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Abstract Background. Detection of circulating tumor DNA (ctDNA) after treatment of early stage breast cancer may identify molecular residual disease. In a prior proof-of-principle study we demonstrated that detection of ctDNA predicted relapse with high accuracy (Garcia-Murillas et al Science Trans Med 2015). We conducted an independent, prospective, multi-centre validation study. Methods. In this validation study, a cohort of 170 early stage breast cancer patients were recruited from five hospitals into two prospective sample collection studies. Patients were scheduled to receive standard chemotherapy, surgery +/- radiotherapy, adjuvant endocrine therapy and HER2 antibodies as appropriate. Plasma samples were collected for ctDNA analysis at baseline, post-surgery, three monthly for the first year of follow-up, and six monthly thereafter and shipped to a central lab for processing. Using previously established criteria, tumor was sequenced to identify somatic mutations that were tracked by digital PCR in DNA extracted from 4mls of plasma at all available time points. Buffy coat DNA was analysed at all time-points to control for clonal haematopoesis of indeterminate potential (CHIP) detection. The primary endpoint was to compare invasive disease free survival between patients with and without detection of ctDNA after treatment. A combined analysis of this validation study, and the prior proof-of-principle study, was also conducted to analyse secondary endpoints. Results. After tumor sequencing, 101 patients from the validation study had at least one mutation to track. At median 35.5 months follow-up, ctDNA was detected in plasma of 15.8% (16/101) patients. Detection of ctDNA strongly predicted relapse, hazard ratio 24.5 (95% CI 6.5 to 93.2, P<0.001 time-dependent Cox model), and was predictive of relapse in all tumor subtypes. In the combined analysis (N=144), lead-time between ctDNA detection and relapse was 10.7 months (95% CI 7.7-17.0). Six patients had a clinical relapse that was not detected by ctDNA prior to relapse. These patients had a distinct pattern of oligo-metastatic relapse, 3 patients with brain-only metastases (P=0.0068), 1 ovarian oligo-metastasis and 2 local disease recurrence. The level of ctDNA in baseline plasma, prior to treatment, was associated with tumor subtype, highest in triple negative breast cancer (P=0.0036). Conclusion. Detection of ctDNA after treatment is associated with a high risk of future relapse in early-stage breast cancer. Prospective studies are required to assess the potential of molecular residual disease detection to guide adjuvant therapy. Citation Format: Turner N, Garcia-Murillas I, Chopra N, Comino-Mendez I, Beaney M, Tovey H, Cutts R, Swift C, Kriplani D, Afentakis M, Hrebien S, Walsh G, Johnston S, Ring A, Russell S, Evans A, Skene A, Wheatley D, Dowsett M, Smith I. Molecular residual disease detection with circulating tumor DNA analysis predicts relapse in patients with early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-01.

Purpose: Invasive lobular carcinomas (ILC) are characterised by loss of the cell adhesion molecule, E-cadherin, most commonly due to somatic CDH1 mutations. These are typically oestrogen receptor (ER)-positive/HER2-negative luminal tumours, which have a similar prognosis to that expected for luminal invasive ductal carcinomas (IDC). However, approximately 15% of ILC will be ER-negative; either at the time of breast cancer diagnosis or at metastatic relapse due to loss of ER expression. Previous studies have suggested that patients with triple-negative ILC have a superior prognosis to matched controls with triple-negative IDC, despite different clinical features, including higher rates of leptomeningeal disease, ovarian and peritoneal metastases [1].

Background Experimental evidence (Fisher et al, 1989) & a small clinical trial (IMPACT) respectively suggested peri-operative endocrine therapy (ET) may improve long-term disease-related outcome in patients undergoing primary surgery for ER positive (ER+) breast cancer (BC) & that tumor Ki67 levels after 2 weeks of peri-operative aromatase inhibitor (POAI) therapy might offer an effective way of predicting outcome & the need for additional adjuvant treatment. POETIC (*Peri-Operative Endocrine Therapy - Individualising Care) is a phase III randomized controlled trial designed to test these hypotheses & provide data to determine whether 2 week Ki67 improves prediction beyond that by baseline Ki67 of the group who have a higher risk of relapse in the first years after diagnosis in spite of best current standard of care. Patients & methods Postmenopausal patients with ER+ BC were randomised 2:1 to either, POAI (centre choice: letrozole 2.5mg or anastrozole 1mg daily) for 14 days prior to & 14 days following surgery or no POAI (Control). Randomization was stratified by treating center; adjuvant treatment was per UK routine practice. Tissue samples were collected at baseline & surgery (FFPE) for blinded Ki67 testing. Primary endpoint was Time to Recurrence (TTR: time from randomization to loco-regional or distant recurrence or BC death). A secondary endpoint was Ki67 at baseline & after 2 weeks of AI. Results Between 2008 & 2014, 4480 patients (2976 AI, 1504 Control) were randomized from 130 UK centers. Median age was 67 (IQR 62-75), 18% had grade 3 tumors, 39% were node positive and 61% had tumor size>2cm. For adjuvant ET 314 patients (7.2%) received tamoxifen (Tam), 3695 (84.6%) an AI, 251 (5.7%) Tam changing to AI and 109 (2.5%) changing from AI to Tam. On 8 August 2017, median follow-up was 60.7 months (IQR 49.5 to 72.2). 408/4480 (9.1%) patients have had a TTR event; 263 (8.8%) allocated to POAI compared to 145 (9.6%) controls: HR=0.91 (95%CI: 0.74, 1.12) Log-rank p=0.37. Adjusted HR=0.91 (95%CI: 0.74, 1.11). The relationship of Ki67 (baseline & after 2 weeks) with TTR in both the POAI & control groups will be presented for the overall ER+ population & HER2 defined sub-groups. Discussion There was no significant evidence that four weeks of POAI improved TTR compared with no POAI. POETIC will provide definitive evidence on the role of 2 week POAI-treated Ki67 to inform future practice & trials in terms of the potential to identify a group of patients for whom current standard of care appears insufficient in the few years post diagnosis. Citation Format: Robertson JFR, Dowsett M, Bliss JM, Morden JP, Wilcox M, Evans A, Holcombe C, Horgan K, Kirwan C, Mallon E, Sibbering M, Skene A, Vidya R, Cheang M, Banerji J, Kilburn L, Dodson A, Smith I. Peri-operative aromatase inhibitor treatment in determining or predicting longterm outcome in early breast cancer – The POETIC* Trial (CRUK/07/015) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-03.

Abstract This abstract was withdrawn by the authors.

BACKGROUND Gene expression (expr) analyses are increasingly used for characterising the pharmacodynamic response of primary BC. This includes assessing ER+ BC9s dependence on estrogen (E) by measuring gene expr changes after AI-treatment. However, differences in tissue sampling and other preanalytic procedures between samples taken at diagnosis (D) and surgery (S), may lead to systematic artifactual changes that are falsely ascribed to the intervention. To identify genes whose expr is truly affected by AI, we measured global gene expr changes from paired core-cut biopsies at D and S from patients in the POETIC presurgical window trial. METHODS In POETIC, 4486 postmenopausal women with primary ER+ BC were randomised 2:1 to receive perioperative AI (2 weeks pre + 2 weeks post surgery, termed Tr) or no perioperative treatment (termed Con), allowing gene expr changes to be compared between Tr and Con. RNA was extracted from paired RNA-later stored core-cuts of 56 Con and 157 Tr patients and arrayed on Illumina whole genome expr BeadChips. Raw data was extracted, transformed, normalised and batch-corrected. Probes not detected (pu003e0.01) in u003e=25% of samples were discarded. Impact of AI on genes was evaluated based on difference of the expr mean changes (log 2 (S/D)) of the Tr and Con samples. RESULTS In the Con group, expr of 73 genes significantly changed (FDR MYC increase) but was unaffected in Tr. Such artifactual gene changes in Con tumors conceal true AI-induced changes that would not be detected in the absence of comparison with Con. 615 genes were down-regulated and 472 up-regulated in Tr but not Con. The majority of down-regulated genes were cell cycle or proliferation-associated or E-regulated, including ESR1, PDZK1, GREB1, HSPB1 . Functional mapping showed changes in the regulation of cyclins and cyclin dependent kinases impacting on G1/S and G2/M. Of note, up-regulated genes included CDK6 (target for CDK4/6 inhibitors) and CCND2, involved in G1/S checkpoint regulation ; SNAI2, TGFB3, TGFBR2, associated with tumour invasion and metastasis ; and other genes involved in aryl hydrocarbon receptor, Glioblastoma Multiforme, HIPPO and p53 signalling. CONCLUSION Expr of certain genes is altered by processes involved in presurgical window studies. In the absence of a Con group, these may be wrongly ascribed to an experimental intervention or wrongly considered as unaffected by the intervention (eg MYC in this study). Down-regulation of E-responsive and proliferation genes was an expected response to AI but increased expr of genes such as SNAI2 , CCND2 and CDK6 indicates immediate tumour re-wiring and provides mechanistic support for benefit from combination therapy with a CDK4/6 inhibitor. Citation Format: Gao Q, Lopez-Knowles E, Cheang MCU, Morden J, Martin L-A, Sidhu K, Evans D, Martins V, Dodson A, Skene A, Holcombe C, Mallon E, Abigail E, Bliss J, Robertson J, Smith I, Dowsett M. True effect of aromatase inhibitor (AI) treatment on global gene expression (expr) changes in postmenopausal ER+ breast cancer (BC) patients: A POETIC study (CRUK/07/015) [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 P2-09-02.

Abstract Background Neoadjuvant endocrine therapy (NAE) is often a good option for postmenopausal (PM) women with estrogen receptor positive (ER+) breast cancers(BC). Fall in Ki67 is widely accepted as valid for predicting favorable tumor response to NAE and improved outcome. We report our planned correlative study to investigate if intrinsic subtype impacts on Ki67 changes (ΔKi67) as measured by immunohistochemistry. We also explored the correlation of several ER+ BC relevant molecular features at baseline(B) with ΔKi67. Patients and methods POETIC is a phase III, randomized 2:1 study for 4486 PM patients with ER+ BC to determine whether peri-operative aromatase inhibitor (AI) followed by standard adjuvant therapy improves outcome compared with standard adjuvant therapy alone. The proliferation rate was estimated as percentage (%) of cancer cells staining for Ki67. Primary biological endpoint was defined as two-week (2wk) change in Ki67 (2wkΔKi67): ln[(2wk Ki67+0.1)/(B Ki67+0.1)]. Secondary endpoint: “responders”, was % change of Ki67 defined as (2wk Ki67 – B Ki67) *100/B Ki67. “Responder” was defined as follows: reduction <50% as poor (PR), 50-75% moderate and >75% as good responder (GR). Human whole genome expression(GE) Illumina BeadChips were performed. Data was obtained from 137 paired samples from the treatment group(T) and 49 pairs from the control(C) group with GE data passing quality check and baseline Ki67≥5% to minimise the impact of extreme values based on proportional ΔKi67. Intrinsic subtype and risk of recurrence(ROR) groups were calculated using PAM50. GE scores from Oncotype Dx, MammaPrint, p53 mutation/wildtype(Troester 2006), ER+ early response (ERE)(Hatzis 2011), estrogen-regulated genes subtypes (Oh 2006) and markers for 23 different immune cell types(Bindea 2013) were calculated. Associations of GE scores to endpoints of response were determined by Spearman correlation and chi-square tests. Bonferroni correction was used to control error rate with p<0.0005 deemed significant. Results At B of the 137 paired T, 64% were Luminal A (LumA), 22% Luminal B (LumB), 9% as HER-2 enriched (HER2-E), 2% as Basal-like (BLBC) and 3% as Normal-like. Subtypes at B were associated with response, with LumA showing the biggest reduction of Ki67 (p=0.0001) and GR. All GE, except ERE, correlated significantly with 2wkΔKi67 and response: higher risk groups associated with lowest reduction rate. None of immune cell types correlated with 2wkΔKi67, except that tumors enriched with T-helper 1 cell type were associated with PR (p < 0.000001). Comparing subtypes between time-points, 85% of LumB and 42% of HER2-E were assigned instead as LumA at 2wk regardless of response. Of the 15 ROR defined high-risk group, only 33% were assigned instead as low-risk at 2wk. Conclusion Both LumA and LumB are endocrine sensitive. A fall of Ki67 was observed in majority of cases. Most tumors estimated as high-risk by molecular profiling showed less response and most remained moderate or high risk of recurrence on endocrine therapy. Whether molecular profiling at 2wk after starting AI predicts for long-term outcome in PM women with ER+ better than at diagnosis will need to be determined. Citation Format: Cheang MCU, Morden J, Gao Q, Parker J, López-Knowles E, Detre S, Hills M, Zabaglo L, Tomiczek M, Mallon E, Robertson J, Smith I, Bliss J, Dowsett M, On Behalf of the POETIC Trialists. The impact of intrinsic subtypes and molecular features on aromatase inhibitor induced reduction of proliferation marker of Ki67 in primary ER+ breast cancer: A POETIC study (CRUK/07/015) [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 P2-10-02.

Abstract Background In a previous proof-of-principle study we demonstrated that detection of circulating tumour DNA (ctDNA) in the adjuvant setting, after completion of surgery and chemotherapy for early stage breast cancer, was associated with a high risk of early relapse. Here we present longer follow-up of the same series, to define the predictive power of ctDNA analysis for disease free survival, and assess the potential to predict overall survival. Methods We recruited a cohort of 55 women presenting with early stage, primary breast cancer, who were all scheduled to receive neo-adjuvant chemotherapy. The primary tumour was sequenced to identify somatic mutations, identifying at least one mutation in 43 patients. Mutations were tracked with digital PCR to identify ctDNA, in plasma samples taken either at a single post-surgical time point (2-6 weeks post-surgery) or with serial plasma samples taken every 6 months in the adjuvant setting. Results At a median 31.7 months follow-up, 42% (18/43) patients had relapsed. Detection of ctDNA at the single post-surgical time point was associated with poor disease free survival, HR=13.6 95%CI (4.5, 41.2) p<0.001, and overall survival HR=84.7 95%CI (9.8, 730.4) p<0.001. All patients with ctDNA detected in a single post-surgical time point relapsed and died in the follow-up period (7/7, 100% specificity), although the single post-surgery time point had modest 39% (7/18) sensitivity for relapse. Detection of ctDNA at any point in serial sampling was associated with poor disease free survival HR=25.7 95%CI (8.3, 79.8) p<0.001 and overall survival HR=47.1 95%CI (6.1, 366.1) p<0.001. All patients with ctDNA detected in a serial mutation tracking relapsed in the follow-up period (14/14, 100% specificity), with 78% (14/18) sensitivity for relapse. Sensitivity was limited by 3 cases of brain only relapse and one case of solitary ovarian relapse. Detection of ctDNA in serial sampling had a median lead-time of 8.1 months over clinical relapse. Conclusion Detection of ctDNA in the adjuvant setting has a high predictive power for future relapse and death from breast cancer. Therapeutic trials are required to determine whether mutation tracking identifies relapse sufficiently early to allow for further adjuvant therapy. Citation Format: Turner NC, Garcia-Murillas I, Chopra N, Beaney M, Kilburn L, Cutts R, Osin P, Nerurkar A, Schiavon G, Hrebien S, Bliss J, Dowsett M, Smith I. Circulating tumor DNA analysis to predict relapse and overall survival in early breast cancer – Longer follow-up of a proof-of-principle 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 P1-02-01.

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 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<0.001). After 2 weeks of AI, variation in extent of ΔKi67 was dominated by PgR, HER2 and grade (B). Centres using L more often supplied excision samples (p<0.001). In spite of this both surgical sample type and choice of AI (both p<0.001) independently influenced ΔKi67 (core cut: A 65.1% L 73.8% excision A 75.2% L 81.3%). Pattern of change in grade between B and S differed for AI and control. Baseline Ki67 %Ki67 change (%) with 2 weeks AIVariableNMedianln(Ki67) P value (MVM)NMedianln (Ki67(S)/Ki67 (B)) P Value (MVM)All patients391315.2 252877.6 PgR (local) 0.01 <0.001Positive221914.6 141180.0 Negative47720.4 32661.1 Not known121715.1 79177.0 HER2 (local) <0.001 <0.001Negative345914.3 224079.2 Positive40226.8 26552.9 Unknown5213.9 2385.5 Tumour grade (B) <0.001 <0.001G15489.1 34378.6 G2227514.5 149479.6 G370330.1 44762.1 Unknown38714.0 24475.4 Tumour size (S) 0.002 0.49<2cm150312.9 98379.1 2-5cm222117.5 142576.4 >5cm17416.9 11474.1 Histological type (B) <0.001 0.58Ductal313816.3 203077.5 Lobular54911.9 35379.0 Other/Not known22613.1 14574.0 Nodes involved 0.03 0.530235714.1 153577.6 1-3110815.8 70878.2 4+43320.3 27876.1 Vascular invasion <0.001 0.03Yes112119.9 71175.9 No260313.6 169578.2 Unknown18914.6 12275.8 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.

Purpose: Leptomeningeal disease (LMD) is an uncommon complication of advanced breast cancer. The prognosis is poor, and although radiotherapy (RT), systemic and intra-thecal (IT) chemotherapy are accepted treatment modalities, efficacy data are limited. This study was designed to evaluate potential predictors of survival in this patient group. Methods: Breast cancer patients with LMD diagnosed by MRI in a 10-year period (2004-2014) were identified from electronic patient records. PFS and OS estimates were calculated using Kaplan-Meier method, with planned sub-group analysis by treatment modality. Cox regression was employed to identify significant prognostic variables. Results: We identified 182 eligible patients; all female, median age at LMD diagnosis 52.5 years (range 23-80). Ninety patients (49.5%) were ER positive/HER2 negative; 48 (26.4%) were HER2 positive, and 27 (14.8%) were triple negative. HER2 status was unknown in 17 (9.3%). Initial management of LMD was most commonly whole or partial brain RT in 62 (34.1%), systemic therapy in 45 (24.7%) or supportive care alone in 37 (20.3%). Fourteen patients (7.7%) underwent IT chemotherapy, of whom two also received IT trastuzumab. From diagnosis of LMD, the median PFS was 3.9 months (95% CI 3.2-5.0) and median OS was 5.4 months (95% CI 4.2-6.6). Patients treated with systemic therapy had the longest OS (median 8.8 months, 95% CI 5.5-11.1), compared to RT; 6.1 months (95% CI 4.2-7.9 months), IT therapy; 2.9 months (95% CI 1.2-5.8) and supportive care; 1.7 months (95% CI 0.9-3.0). On multivariable analysis, triple negative histology, concomitant brain metastases, and LMD involving both the brain and spinal cord were associated with poor OS. Conclusions: Breast cancer patients with triple negative LMD, concomitant brain metastases or LMD affecting both the spine and brain have the poorest prognosis. Clinical trials to identify more effective treatments for these patients are urgently needed. (C) 2017 Elsevier Ltd. All rights reserved.

Abstract BACKGROUND Gene expression (expr) analyses are increasingly used for characterising the pharmacodynamic response of primary BC. This includes assessing ER+ BC's dependence on estrogen (E) by measuring gene expr changes after AI-treatment. However, differences in tissue sampling and other preanalytic procedures between samples taken at diagnosis (D) and surgery (S), may lead to systematic artifactual changes that are falsely ascribed to the intervention. To identify genes whose expr is truly affected by AI, we measured global gene expr changes from paired core-cut biopsies at D and S from patients in the POETIC presurgical window trial. METHODS In POETIC, 4486 postmenopausal women with primary ER+ BC were randomised 2:1 to receive perioperative AI (2 weeks pre + 2 weeks post surgery, termed Tr) or no perioperative treatment (termed Con), allowing gene expr changes to be compared between Tr and Con. RNA was extracted from paired RNA-later stored core-cuts of 56 Con and 157 Tr patients and arrayed on Illumina whole genome expr BeadChips. Raw data was extracted, transformed, normalised and batch-corrected. Probes not detected (p>0.01) in >=25% of samples were discarded. Impact of AI on genes was evaluated based on difference of the expr mean changes (log2(S/D)) of the Tr and Con samples. RESULTS In the Con group, expr of 73 genes significantly changed (FDR<5%); 70 of these changed by a similar magnitude in the Tr group, indicating their change was independent of AI therapy but would have been artifactually discovered as changed by AI in the absence on Con. The 8 genes most up-regulated in Tr were all among the 20 genes most up-regulated in Con: many were early-response or stress-associated genes. Three of the 8 most down-regulated in AI were the most down-regulated in Con: all were haemoglobin-related. Expr of some genes was changed in Con (eg MYC increase) but was unaffected in Tr. Such artifactual gene changes in Con tumors conceal true AI-induced changes that would not be detected in the absence of comparison with Con. 615 genes were down-regulated and 472 up-regulated in Tr but not Con. The majority of down-regulated genes were cell cycle or proliferation-associated or E-regulated, including ESR1, PDZK1, GREB1, HSPB1. Functional mapping showed changes in the regulation of cyclins and cyclin dependent kinases impacting on G1/S and G2/M. Of note, up-regulated genes included CDK6 (target for CDK4/6 inhibitors) and CCND2, involved in G1/S checkpoint regulation; SNAI2, TGFB3, TGFBR2, associated with tumour invasion and metastasis; and other genes involved in aryl hydrocarbon receptor, Glioblastoma Multiforme, HIPPO and p53 signalling. CONCLUSION Expr of certain genes is altered by processes involved in presurgical window studies. In the absence of a Con group, these may be wrongly ascribed to an experimental intervention or wrongly considered as unaffected by the intervention (eg MYC in this study). Down-regulation of E-responsive and proliferation genes was an expected response to AI but increased expr of genes such as SNAI2, CCND2 and CDK6 indicates immediate tumour re-wiring and provides mechanistic support for benefit from combination therapy with a CDK4/6 inhibitor. Citation Format: Gao Q, López-Knowles E, Cheang MCU, Morden J, Martin L-A, Sidhu K, Evans D, Martins V, Dodson A, Skene A, Holcombe C, Mallon E, Abigail E, Bliss J, Robertson J, Smith I, Dowsett M. True effect of aromatase inhibitor (AI) treatment on global gene expression (expr) changes in postmenopausal ER+ breast cancer (BC) patients: A POETIC study (CRUK/07/015) [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 P2-09-02.

Background: Trastuzumab (T), a recombinant monoclonal antibody against HER-2 receptor, significantly improves overall (OS) and disease-free survival (DFS) in women with HER2 positive (HER2+) early breast cancer (EBC) when administered concurrent with or sequentially after adjuvant chemotherapy. Material and Methods: HERA (BIG 1-01) is an international, multicenter, phase III randomized trial involving 5102 women with HER2-positive (HER2+) EBC either nodal negative (tumor-size ≥ 1cm) or nodal positive. After completion of primary therapy, including surgery, chemotherapy and radiotherapy as indicated, patients (pts.) were randomized to T every 3 weeks for 1 yr, 2 years (yrs), or observation. Primary endpoint is DFS and secondary endpoints are OS and time to distant recurrence (TTDR). Here, we are presenting the HERA final analysis after 10 years of follow-up. Results: The clinical data cut-off for this final analysis of the HERA trial is June 2015. Data are being cleaned and final safety and efficacy analyses will be available for presentation at the meeting. Cardiac toxicity remained low and occurred mostly during the treatment phase. Citation Format: Jackisch C, Piccart MJ, Gelber RD, Procter M, Goldhirsch A, DeAzambuja E, Castro Jr G, Untch M, Smith I, Gianni L, Baselga J, Al-Sakaff N, Lauer S, Mcfadden E, Leyland-Jones B, Bell R, Dowsett M, Cameron D. HERA trial: 10 years follow up of trastuzumab after adjuvant chemotherapy in HER2 positive early breast cancer – Final analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD5-01.

Background: There is great interest in the mutational landscape of metastatic breast cancer (BC) for personalised medicine. Knowledge about how this landscape differs in recurrent lesions from primary BC as a result of metastatic selection or the impact of treatment will extend our knowledge of mechanisms of endocrine resistance and determine the manner in which diagnostics and treatment are integrated. A small number of limited series have reported an increased presence of ESR1 mutations in metastatic lesions after endocrine treatments including aromatase inhibitors (AIs) which are the most common agents for ER+ postmenopausal BC. Aim: To determine the change in mutational profile of 16 genes affected by driver mutations in patients with metastatic BC at the time of progression after an AI. Methods: We conducted targeted sequencing with a custom AmpliSeq panel in 48 matched pairs of FFPE archival blocks of pre-treatment diagnostic and recurrent lesions. 24 of these were metastatic, 24 local recurrences. The genes were AKT1, BRAF, CDH1, ERBB2, ESR1, GATA3, KIT, KRAS, MAP2K4, MAP3K1, PIC3CA, PIK3R1, PTEN, RUNX, SF3B1 and TP53. Only mutations with high depth (u003e250x) were retained. Germline mutations were identified by 1000 Genomes Project data. C:Gu003eT:A transitions with low frequency ( Results: Median coverage was 782-fold. Good quality data was available on 42 pairs. At least one mutation was found in 34 pairs. Three cases had lost IHC expression of ER in the recurrence. A total of 115 mutations in coding regions or splice sites were identified, the largest number in PIK3CA (in 20 samples), CHD1 (18), MAP3K1 (12), TP53 (12) and PTEN (9). In 47 instances the mutation was private to one or other sample. There was no difference in the number of mutations between the presentation and recurrent lesions. Six ESR1 mutations were found, all private to 5 recurrent lesions (all IHC ER+). Four of these mutations have been previously reported (2x E380Q, 2x D538G), one was novel (D484G) and also in the ligand-binding domain. One had an additional previously reported mutation (H524L). Four of these lesions were metastatic but one in a local recurrence suggesting that the metastatic process is more likely, but not essential in selecting the emergence of ESR1 mutations. HER2 mutations were identified in 3 patients; in two cases private in the presentation tumour while in the other case the mutation in the recurrent lesion (L755S) differed from that in the primary. L755S has been reported to be sensitive to neratinib in experimental systems. CDH1 (e-cadherin) mutations were numerically higher than reported in other series. The mutations were across the gene and private in 4 pretreatment and 2 recurrent lesions (6 had identical mutations). They were not associated with lobular phenotype. Conclusions: These data confirm that recurrences after AI but not primary ER+ tumours often contain ESR1 mutations that could influence clinical decision making. The high number of CDH1 mutations at diagnosis may be at least in part because of the selection of recurrent ER+ cases. The data stress the individuality of mutational profiles in recurrent BC and the need for individual interpretation of the data. Citation Format: Gellert P, Ribas R, Pancholi S, Lopez-Knowles E, Yeo B, Garcia-Murillas I, Pearson A, Smith I, Turner N, Dowsett M, Martin L-A. Occurrence of natural ESR1 mutations during acquisition of endocrine resistance in breast cancers and widely used ER+ cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S6-02.

Abstract Background: There is great interest in the mutational landscape of metastatic breast cancer (BC) for personalised medicine. Knowledge about how this landscape differs in recurrent lesions from primary BC as a result of metastatic selection or the impact of treatment will extend our knowledge of mechanisms of endocrine resistance and determine the manner in which diagnostics and treatment are integrated. A small number of limited series have reported an increased presence of ESR1 mutations in metastatic lesions after endocrine treatments including aromatase inhibitors (AIs) which are the most common agents for ER+ postmenopausal BC. Aim: To determine the change in mutational profile of 16 genes affected by driver mutations in patients with metastatic BC at the time of progression after an AI. Methods: We conducted targeted sequencing with a custom AmpliSeq panel in 48 matched pairs of FFPE archival blocks of pre-treatment diagnostic and recurrent lesions. 24 of these were metastatic, 24 local recurrences. The genes were AKT1, BRAF, CDH1, ERBB2, ESR1, GATA3, KIT, KRAS, MAP2K4, MAP3K1, PIC3CA, PIK3R1, PTEN, RUNX, SF3B1 and TP53. Only mutations with high depth (>250x) were retained. Germline mutations were identified by 1000 Genomes Project data. C:G>T:A transitions with low frequency (<10%) were removed as artefacts from formalin cross-linking. Results: Median coverage was 782-fold. Good quality data was available on 42 pairs. At least one mutation was found in 34 pairs. Three cases had lost IHC expression of ER in the recurrence. A total of 115 mutations in coding regions or splice sites were identified, the largest number in PIK3CA (in 20 samples), CHD1 (18), MAP3K1 (12), TP53 (12) and PTEN (9). In 47 instances the mutation was private to one or other sample. There was no difference in the number of mutations between the presentation and recurrent lesions. Six ESR1 mutations were found, all private to 5 recurrent lesions (all IHC ER+). Four of these mutations have been previously reported (2x E380Q, 2x D538G), one was novel (D484G) and also in the ligand-binding domain. One had an additional previously reported mutation (H524L). Four of these lesions were metastatic but one in a local recurrence suggesting that the metastatic process is more likely, but not essential in selecting the emergence of ESR1 mutations. HER2 mutations were identified in 3 patients; in two cases private in the presentation tumour while in the other case the mutation in the recurrent lesion (L755S) differed from that in the primary. L755S has been reported to be sensitive to neratinib in experimental systems. CDH1 (e-cadherin) mutations were numerically higher than reported in other series. The mutations were across the gene and private in 4 pretreatment and 2 recurrent lesions (6 had identical mutations). They were not associated with lobular phenotype. Conclusions: These data confirm that recurrences after AI but not primary ER+ tumours often contain ESR1 mutations that could influence clinical decision making. The high number of CDH1 mutations at diagnosis may be at least in part because of the selection of recurrent ER+ cases. The data stress the individuality of mutational profiles in recurrent BC and the need for individual interpretation of the data. Citation Format: Gellert P, Ribas R, Pancholi S, Lopez-Knowles E, Yeo B, Garcia-Murillas I, Pearson A, Smith I, Turner N, Dowsett M, Martin L-A. Occurrence of natural ESR1 mutations during acquisition of endocrine resistance in breast cancers and widely used ER+ cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S6-02.

Introduction and hypothesis Spinal anaesthesia (SA) and general anaesthesia (GA) are widely used techniques for vaginal surgery for pelvic floor disorders with inconclusive evidence of the superiority of either. We conducted a randomised controlled trial (RCT) to assess the feasibility of a full scale RCT aiming to examine the effect of anaesthetic mode for vaginal surgery on operative, patient reported and length of hospital stay (LOHS) outcomes.Methods Patients undergoing vaginal surgery, recruited through a urogynaecology service in a University teaching hospital, were randomised to receive either GA or SA. Patients were followed up for 12 weeks postoperatively. Pain was measured on a visual analogue scale; nausea was assessed with a four-point verbal rating scale. Patient's subjective perception of treatment outcome, quality of life (QoL) and functional outcomes were assessed using the International Consultation on Incontinence Modular Questionnaire (ICIQ) on vaginal symptoms and the SF-36 questionnaire.Results Sixty women were randomised, 29 to GA and 31 to SA. The groups were similar in terms of age and type of vaginal surgery performed. No statistically significant differences were noted between the groups with regard to pain, nausea, quality of life (QoL), functional outcomes as well as length of stay in the postoperative recovery room, use of analgesia postoperatively and LOHS.Conclusion This study has demonstrated that a full RCT is feasible and should focus on the length of hospital stay in a subgroup of patients undergoing vaginal surgery where SA may help to facilitate enhanced recovery or day surgery.

Bisphosphonates have been studied in randomised trials in early breast cancer to investigate their ability to prevent cancer treatment-induced bone loss (CTIBL) and reduce the risk of disease recurrence and metastasis. Treatment benefits have been reported but bisphosphonates do not currently have regulatory approval for either of these potential indications. This consensus paper provides a review of the evidence and offers guidance to breast cancer clinicians on the use of bisphosphonates in early breast cancer. Using the nominal group methodology for consensus, a systematic review of the literature was augmented by a workshop held in October 2014 for breast cancer and bone specialists to present and debate the available pre-clinical and clinical evidence for the use of adjuvant bisphosphonates. This was followed by a questionnaire to all members of the writing committee to identify areas of consensus. The panel recommended that bisphosphonates should be considered as part of routine clinical practice for the prevention of CTIBL in all patients with a T score of <-2.0 or ≥2 clinical risk factors for fracture. Compelling evidence from a meta-analysis of trial data of >18 000 patients supports clinically significant benefits of bisphosphonates on the development of bone metastases and breast cancer mortality in post-menopausal women or those receiving ovarian suppression therapy. Therefore, the panel recommends that bisphosphonates (either intravenous zoledronic acid or oral clodronate) are considered as part of the adjuvant breast cancer treatment in this population and the potential benefits and risks discussed with relevant patients.

Hintergrund: Ein Defekt in der homologen Rekombination (HR) ist ein Indikator für das Ansprechen auf Platinderivate und Poly(ADP-ribose)polymerase (PARP) Inhibitoren. Wir haben als Marker die RAD51 Foci Formation untersucht und mit den klinisch-pathologischen Daten korreliert. Zusätzlich untersucht ob die niedrige BRCA1 und PTEN Expression als Surrogatmarker für die defekte HR dienen kann.