Abigail Evans

Endocrine Therapy, New Biologicals, and New Study Designs for Presurgical Studies in Breast Cancer

The preoperative setting is increasingly popular for the clinical investigation of hormonal agents and new biological drugs. The effectiveness of endocrine agents is well established for estrogen receptor-positive disease, and the emphasis in preoperative studies is on their combination with agents targeted at resistance mechanisms over 3 or more months. New agents are also being assessed for early evidence of clinical efficacy in shorter-term window-of-opportunity studies. The establishment of Ki67 as an intermediate marker of treatment benefit and of long-term outcome, with endocrine drugs, provides the opportunity for new trial designs with Ki67 as the primary endpoint. The PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial is randomizing (2:1) 4000 estrogen receptor-positive patients to 2 weeks presurgical treatment with a nonsteroidal aromatase inhibitor or no presurgical treatment. It provides a unique opportunity for detailed study of the determinants of response and resistance to estrogen deprivation as well as testing the role of presurgical therapy for improved biomarker-based estimates of prognosis.

EGFR/HER2 inhibitor AEE788 increases ER-mediated transcription in HER2/ER-positive breast cancer cells but functions synergistically with endocrine therapy

Background:Cross-talk between receptor tyrosine kinases and the oestrogen receptor (ER) is implicated in resistance to endocrine therapy. We investigated whether AEE788 (a combined inhibitor of EGFR, HER2 and VEGFR) plus tamoxifen or letrozole enhanced the individual anti-tumour effects of these agents.Methods:Breast cancer cell lines modelling endocrine-resistant and -sensitive disease were engineered to express aromatase (A) and examined using proliferation, western blotting and ER-α transcription assays.Results:AEE788 enhanced the anti-proliferative effect of tamoxifen and letrozole in ER+ cell lines (MCF-7 2A, ZR75.1 A3 and BT474 A3). This associated with an elevated G1 arrest and nuclear accumulation of p27. It is noteworthy that AEE788 alone or in combination with endocrine therapy increased the expression of progesterone receptor (PGR) and TFF1 in BT474 A3 cells. This may indicate a mechanism of resistance to AEE788 in ER+/HER2+ breast cancers. In a ZR75.1 A3 xenograft, AEE788 alone or in combination with tamoxifen provided no further benefit compared with letrozole. However, letrozole plus AEE788 produced a significantly greater inhibition of tumour growth compared with letrozole alone.Conclusion:These data suggest that AEE788 plus letrozole in breast cancer overexpressing HER2 may provide superior anti-tumour activity, compared with single agents.

Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy.

Antiproliferative Effect of Lapatinib in HER2-Positive and HER2-Negative/HER3-High Breast Cancer: Results of the Presurgical Randomized MAPLE Trial (CRUK E/06/039)

Purpose: Not all breast cancers respond to lapatinib. A change in Ki67 after short-term exposure may elucidate a biomarker profile for responsive versus nonresponsive tumors. Experimental Design: Women with primary breast cancer were randomized (3:1) to 10 to 14 days of preoperative lapatinib or placebo in a multicenter phase II trial (ISRCTN68509377). Biopsies pre-/posttreatment were analyzed for Ki67, apoptosis, HER2, EGFR, ER, PgR, pAKT, pERK, and stathmin by IHC. Further markers were measured by RT-PCR. Primary endpoint was change in Ki67. HER2+ was defined as 2+/3+ by IHC and FISH+. Results: One hundred twenty-one patients (lapatinib, 94; placebo, 27) were randomized; of these, 21% were HER2+, 78% were HER2− nonamplified, 26% were EGFR+. Paired samples containing tumor were obtained for 98% (118 of 121). Ki67 fell significantly with lapatinib (−31%; P < 0.001), but not with placebo (−3%). Whereas Ki67 reduction with lapatinib was greatest in HER2+ breast cancer (−46%; P = 0.003), there was a significant Ki67 decrease in HER2− breast cancer (−27%; P = 0.017) with 14% of HER2− breast cancer demonstrating ≥50% Ki67 reduction with lapatinib. Among HER2+ patients, the only biomarker predictive of Ki67 response was the EGFR/HER4 ligand epiregulin (EREG) (rho = −0.7; P = 0.002). Among HER2− tumors, only HER3 mRNA levels were significantly associated with Ki67 response on multivariate analysis (P = 0.01). In HER2− breast cancer, HER2 and HER3 mRNA levels were highly correlated (rho = 0.67, P < 0.001), with all Ki67 responders having elevated HER3 and HER2 expression. Conclusions: Lapatinib has antiproliferative effects in a subgroup of HER2− nonamplified tumors characterized by high HER3 expression. The possible role of high HER2:HER3 heterodimers in predicting response to lapatinib merits investigation in HER2− tumors. Clin Cancer Res; 21(13); 2932–40. ©2014 AACR. See related commentary by Campbell and Moasser, p. 2886

Abstract P2-05-01: Clinico-pathological relationships with Ki67 in POETIC (CRUK/07/015) – Critical lessons for assessing Ki67 for prognosis and as a pharmacodynamic marker

Background Higher levels of the proliferation marker Ki67 at breast cancer (BC) diagnosis are increasingly recognised to indicate poorer prognosis. Change in ΔKi67 in response to endocrine treatment reflects response. The precise relationship between Ki67 and other clinico-path factors and how associations are affected by short exposure to aromatase inhibitor (AI) has been unclear. Methods POETIC was a UK-wide, phase III, randomised trial which tested perioperative use of AI (anastrozole (A), letrozole (L)) in postmenopausal women with early BC (Dowsett JNCI Monogr 2011). Ki67 was measured in a single central lab at diagnosis (B=baseline) and 2 weeks later at surgery (S) allowing in vivo assessment of AI sensitivity. POETIC recruited 4483 women from 130 UK centres. Paired biopsies were available for 96%. Relationship between Ki67 and clinico-path factors is described by summary statistics (median) and independent associations explored in multivariable linear regression models (MVM). Analyses of Ki67 at S and ΔKi67 (reduction) were adjusted for B Ki67 and surgical sample type. Results Absolute Ki67 (B) was associated with each classic prognostic factor. Factors affecting ΔKi67 in control pts were Ki67 (B), grade (B) and surgical sample type (core cut 4.1%; excision 17.7% p Summary POETIC provides the largest multi-centre series of women in whom centrally assessed Ki67 has been correlated with classic clinico-path factors and impact of short term AI exposure explored. Choice of AI and surgical sample type are both dictated by participating site and their inter-relationship requires further review. Whether the greater suppression of Ki67 following L, a drug associated with better E2 suppression and aromatisation than A has clinical consequences is beyond the scope of this work. Relationships found (e.g. sample type, grade) are critical for interpretation of studies using Ki67 for prognosis and ΔKi67 as a pharmacodynamic response marker. Citation Format: Bliss JM, Morden J, Evans A, Holcombe C, Horgan K, Mallon E, Raghavan V, Skene A, Dodson A, Hills M, Detre S, Zabaglo L, Graf M, Banerji J, Gillman A, Robertson J, Dowsett M, Smith I, On Behalf of the POETIC Trialists. Clinico-pathological relationships with Ki67 in POETIC (CRUK/07/015) – Critical lessons for assessing Ki67 for prognosis and as a pharmacodynamic marker [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-01.

Abstract PD07-07: Prediction of antiproliferative response to lapatinib by HER3 in an exploratory analysis of HER2-non-amplified (HER2−) breast cancer in the MAPLE presurgical study (CRUK E/06/039)

Aim: To identify pretreatment biomarker predictors of Ki67 response to lapatinib in women with HER2− primary breast cancer. Background: Lapatinib is an EGFR/HER2 inhibitor. Its clinical use is restricted to HER2 overexpressing disease. The MAPLE (Molecular Antiproliferative Predictors of Lapatinib9s Effects) presurgical window of opportunity study of lapatinib vs placebo was conducted in women with HER2-amplified (HER2+) or HER2− primary disease. Ki67 (primary end-point) was reduced by a geomean 46% (95%CI 23–63%, p = 0.002) and 27% (95%CI 8–42%, p = 0.008) in HER2+ and HER2− disease, respectively (Leary et al, AACR 2012). We have now assessed whether predictive biomarkers of the antiproliferative response in HER2− disease could be identified. Methods: 121 primary breast cancer patients were randomized (3:1) to 14 days of 1500mg/d lapatinib or placebo before surgery. Biopsies were taken before treatment and at surgery. Ki67 responders were defined as having a >/=50% reduction in Ki67 compared to baseline (Ellis, P et al, Breast Cancer Res Treat 1998, 48, 107). ER, PgR, HER2, EGFR, pAKT, pERK1/2 (nuclear and cytoplasmic), stathmin and apoptosis (TUNEL) were assessed by IHC (+FISH for HER2[all cases]) and scored visually by continuous methods. HER2, HER3, epiregulin (epir), amphiregulin (amphir) and neuregulin (neur) were assessed by qrtPCR. Results: Three of the 121 patients were excluded because of inadequate biopsy material. Ninety-one of the remaining 118 patients received lapatinib: 7/19 (37%) HER2+ cases and 10/72 (14%) HER2− cases were Ki67 responders. Thus while the proportion of Ki67 responders was higher for HER2+ disease there was a similar or higher absolute number of responders with HER2− disease. All of the following relates to patients with HER2− disease. None of the pretreatment levels of ER, PgR, pAKT, pERK1/2, EGFR, epir, amphir or neur were associated with Ki67 response (p > 0.20). However, HER3 (p = 0.01) and HER2 (p = 0.06) mRNA levels were associated with greater Ki67 response. There was a tendency for Ki67 response to be greater with lower baseline Ki67 (p = 0.07). Multivariate analysis showed only HER3 mRNA levels to be independently significant. HER2 and HER3 mRNA levels were highly correlated (rho = 0.67, p Conclusions: Lapatinib is antiproliferative in a subgroup of HER2− tumours. This exploratory analysis indicates that they are characterized by high HER3 expression. The possible importance of high HER2:HER3 heterodimers in predicting this response is supported by the relationship between HER2 and HER3 expression. Further exploration of lapatinib is merited in HER2− cases with high HER3 expression. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD07-07.

Major Impact of Sampling Methodology on Gene Expression in Estrogen Receptor–Positive Breast Cancer

Abstract To investigate the impact of sampling methodology on gene expression data from primary estrogen receptor–positive (ER+) breast cancer biopsies, global gene expression was measured in core-cut biopsies at baseline and surgery from patients randomly assigned to receive either two weeks of presurgical aromatase inhibitor (AI; n = 157) or no presurgical treatment (n = 56). Those genes most markedly altered in the AI group (eg, FOS, DUSP1, RGS1, FOSB) were similarly altered in the no treatment group; some widely investigated genes that were apparently unaffected in the AI group (eg, MYC) were counter-altered in the control group, masking actual AI-dependent changes. In the absence of a control group, these artefactual changes would likely lead to the most affected genes being the erroneous focus of research. The findings are likely relevant to all archival collections of ER+ breast cancer.

Abstract PD4-02: A study to determine the optimal method of detection and threshold for lymphoedema intervention: a multi-centre prospective study

Introduction Lymphoedema, a complication of nodal surgery in 30-40% of patients, reduces quality of life for sufferers. This prospective, multi-centre study compared multi-frequency bioimpedance spectroscopy (BIS, ImpediMed) with a validated perometer method to determine which test is more sensitive for detecting lymphoedema after axillary clearance and identify the factors predicting lymphoedema development. Material and methods Participants (n = 629) undergoing axillary clearance at 9 UK centres underwent pre-operative and arm volume measurements post-surgery (1, 3, 6, 9 & 12 months, then 6 monthly) by arm perometry, BIS measurements (L-Dex) and recorded self-reported symptoms via questionnaires. Follow-up was a minimum of two years from surgery. Change in arm volume was calculated using relative arm volume change (RAVC) with >10% increase defined as lymphoedema. The predictors of lymphoedema development and optimal method for its detection were assessed using Cox Regression, Log Rank and Kaplan-Meier survival analyses. Results In total, 629 women underwent axillary surgery, with a median age of 56 (range 22 to 90) years; 80% were ER positive and received endocrine therapy, 78% received radiotherapy and 65% received chemotherapy. Lymphoedema was detected by 24 months in 124 (20%) women by perometry. Using the LDex >10 cut-off score, bioimpedance sensitivity was 71% and specificity was 89% (PPV 47%) compared to RAVC changes. Women who had an RAVC >5%- 0.000001). Twenty-six per cent of ER negative patients developed lymphoedema compared to 19% ER positive cancer patients. The type (taxane versus no taxane) and whether chemotherapy was neo-adjuvant or adjuvant did not predict lymphoedema development. Univariate analysis revealed BMI (p=0.003), ER negativity (p= 5%- 5%- Conclusions This is the first report; ER negative cancer is associated with an increased risk of lymphoedema after axillary node clearance. Arm measurements should be taken from baseline in all patients undergoing axillary surgery and increases greater than 3% should lead to further surveillance to prevent lymphoedema development. Perometer measurement is the optimal technique for measuring and predicting the development of lymphoedema. A threshold RAVC of >5%- (Funded by NIHR Programme Grant). Citation Format: Bundred NJ, Ashton S, Riches K, Ashcroft L, Evans A, Todd C, Bramley M, Hodgkiss T, Purushotham A, Keeley V. A study to determine the optimal method of detection and threshold for lymphoedema intervention: A multi-centre prospective study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD4-02.

A poetic story: lessons learnt from the world's largest breast cancer window of opportunity study

Hormone sensitive breast cancer (BC) is a common disease in postmenopausal women. Generally seen as less aggressive than other BC subtypes patients have a continued risk of relapse for 15+ years (EBCTCG, 2011) thus the cumulative risk is not insubstantial. Efforts continue to identify patients who are at continued residual risk of relapse in order to develop new treatment strategies. Previous trials (IMPACT, 2007) suggested that aromatase inhibitor (AI) treatment for 2 weeks in the peri-surgical window-of-opportunity results in detectable biomarker changes (Ki67) and predicts long term outcome. Gene expression profiling offers opportunities to identify patients demonstrating early resistance to endocrine therapy. POETIC was a UK-wide RCT devised to provide definitive results on the role of perioperative (AI) treatment. Biopsies were taken at diagnosis and 2 weeks later at surgery thus allowing an in vivo assessment of AI sensitivity. To succeed, POETIC needed to overcome substantial barriers in relation to compliance with cancer wait times, recruitment of women at diagnosis, varied clinical practice and to ensure receipt of sufficient quality tissue samples for analysis of biomarker endpoints. Patient advocates were involved from inception. POETIC succeeded in recruiting 4486 women from 130 UK centres. Paired tissue samples were received for 96% patients. Lessons learnt in rolling out the worlds largest window-of-opportunity BC trial illustrate the viability and potential of this experimental model as a vehicle for testing early biological effects of novel agents and to identify women most likely to be at long term residual risk of relapse.

Abstract P6-08-07: Optimal method of detection and threshold for early intervention to prevent lymphoedema: A multi-centre prospective study

Introduction Women who undergo axillary surgery are at risk of developing lymphoedema. Early detection is recommended by measuring arm volume from a baseline before surgery to enable early intervention. The optimal measurement method to enable early detection and time to intervention are unclear. This prospective multi-centre study compares multi-frequency bioimpedance spectroscopy (BIS, ImpediMed) with the validated perometer method to determine which test is more sensitive for detecting the optimal threshold to prevent lymphoedema. Methods Participants (N = 960) undergoing axillary clearance at 9 UK centres have pre-operative and regular arm volume measurements post-surgery (1, 3, 6, 9 & 12 months, then 6 monthly), by the validated arm perometry compared with BIS (L-Dex) measurements as well as self-reported symptoms questionnaire. Change in arm volume was calculated using relative arm volume change (RAVC). The predictors of lymphoedema development and optimal method were assessed. Results Currently 612 patients, median age 55 (range 24 to 90) years, have 6 month follow-up data and 327 have 18 month follow-up data. Seventy six percent were ER positive and received endocrine therapy, 84% percent received radiotherapy and 67% received chemotherapy in addition to surgery. Lymphoedema by 18 months was detected in 19% (n=79) of women by perometry (≥10% RAVC) and a change in L-Dex of 10 was observed in 31% of women. A moderate correlation between perometer and BIS at 3 months (r=0.40) and 6 months (r=0.60), with a sensitivity of 73% and specificity of 84% was found. Univariate analysis revealed a threshold for early intervention to prevent lymphoedema was RAVC ≥5%- Conclusions The optimal threshold for early intervention to prevent progression to lymphoedema is ≥5%- (Funded by NIHR Programme Grant). Citation Format: Nigel J Bundred, Charlotte Stockton, Katie Riches, Linda Ashcroft, Abigail Evans, Anthony Skene, Maria Bramley, Tracey Hodgkiss, Arnie Purushotham, Vaughan Keeley, BEA Investigators. Optimal method of detection and threshold for early intervention to prevent lymphoedema: A multi-centre prospective study [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 P6-08-07.