of the signatures on distant relapse-free survival (DRFS). Cox-regression models tested independent prognostic value on DRFS in the presence of treatment, age, tumour size, nodal status, ER status and grade, and treatment by marker interactions. Results: Of the 761 samples available from the BR9601 and MA5 cohorts we successfully analysed 703 (92.4%). High CIN25 and CIN70 scores were associated with age (p A more limited set of genes that reflected CIN was established by examining the expression profile of the genes and clustering them. The combined cohort was split into a 60% training and 40% validation set. The area under the curve (AUC) was calculated and the gene signature with the greatest AUC was selected and termed CIN4. Patients with low CIN4 score benefited from anthracycline treatment compared to those that had high CIN4 score (HR 2.72, 95% CI 1.48-5.02, p=0.001). No significant benefit with CMF treatment was observed in (HR: 1.02, 95% CI 0.58-1.82, p=0.92). After multivariate analysis the treatment by marker interaction for CIN4 had a hazard ratio of 2.10 (95% CI 2.18-30.38, p= 0.001). Conclusion: High CIN70 and CIN25 scores were associated with an aggressive phenotype and showed a potential increased sensitivity to anthracycline therapy compared to those with low CIN scores. CIN4 was an independent predictor of anthracycline benefit for DRFS. However, further work in larger patient cohorts such as NEAT is warranted. Citation Format: Melanie Spears, Nicola S Lyttle, Fouad Yousif, Alison F Munro, Christopher Twelves, Kathleen I Pritchard, Mark N Levine, Lois Shepherd, John MS Bartlett. A four gene signature predicts anthracycline benefit: Evidence from the BR9601 and MA5 breast cancer trials [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-05.