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Background: The ER+/HER2+ subtype accounts for up to 10% of all breast cancers (BCs) and most are treated with surgery followed by adjuvant chemotherapy with Herceptin +/- radiotherapy then adjuvant endocrine therapy (ET) to reduce the recurrence risk. Despite this it is clear that not all ER+/HER2+ patients gain benefit from the addition of chemotherapy and Herceptin. In particular, given the significant side effects associated with the chemotherapy, the risks may out way the benefit in some older patients and in those with co-morbidities. Currently there are no clinically validated tools to identify women with ER+/HER2+ BC whose risk of recurrence remains unchanged with the addition of chemotherapy plus Herceptin and who can be effectively managed with adjuvant ET alone. Using levels of IL6ST, a biomarker for ET response, ER+/HER2+ patients who gain benefit from ET can be differentiated from those for whom ET alone is not sufficient to reduce the risk of recurrence. The latter group were characterised by inactive ER signalling and active MAPK and PI3K signalling. The aim is to show that ER+/HER2+ patients predicted to respond less well to ET alone using IL6ST levels, gain additional benefit from chemotherapy plus Herceptin. Patients: • Cohort A - 32 post-menopausal women (PMW) with large ER+/HER2+ BC treated with neoadjuvant (3-6 months) then adjuvant letrozole. Neoadjuvant clinical response was assessed by changes in tumour volume. Tumour core biopsies were taken at 0, 14 days and 3 months. Gene expression analysis using Illumina HT12 whole-genome beadarrays was performed on a subset (n=17) where fresh tissue was available. Median follow-up was 7.5 years. • Cohort B - 362 women with ER+/HER2+ BC treated with surgery +/- radiotherapy followed by adjuvant endocrine therapy between 2005 and 20010. 219 also received chemotherapy plus Herceptin. Median follow-up is 9.5 years. Results: In cohort A, half (16/32) of the patients responded to ET with tumour volume reductions of >70% with neoadjuvant treatment. Innate resistance was apparent in 3 patients with continued tumour growth on ET, whereas 13 patients acquired resistance after a period of response. Neoadjuvant clinical response was predicted with 92% accuracy using levels of IL6ST. Gene expression analysis in 17 patients showed increased MAPK and PI3K pathway activity in the 9 NR compared with the 8 R tumours. In the 16/32 patients who responded well to neoadjuvant ET the actuarial recurrence rate was 0% at 5 and 10 years. The rate of recurrence in the NR was 30% at both 5 and 10 years. Samples from cohort B are currently being profiled using IL6ST in tandem with custom gene expression assays for ER, MAPK and PI3K pathway activity. Conclusions: • IL6ST levels can differentiate ER+/HER2+ BCs who respond well to ET alone and those with a poor clinical response who have a higher risk of recurrence. • NR to ET have increased expression of PI3K and MAPK pathways, consistent with active HER2 signalling. • Analysis of cohort B is underway and will elucidate the benefit in terms of recurrence-free and overall breast cancer specific survival from the addition of chemotherapy plus Herceptin to the group predicted to respond less well to ET using IL6ST. • There is potential role for IL6ST in selecting ER+/HER2+ patients that require and benefit from HER2-targeted therapies. Citation Format: Arran K Turnbull, Victoria Webber, Daniel McStay, Laura M Arthur, Carlos Martinez-Perez, James Meehan, Mark Gray, Charlene Kay, Lorna Renshaw, Jane Keys, Robert Clarke, Andrew H Sims, Olga Oikonomidou, J Michael Dixon. Can some ER+/HER2+ patients be safely spared from treatment with chemotherapy plus herceptin? [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-07.

Background: The majority of patients with early-stage estrogen receptor positive (ER+) breast cancer (BC) are treated with adjuvant endocrine therapy (ET) after surgery to reduce the risk of recurrence. Recently, we have developed and validated an immunohistochemistry (IHC) based assay (EndoAdjuvant2 Clinical; EA2Clin) that measures pre-treatment IL6ST level together with clinical variables and on-treatment MCM4 to assess proliferation. We have previously shown that it can accurately identify responders and non-responders to ET and predicts recurrence-free survival (RFS) and BC-specific overall survival (BCSS). We postulated that measuring on-treatment proliferation in lymph node metastases (LN+) rather in the primary cancer might further improve the accuracy of the test for these patients. The aim was to test and validate this in cohorts of pre- and post-menopausal women (preMW & PMW) treated with preoperative ET (tamoxifen (T), fulvestrant (F), letrozole (L) or anastrozole (A)) and subsequent adjuvant ET. Methods: Cohorts: (1) 137 PMW with ER+ BC, 59 were LN+, treated with neoadjuvant L (median duration 4.8 months, range 1-33), then surgery followed by adjuvant L (n=109) or other ET (n=28); (2) 148 PMW with ER+ BC, 55 were LN+, treated with 2 weeks of preoperative L (n=76) or A (n=72), then surgery followed by adjuvant L (n=69) or T (n=79); (3) 52 preMW with ER+ BC, 24 were LN+, treated with 2 weeks of preoperative T (n=26) or 1x750mg dose of F (n=26), then surgery followed by adjuvant T. All LN+ patients had sentinel node biopsies or clearance. The median follow-up was 6.5 years (cohort 1), 6.3 years (cohort 2) and 10.2 years (cohort 3). EA2Clin: Patients are classified as: · Low risk: ER+ and LN-negative and · High risk: ER+ LN+ grade 3 BCs >2cm or pre-treatment IL6ST is 0 or 1+, or IL6ST is 2+ or 3+ and MCM4 in the primary has >10% positive nuclear staining. EA2CliN uses the post-treatment level of MCM4 in the nodes, rather than the primary cancer. Results: In cohort 1, EA2Clin (using primary tumour MCM4) was significantly associated with both RFS (P=0.0003, HR=13.17, 95%CI=5.48-13.61) and BCSS (P=0.005, HR=11.91, 95%CI=8.73-31.42). The 5 and 10 year actuarial recurrence rates were 5%/5% and 48%/64% for the low and high-risk groups respectively. In the same cohort, using the MCM4 level in the node (EA2CliN) there was an even more significant association with both RFS (P Discussion: · Direct measurement of on-treatment proliferation biomarkers in LN metastases improves prediction of outcomes to ET in women with BC. · This tests identifies a group of low risk women that are node negative and node positive with a 100% RFS and BCSS. · This is the most impressive predictive test for patients with ER+ breast cancer yet developed. Citation Format: Turnbull AK, Mok S, Martinez-Perez C, Fernando A, Renshaw L, Keys J, Sims AH, Dixon JM. Measurement of on-treatment proliferation biomarkers in nodal metastasis improves prediction of endocrine therapy response using the EA2CliN test [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 P5-11-03.

Background: ER+/HER2+ accounts for up to 10% of all breast cancers (BCs) and most are treated with endocrine therapy (ET) after surgery to reduce the recurrence risk. We developed and validated an immunohistochemistry (IHC) based test (EA2Clin) that incorporates baseline IL6ST, clinical variables and on-treatment measurement of MCM4. Responders (Rs) and non-responders (NRs) to ET are identified and it accurately estimates recurrence-free survival (RFS) and BC-specific overall survival (BCSS). The aim was to determine if EA2Clin could accurately predict ER+/HER2+ patients likely to benefit from ET and to determine if it can identify those for whom HER2-targeted therapies are required. Methods: 3 cohorts were studied: A: 32 post-menopausal women (PMW) with large ER+/HER2+ BC treated with neoadjuvant (3-6 months) then adjuvant letrozole. 5 also received adjuvant chemotherapy plus Herceptin. Neoadjuvant clinical response was assessed by changes in tumour volume. Tumour core biopsies were taken at 0, 14 days and 3 months. Gene expression analysis using Illumina HT12 whole-genome beadarrays was performed on a subset (n=17) where fresh tissue was available. B: 13 PMW with ER+/HER2+ BC who were treated by surgery without neoadjuvant therapy. RNA was extracted from excision tissues and analysed using whole-genome Affymetrix U133A microarrays. C: 15 PMW with ER+/HER2+ BC treated with 2-weeks of pre-operative letrozole (n=7) or anastrozole (n=8). All received adjuvant letrozole. Tissues were collected at pre-treatment and at surgery. None received Herceptin or chemotherapy. All patients were followed-up after surgery (median follow-up = 6.4 years). Results: In cohort A, half (16/32) of the patients responded to ET with tumour volume reductions of >70% with neoadjuvant treatment. Innate resistance was apparent in 3 patients with continued tumour growth on ET, whereas 13 patients developed resistance after a period of response. EAClin2 predicted neoadjuvant response with a 92% accuracy. There was increased expression of phospho-AKT and phospho-ERK in NRs, not seen in Rs. Half (8/16) of the NR cancers expressed phospho-ER; but was not seen in any responsive cancer. Gene expression analysis in 17 patients showed increased MAPK and PI3K pathway activity in the 9 NR compared with the 8 R tumours. These results were recapitulated in cohort B where MAPK and PI3K activity were associated with low levels of IL6ST. In the 16/32 patients who responded well to neoadjuvant ET the actuarial recurrence rate was 0% at 5 and 10 years. The rate of recurrence in the NR was 30% at both 5 and 10 years. Of the 5 patients who received chemotherapy plus Herceptin, none recurred despite a poor response to neoadjuvant letrozole (median length to last follow-up was 6.1 years). Initial data suggest that in cohort B EA2Clin identifies a group of ER+/HER2+ cancers that can be managed by ET alone. Conclusions: · The EA2Clin test identifies ER+/HER2+ BCs who respond well to ET alone and those with a poor clinical response who have higher risk of recurrence. · NR to ET have increased expression of PI3K and MAPK pathways, consistent with active HER2 signalling. · There is potential role for EA2Clin in selecting ER+/HER2+ patients that require and benefit from HER2-targeted therapies. Citation Format: Turnbull AK, Webber V, McStay D, Arthur L, Martinez-Perez C, Fernando A, Renshaw L, Keys J, Clarke R, Sims AH, Dixon JM. Predicting benefit from HER2-targeted therapies in patients with ER+/HER2+ 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 P3-10-26.

Background: The majority of patients with early-stage estrogen receptor positive (ER+) breast cancer (BC) are treated with adjuvant endocrine therapy (ET) after primary surgery to reduce the risk of recurrence. A variety of tests are available to predict outcomes on ET but most require gene-level measurements and are expensive. Recently, we developed an immunohistochemistry (IHC) based test (EA2Clin) using levels of pre-treatment IL6ST together with clinical variables and on-treatment proliferation. The aim was to validate this test in cohorts of both pre- and post-menopausal women treated with two weeks of a variety of endocrine treatments (tamoxifen, fulvestrant or an aromatase inhibitor) prior to surgery. Methods: The cohorts are: (A) 186 post-menopausal women (PMW) with ER+ BC treated with at least 2 weeks of preoperative or neoadjuvant letrozole or anastrozole, then surgery followed by adjuvant letrozole (n=132) or tamoxifen (n=54); (B) 51 pre-menopausal women (preMW) with ER+ BC treated with 2 weeks of either neoadjuvant tamoxifen (n=24) or one 750mg dose of faslodex (n=27), then surgery followed by adjuvant tamoxifen. The median follow-up was 5.4 years for cohort A and 10.2 years for cohort B. IHC analysis was performed using a Leica BOND III autostainer and the EA2Clin algorithm was used to stratify patients in binary high or low-risk groups. Results: In the cohort of PMW, EA2Clin was highly significantly associated with both recurrence-free survival (RFS) (P In the cohort of preMW, our test was significantly associated with both RFS (P=0.002, HR=5.71, 95%CI=1.91-17.05) and BCSS (P=0.016, HR=4.81, 95%CI=1.34-17.26). The 5 and 10 year actuarial recurrence rates were 12%/29% and 27%/77% for the low and high risk groups, respectively. The 5 and 10 year actuarial breast cancer-related death rates were 7%/19% and 9%/58% for low and high risk groups, respectively. Discussion: · This study has validated EA2Clin as the first predictive tool to incorporate clinical data with pre and on-treatment immunohistochemical biomarkers to predict accurately the outcome of patients with ER positive breast cancer treated with adjuvant ET. · This test predicts both RFS and BCSS in pre- and PMW treated with a variety of endocrine agents. · Because this test incorporates clinical variables with simple IHC, it can be performed locally in any pathology lab. Citation Format: Turnbull AK, Fernando A, Renshaw L, Keys J, Thomas JS, Sims AH, Dixon JM. EA2Clin: A novel immunohistochemical prognostic and predictive test for patients with estrogen receptor-Positive breast cancer [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 P4-08-03.

Background:Neoadjuvant therapy (NET) in women with large or locally advanced estrogen receptor(ER)-rich breast cancers (BC) allows the option of breast-conserving surgery (BCS) + radiotherapy (RT). The aim was to study the long term safety of this strategy. Methods: 280 postmenopausal women (median age 77, range 50-95, table 1) with ER-rich BC had BCS after NET (median duration 4.8 mths, range 1.7-42.8 mths). 221 (79%) received only letrozole. 59 (21%) began on letrozole and were switched to anastrozole, exemestane or tamoxifen due to adverse events or lack of clinical response. 200 patients (71%) had adjuvant RT (RTgroup) and 25 (9%), adjuvant chemotherapy. Median follow-up = 5.5 years. Table 1: Clinical characteristics of patients.CharacteristicNo. PatientsTumour SizeT131 (11%)T2169 (60%)T314 (5%)T456 (20%)Unknown10 (4%)Grade136 (13%)2153 (55%)370 (25%)Unknown21 (7%)Nodes+ve101 (36%)-ve177 (63%)Unknown3 (1%)ER Allred615 (5%)762 (22%)8203 (73%) Results: 254 patients had NET response data. 74% had a clinical response. NET response was higher for grade 1 (84%) than for 2 (73%) or 3 (72%) cancers. Actuarial local recurrence rates (LRR) were 8% (95%CI±0.04) and 12% (95%CI±0.06) at 5 & 10 years. Actuarial overall BC recurrence rates were 14% (95%CI±0.04) and 27% (95%CI±0.12) at 5 & 10 years, with BCS death rates of 7% (95%CI±0.04) and 14% (95%CI±0.10) at 5 & 10 years, showing only half with recurrence died from BC. Crude all-cause mortality but not BC-specific survival (BCSS) favoured those who had adjuvant RT (P Positive nodes were associated with worse overall recurrence free survival (RFS) (P=0.007) but not local RFS or BCSS. Tumour size was not associated with RFS or BCSS. Tumour grade was not associated with RFS but grade 3 patients had a lower BCSS (P=0.002) compared to patients grade 1/2 cancers. RT was associated with improved LRR (P 16/67 patients with T3/4 cancers with no RT had lower overall RFS (P=0.018) but no difference in local RFS. 13/98 patients with node +ve disease with no RT had lower LRR (P=0.002) and overall RR (P=0.024). 54/169 node -ve patients with no RT had lower LRR (P=0.019) but similar overall RR. 50/183 patients with grade 1/2 cancers had no RT and had lower LRR (P Discussion: · Response to NET is not worse in ER rich grade 3 or node positive cancers. · After NET, BCS and RT provides excellent LRR. · BCSS rates were low; most died of other causes. · NET followed by BCS and RT is safe even for grade 3 and node positive cancers. · BCS alone provides adequate disease control for majority with significant co-morbidities. Citation Format: Yau JD, Turnbull AK, Renshaw L, Keys J, Leeper A, Thomas JS, Dixon JM. Long-term outcome of neoadjuvant endocrine therapy followed by breast conserving surgery [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 P3-13-05.

Background Identifying breast cancer (BC) patients likely to recur on endocrine therapy (ET) is a challenge. Several methods and tests based on clinical parameters and multi-gene or protein classifiers have been shown to predict those likely to recur. Tests that incorporate baseline and on-treatment markers are likely to be more accurate than tests based on baseline characteristics alone. 4 genes were identified by microarray that predicted for to ET: 2 at diagnosis and 2 at 14 days. The EndoAdjuvant2 (EA2) test uses 2 of these genes: IL6ST at diagnosis and on-treatment MCM4 at transcript level or by graded immunohistochemistry (IHC). The aim of this study was to compare EA2 with currently used clinical parameters in 4 different cohorts of pre and postmenopausal women. Patients The cohorts are (1) 73 post-menopausal women (PMW) with ER+ BC treated with neoadjuvant letrozole (L) then surgery, (2) 39 PMW with ER+ BC treated with neoadjuvant anastrozole (A) then surgery, (3) 108 PMW who received 2-weeks of A or L prior to surgery (4) 25 preMW with ER+ BC who received one 750mg dose of fulvestrant prior to surgery. All had adjuvant ET and 5-10 years follow up. Neoadjuvant response was assessed by periodic 3D ultrasound. Results The 4 gene assay had 96% (training; cohort 1) and 93% (validation; cohort 2) accuracy of response prediction to neoadjuvant L or A. In cohort 1, clinical parameters were available. On univariate regression analysis intrinsic subtype (luminal A/B; defined using PAM50) (P=0.002), histological grade (P=0.033) and HER2 status (P=0.001) significantly predicted clinical response. EA2 out-performed all these in both univariate (P EA2 predicted recurrence free in cohorts 1 and 3 combined: RFS (P=0.0004, HR=17.63 95%CI: 4.95-17.6), BCSS (P=0.0007, HR=16.60: 3.36-45.7). The Nottingham Prognostic Index (NPI) also predicted RFS (P=0.0002) and BCSS (P=0.0017) in univariate analysis but in the Cox analysis NPI was not found to be significant, although in the low risk group there were only 1/46 events compared to 19/62 in the moderate/high risk group. Histological grade (P Conclusions • EA2 predicts clinical response, RFS and BCSS • EA2clin combines NPI and EA2, outperforms either alone and predicts outcome in a new validation cohort of preMW treated with Fulvestrant • EA2clin works in preM and PM women regardless of ET. Citation Format: Turnbull AK, Pearce DA, Arthur LM, Martinez-Perez C, Thomas JS, Fernando A, Renshaw L, Sims AH, Dixon JM. The EA2clin test significantly predicts response and survival in both pre and post-menopausal women with ER-positive breast cancers [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 P6-09-27.

Introduction Currently the primary breast tumour is used for prognostic profiling and as a monitor of response to therapy but how often does the molecular profile of the primary cancer reflect the molecular profile of nodal metastases? No previous study has investigated in detail the genomic profile of matched primary breast cancer (P) and nodal metastases (N) and correlated these with outcome. The aim of this study was to investigate whether the mRNA profiles of matched P and N differ significantly. Methods RNA was extracted from core biopsies from primary breast tumours and paired metastatic axillary lymph node samples from both FFPE blocks and fresh frozen samples. RNA was labelled and hybridised to Illumina HT-12 BeadChips to create a dataset consisting of one primary and one or two matched nodal metastasis, totalling 68 samples from 31 patients. Data was processed and corrected for batch effects, then analysed using the statistical programming language R. Clinical data on progression free and overall survival was collected from electronic and medical case note review. Results Unsupervised hierarchical clustering of the 500 most variable genes in each sample grouped only 12 of 31 P&Ns (39%) together, meaning in the majority of patients their P or N more resembled a cancer from another patient than its own paired P or N. The number of genes with greater than 2 fold change (>2FC) between P&N was used to categorise paired samples into 'least changed' (2 FC) and 'most changed' (>370 genes with >2FC) groups. Multidimensional scaling of the 500 most variable genes in the most changed group (n-=10) showed consistently that nodal metastases differed molecularly from the primary cancer. When categorised by Sorlie centroid, 12 of 31 patients (39%) had a different molecular subtype in N compared with P. N tended to be a poorer prognostic subtype than P. 50% had luminal A primaries paired with luminal B nodes. The remaining 50% changed in other non-consistent patterns. 6 patients had 2 N samples to analyse alongside P. 4 of these (67%) had the same subtype in all 3 samples, and a further 1 the same 2Ns (luminal B) which differed from P (luminal A). The final had luminal A P paired with 1 luminal A and 1 luminal B Ns. There was no evident correlation between the least changed and most changed groups and progression free and overall survival. This may however reflect the short term follow up. Discordance between P and N in expression of ESR1 was 32%; PGR 19% and ERBB2 16%. Conclusions This study of gene expression change in matched primary breast cancers and synchronous metastatic paired axillary lymph nodes shows that molecular subtype differs in 39%. 50% of nodes had a poorer prognostic subtype than their primary. Expression of ESR, PGR and ERBB2 differs in up to 32% Classifying cancer molecular phenotype and estimating prognosis based only on the primary cancer misclassifies significant numbers of patients. Classification of prognosis, and treatment based on the nodal metastasis may provide better information on which to base treatment. Citation Format: Arthur LM, Turnbull AK, Pearce DA, Renshaw L, Thomas JS, Sims AH, Dixon JM. Molecular characterisation, subtype concordance and prognostic group assignment between patient-matched primary breast tumours and axillary lymph node metastases [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 P1-06-04.

Accurate assessment of estrogen receptor (ER) expression is crucial to ensure that patients with early breast cancer are accurately identified for appropriate treatment with endocrine therapy. Reverse transcriptase polymerase chain reaction (RT-PCR), compared with immunohistochemistry (IHC), may provide a more precise indication of ER status. Data were pooled and analyzed from two independent, but similarly designed, studies that examined ER status by IHC and the 21-gene Recurrence Score that employs RT-PCR-based methodology. Tumor tissue from patients with early stage breast cancer where ER status could be determined by both IHC and RT-PCR was included. ER status by IHC staining was defined as ER-negative ( 10%). ER status by RT-PCR was defined as ER-negative (≤ 6.5) or ER+ (> 6.5). Recurrence Score results from the 21-gene assay were reported on a continuous scale from 0 to 100. A sub-analysis examined the association between ER expression (Allred score 2–7) and response to a 14-day pre-surgery pulse with an aromatase inhibitor. A separate sub-analysis examined the association between ER expression and human epidermal growth factor receptor 2 (HER2) expression. Tumor specimens from 192 patients (aged 25–92 years) were included in the pooled analysis. Correlation between IHC- and RT-PCR-measured ER was strong for IHC-defined ER-negative and ER+ samples (r = 0.646 [95% CI 0.553–0.720]). There was 100% concordance for ER+ tumors; however, 56% of the ER-low+ tumors were negative by RT-PCR. Allred score correlated better with ER status measured by RT-PCR at pre-treatment (r = 0.83) than at post-treatment (r = 0.76). The majority (77%) of ER-negative and ER-low+ tumors were HER2-negative. RT-PCR provided a more accurate assessment of ER expression in patients with ER-low+ tumors, and data support dual testing for patients with ER-low+ status to ensure appropriate treatment planning as it pertains to endocrine therapy. Genomic Health, Inc.

WOS: 000455112600001, PubMed ID: 30616553, BackgroundThe risk of recurrence for endocrine-treated breast cancer patients persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate; previous efforts have been limited to in-vitro or in-vivo approaches. In this study, sequential tumour samples from patients receiving extended neoadjuvant aromatase inhibitor therapy were characterised as a novel clinical model.MethodsConsecutive tumour samples from 62 patients undergoing extended (4-45months) neoadjuvant aromatase inhibitor therapy with letrozole were subjected to transcriptomic and proteomic analysis, representing before (0), early (13-120days), and long-term (>120days) neoadjuvant aromatase inhibitor therapy with letrozole. Patients with at least a 40% initial reduction in tumour size by 4months of treatment were included. Of these, 42 patients with no subsequent progression were classified as dormant, and the remaining 20 patients as acquired resistant.ResultsChanges in gene expression in dormant tumours begin early and become more pronounced at later time points. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to the resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. The DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared with dormant tissues after extended letrozole treatment.ConclusionsThis is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumours continue to change during treatment whereas acquired resistant tumours more closely resemble their diagnostic samples. Global loss of DNA methylation was observed in resistant tumours under extended treatment. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients, supporting a potential role for therapy targeted at these epigenetic alterations in the management of resistance to oestrogen deprivation therapy., Marie Sklodowska-Curie Individual Fellowship (H2020-MSCA-IF) [658170]; Breast Cancer Now; Welcome Trust Institutional Fund (ISSF3), The work was supported Marie Sklodowska-Curie Individual Fellowship (H2020-MSCA-IF, 658170) to CS. AHS and JMD are very grateful for funding provided by Breast Cancer Now. The work was partly supported by Welcome Trust Institutional Fund (ISSF3) to CS and AHS.

Background: Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive post-menopausal breast cancer. Recently we have developed and validated a microarray-derived 4-gene test (Edinburgh EndoResponse4) to predict response to AIs in the neoadjuvant setting. We have also demonstrated the translational potential of this test in predicting accurately clinical response when mRNA is measured for these genes by polymerase chain reaction (PCR) or the gene protein is measured by immunohistochemistry (IHC). There is a major clinical need for biomarkers to predict which patients are likely to recur on adjuvant endocrine therapy so alternative or additional treatments can be provided to reduce recurrence and improve outcome. The aim of this study was to determine if Endoresponse4 and IHC of these gene proteins could do this. Methods: The original microarray assay used pre- and on-treatment (14-days) biopsies from 73 post-menopausal women with ER-rich breast cancer receiving 3 months of neoadjuvant letrozole prior to surgery with 10 years follow-up after adjuvant letrozole. Matched formalin-fixed paraffin embedded (FFPE) tissue sections from 42 of these patients were used for IHC and antibodies were optimised against 3 of the 4 proteins (where validated antibodies were available) using Envision technology. The ability of our test to estimate recurrence-free (RFS) and breast cancer specific overall survival (OS) using both PCR and IHC was then tested in a unique validation cohort of 140 post-menopausal women with ER-rich breast cancer treated with 2 weeks of neoadjuvant letrozole or anastrozole prior to surgery followed by adjuvant endocrine therapy and 10 years of follow up.. Results: Within our training cohort (n=73) using Kaplan-Meier analysis our 4-gene test predicted neoadjuvant clinical response and demonstrated a significant association with both RFS (P=0.029) and OS (P=0.009). This approach predicts outcomes within 2-weeks rather than 4-months of treatment required in other studies such as P024. Using IHC in the training cohort (n=42), two gene markers in combination (IL6ST at diagnosis and MCM4 after 2-weeks treatment) predicted both RFS (P=0.017) and OS (P=0.009) with great accuracy. The 140 patient group is being analysed and the findings are so far are consistent with the initial training cohort and indicate a significant association with outcomes. Conclusion: • A 4 gene model with clinical potential has been developed and validated to predict response to neoadjuvant aromatase inhibitors. • This 4 gene model predicts for response and also predicts relapse free and overall survival. • Proteins encoded by 2 of these 4 genes measured by IHC in an initial test set of 42 patients predict accurately both EFS and OS • A validation cohort (n=140) with over 10-years of follow-up will be available at SABCS 2015 to determine if this 2 biomarker test can predict outcome on adjuvant endocrine therapy. Citation Format: Turnbull AK, Arthur LM, Webber V, Thomas J, Uddin S, Webb H, Dunbier A, Dowsett M, Renshaw L, Sims AH, Dixon JM. A validated test for neoadjuvant clinical response to endocrine therapy in breast cancer that estimates accurately recurrence-free and overall survival. [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 P3-07-20.

Purpose Aromatase inhibitors (AIs) have an established role in the treatment of breast cancer. Response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Accurate biomarkers are urgently needed to predict response in these settings and to determine which individuals will benefit from adjuvant AI therapy. Patients and Methods Pretreatment and on-treatment (after 2 weeks and 3 months) biopsies were obtained from 89 postmenopausal women who had estrogen receptor–alpha positive breast cancer and were receiving neoadjuvant letrozole for transcript profiling. Dynamic clinical response was assessed with use of three-dimensional ultrasound measurements. Results The molecular response to letrozole was characterized and a four-gene classifier of clinical response was established (accuracy of 96%) on the basis of the level of two genes before treatment (one gene [IL6ST] was associated with immune signaling, and the other [NGFRAP1] was associated with apoptosis) and the level of two proliferation genes (ASPM, MCM4) after 2 weeks of therapy. The four-gene signature was found to be 91% accurate in a blinded, completely independent validation data set of patients treated with anastrozole. Matched 2-week on-treatment biopsies were associated with improved predictive power as compared with pretreatment biopsies alone. This signature also significantly predicted recurrence-free survival (P = .029) and breast cancer –specific survival (P = .009). We demonstrate that the test can also be performed with use of quantitative polymerase chain reaction or immunohistochemistry. Conclusion A four-gene predictive model of clinical response to AIs by 2 weeks has been generated and validated. Deregulated immune and apoptotic responses before treatment and cell proliferation that is not reduced 2 weeks after initiation of treatment are functional characteristics of breast tumors that do not respond to AIs.

Intro Accurate measurement of ER in early stage invasive breast cancer (EBC) is important to identify patients likely to benefit from HT. While immunohistochemistry (IHC) is the most common method to quantify ER, other methods can also accurately measure ER, such as RT-PCR. ER is one of the genes included in the RT-PCR based 21-gene Recurrence Score assay (Oncotype DX®, Genomic Health, Redwood City, CA) and is also reported separately as a single gene expression. Additionally, the association between ER expression by RT-PCR (ER-PCR) and tamoxifen benefit has been reported by Kim, et al (2011). A recent study reported that patients with ER levels Aim The study aims are: (1) To correlate quantification of ER in EBC as assessed by Allred Score (AS) and ER as measured by RT-PCR in the 21-gene assay; (2) To describe changes in ER, Recurrence Score, and measures of proliferation after 2wks of an aromatase inhibitor (AI); (3) To perform exploratory analyses of factors associated with changes in proliferation. Methods 55 postmenopausal EBC patients with lower ER (AS 2-7) were treated with 2wks of an AI followed by wide excision. All patients had a 21-gene assay on a pre-and post-treatment (Tx) sample. Proliferation was measured by both Ki67 by IHC (in 45 patients) and by the proliferation gene group score (PGS) in the RT-PCR based 21-gene assay (in all patients). Proliferation response was defined by a 20% relative decrease in Ki67 or a decrease in PGS. Changes in proliferation were correlated with AS, ER-PCR and Recurrence Score result. Results The Table shows the correlation of AS with ER-PCR measured in the pre-Tx (r=0.83) samples. 94% of AS (2-3) patients and 56% of AS (4-5) were ER(-) by RT-PCR There was a significant change (pre to post) in the average Ki67 level (18% to 11%; p Pre-Tx ASER-PCR Status (Pre-Tx)NegativePositiveTotalAS 2-317 (94%)1(6%)18AS 4-59(56%)7(44%)6AS 6-70 (0%)21(100%)21Total262955 Conclusions • Results confirm earlier reports showing substantial disagreement in ER measured by IHC vs RT-PCR in patients with lower ER-expressing tumors • The clinical implications are that a substantial number of patients with low ER by IHC may have little to no benefit from HT • The 21-gene assay may be useful in selecting patients likely to benefit from HT • Further studies in larger cohorts are required to confirm these findings. Citation Format: J Michael Dixon, Arran Turnbull, Lorna Renshaw, Megan P Rothney, Cynthia A Loman, Laura Arthur, Jeremy S Thomas, Oliver Young, Juliette Murray, Linda Williams, Amy P Sing, David Cameron. Association of estrogen receptor (ER) levels and prediction of antiproliferative effect of hormone therapy (HT) in lower ER-expressing tumors [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 P3-06-35.

Background: Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive (ER+) post-menopausal breast cancer. Response rates are only 50-70% in the neoadjuvant setting and up to 40-50% of all adjuvantly treated patients will eventually relapse. Mutations in certain genes have been previously shown to confer resistance to therapy, and molecular subtype has associations with poor outcome (i.e. LumB and HER2E). In order to improve the outcomes of non-responders or patients who become resistant to endocrine treatment, the identification of key mutations, and their interaction with subtype, is crucial. Dynamic profiling of the same tumour demonstrating de novo or developing resistance after responding to one or more lines of endocrine treatment in the neoadjuvant setting provides a unique opportunity to identify such genomic changes. Methods: This series is unique in that it includes 17 post-menopausal women with ER+ breast cancer treated with neoadjuvant letrozole. 13 of these patients progressed on treatment or initially responded to treatment and then developed acquired resistance and 4 responded well. Dynamic clinical response was assessed for each patient using periodic 3D ultrasound measurements performed during treatment. Fresh tissue was taken before treatment and when the tumor was resistant to treatment (4 patients had 2 biopsies, 9 patients had 3 and 4 patients had 4 available biopsies taken). RNA and DNA were extracted from tumour and normal DNA obtained from either matched blood or normal lymphatic tissue. In total, 51 tumour samples were available and have completed RNA-Seq, with exome sequencing shortly to be completed. Results: From the RNA-seq data, the intrinsic subtype distribution was 9 LumA, 7 LumB, and 1 HER2-enriched; when stratified according to response, the "progressors" were 7 LumB, 5 LumA and 1 HER2-enriched, while "responders" were 3 LumA and 1 LumB. When examined in an unsupervised hierarchical clustering analysis along with >800 TCGA Breast tumor samples, 13/17 patients had all of their samples grouped immediately together, suggesting that the overall tumor phenotype was maintained. Interestingly, the most dominant change in gene expression was the observation that there were 5 "progressor" patients where the pre-treatment sample was LumB and all subsequent samples were LumA; we only observed one instance of a patient starting as LumA and changing to a LumB, who was also labelled as a "progressor". Full exome sequencing is underway and these results will be presented. Conclusion: Genomic analysis of progression suggests that an apparent "subtype shift" appears in a number of patients where a shift to LumA is seen; this apparent change may be reflective of decreased proliferation rates caused by therapy, or the acquisition of a true LumA phenotype. We cannot differentiate between these two hypotheses at this time, but expect that the exome sequencing will help to differentiate between these two hypotheses given the large number of mutations and copy number alterations that can differentiate between LumA vs. LumB. Citation Format: J Michael Dixon, Arran K Turnbull, Chris Fan, Joel S Parker, Xiaping He, Laura Arthur, Carlos Martinez-Perez, Lorna Renshaw, Charles Perou. In-depth genomic analysis of ER+ breast cancers during development of endocrine resistance [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 S1-05.

The phosphatidylinositol-3-kinase pathway plays an important role in proliferation, migration and survival in breast cancer and may play a role in resistance to endocrine therapy. Pathway activation occurs as a result of mutations in PIK3CA or loss of functional PTEN. Matched primary and recurrent samples from 120 breast cancer patients treated with endocrine therapy were profiled with a qPCR-based mutation assay covering eight mutational hotspots in PIK3CA. PTEN was assayed by immunohistochemistry. Samples were well characterized with respect to anatomic location of recurrence (metastatic nodal or local recurrence as opposed to contralateral or ipsilateral new primary cancers). In total, 43 % of patients had at least one PIK3CA mutation at diagnosis, and 41 % had a mutation at the time of recurrence. Only 8 % of patients with local recurrence, metastatic disease or progression on primary endocrine treatment changed their PIK3CA mutation status (four gains, two losses, total 76). The most common changes in PIK3CA mutation status were seen in patients who developed a new cancer either in the treated or contralateral breast (64 %, three gains, four losses, total 11). PIK3CA mutation status does not change in the majority of patients with breast cancer and the acquisition of mutations in PIK3CA is not responsible for the development of endocrine resistance. PTEN loss at diagnosis is associated with a significantly shorter time to progression compared with tumours in which PTEN was retained. These are the most comprehensive data currently available correlating PIK3CA status, site of recurrence and endocrine resistance.

Background: Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive post-menopausal breast cancer. Response rates are only 50-70% even in patients with ER-rich cancers in the neoadjuvant setting and are lower in advanced disease. Recently we developed and validated a microarray-derived 4-gene test to predict response to AIs in the neoadjuvant setting. Whole-genome expression analysis is impractical for clinical utility. There is a need to translate and validate any test utilising clinically accessible and reproducible material and technologies such as polymerase chain reaction (PCR) and immunohistochemistry (IHC). Methods: The original microarray experiment used pre- and on-treatment (at 14 days and 3-months) biopsies from 89 post-menopausal women with ER-rich breast cancer receiving 3 months of neoadjuvant letrozole. Dynamic response was based on periodic 3D ultrasound measurements performed during treatment. The derived 4-gene model was independently validated in a cohort of 44 post-menopausal women with ER-rich breast cancer treated with neoadjuvant anastrozole [table 1]. RNA was extracted from the original biopsies for RT-qPCR analysis using validated primers for the 4 genes with SYBR-green technology normalised to the geometric mean of 3 housekeeping genes. Matched formalin-fixed paraffin embedded (FFPE) tissue sections were used for IHC with optimised antibodies against 3 of the 4 proteins (where validated antibodies were available) using Envision technology. Results: PCR: Microarray and PCR expression levels for each of the four genes were well correlated (Pearson r=0.87-0.65, p Conclusion: Table 1NAccuracySensitivitySpecificityPPVNPVMicroarray (Training)890.960.890.980.980.96Microarray (Validation)440.910.800.970.920.90PCR260.960.951.001.000.86IHC280.890.861.001.000.89Sensitivity and specificity of model in training (microarray, PCR, IHC) and validation datasets (microarray). PPV/NPV = postivie/negative predictive value. •A 4 gene model has been developed and validated to predict response to neoadjuvant aromatase inhibitors. •This model has been shown to work with a high degree of accuracy using both PCR and IHC technologies. Further independent validation is currently underway. •This new test has the potential to predict accurately the benefit of endocrine therapy and has huge potential clinical value. Citation Format: Arran K Turnbull, Laura Arthur, Victoria Webber, Jeremy Thomas, Charlotte Heerlyn, Phoebe Thornton, Anita Dunbier, Mitch Dowsett, Lorna Renshaw, Andrew H Sims, J Michael Dixon. Development for clinical utility of a validated predictive test of clinical response to aromatase inhibitors [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 P3-05-01.

Background 10% of all breast cancers are HER2+/ER+ and these cancers exhibit both intrinsic and acquired resistance to endocrine therapy. They have a worse prognosis than HER2-/ER+ cancers. Aims 1. To investigate the role of HER2 in response to neoadjuvant Letrozole. 2. To predict which HER2+ cancers do not respond to Letrozole. Methods 23 postmenopausal women with large, operable, locally advanced HER2+/ER+ breast cancer treated with neoadjuvant Letrozole had response assessed by periodic 3D ultrasound. Core biopsies taken at 0, 14 days and 3 months of treatment. RNA were extracted, amplified, labelled and hybridised to Illumina HT-12 whole genome beadarrays. A group of patients with ER+/HER2- disease were identified to compare clinical and molecular response. Results 13 (57%) HER2+/ER+ patients responded (R) and 10 (43%) patients did not (NR). HER2 expression was significantly higher at baseline in the NR group (p = 0.005). There were differences in gene expression between HER2+/ER+ R and NR and between the HER2+/ER+ and HER2-/ER+ NR groups. In the HER2+/ER+ NR group, there was virtually no change in gene expression during treatment with Letrozole. Table 1 Up Regulated GenesDown Regulated Genes n0-14 days14 days-3 months0-3 months0-14 days14 days-3 months0-3 monthsHER2-ve Responders43122833505127213HER2-ve Non Responders1588361432340129HER2+ve Responders13541272703831125HER2+ve Non Responders10851117106 Considerable gene changes with overlap in the genes that changed most was evident in HER2+/ER+ and HER2-/ER+ responders. Analysis of 55 estrogen sensitive proliferation genes revealed significantly less reduction in the HER2+/ER+ NR group than in the HER2+/ER+ R group (AUC = 10.57 vs 27.93 respectively; p The HER2+/ER+ NR group had significantly less reduction in these 55 estrogen sensitive proliferation genes when compared to the HER2-/ER+ NR group at 14 days (AUC = 17.94 vs 18.05 respectively; p = 0.0007) and by 3 months (AUC = 10.57 vs 26.26; p In logistic regression analysis, HER2 status was predictive of disease progression (p = 0.048). A single gene classifier predicted response to endocrine therapy in the ER+/HER2+ group. This was accurate in 94% of patients in the test set (n = 23). This classifier was also predictive of progression free survival (p = 0.006) in HER2+/ER+ patients. Conclusions In this large cohort of patients treated with neoadjuvant letrozole: • HER2+/ER+ cancers have a low rate of response. • Changes in gene expression are similar in HER2+/ER+ and HER2-/ER+ responders. • HER2+/ER+ cancers that do not respond to letrozole have few gene changes and little reduction in estrogen signalling pathways. • In contrast HER2-/ER+ non responders show significant gene changes and reduced expression of estrogen sensitive proliferation genes. • A single gene classifier at diagnosis in ER+/HER2+ has been identified which predicts response to Letrozole with 94% accuracy. • Validation continues. This single gene classifier may provide a simple test to predict which HER2+/ER+ cancers are endocrine resistant. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-01.

Background: Approximately 75% of older postmenopausal patients with large operable or locally advanced oestrogen receptor positive breast cancer respond to treatment with neoadjuvant letrozole. Prediction of response is required as approximately 25% fail to respond. The aim of this study was to design a response predicting classifier with two data sets, one published previously [1] and secondly a new dataset. Methods: Patients were treated with Letrozole (Femara, 2.5 mg daily) and tissues were collected before treatment, 2 or 3 weeks on treatment, and between 3 and 5 months on treatment. Response was based on a 3D ultrasound, with response being classified as a 50% reduction in tumour volume at 3 months. To ensure biological relevance of the response groups we also separated patients into Quick Stable Responders (QSR) and Long-Term Non-responders (LNR), as summarised in Table 1. Illumina HT-12 BeadArrays were used for gene expression profiling, allowing platform-independent cross-validation with our previous study using Affymetrix HGU-133 chips [1]. The probe IDs were re-annotated to Ensemble gene IDs and XPN batch correction has been applied to make the datasets directly comparable. A combination of Rank-Product, SAM and Random Forrest analyses have been used to design several alternative signatures separating extreme response groups, the patients who responded rapidly vs those who did not respond for a long time. One of the best performing signatures included 48 informative features: 24 at baseline, 12 at 2-weeks and 12 changes between 0 and 2 weeks. This signature was validated on the independent previously published test set by classifying extreme responses (quick stable responders vs long term non responders) using Support vector machines (SVM) with different kernel functions. Results Are summarised in Table 2. Conclusions – A new sample series has been collected to study neo-adjuvant endocrine response. It can be used for independent validation of previous results [1] and for design of new platform-independent response predictors.– Multi-gene predictors based upon change in gene expression outperform those from baseline or 14 day measurements alone. The best classifiers include all 3 time points.– A classifier has been derived with a 91% accuracy rate of predicting extreme response group in 21 of 23 patients. References [1] Miller 2009 J Clin Oncol 27:1382. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-23.

Purpose To investigate whether suppression of plasma estradiol and estrone sulfate levels by the aromatase inhibitors (AIs) anastrozole and letrozole is related to body mass index (BMI) in postmenopausal women with early estrogen receptor (ER) –positive breast cancer. Recent studies have reported that the AI anastrozole has lower effectiveness than tamoxifen in women with high BMI. This effect with high BMI might hypothetically be a result of reduced inhibition of aromatase and suppression of plasma estrogen levels and might be overcome by the use of an increased dose of anastrozole or, alternatively, the use of a more potent AI such as letrozole. Patients and Methods Plasma estradiol and estrone sulfate levels from a highly sensitive radioimmunoassay were available for 44 postmenopausal patients who received anastrozole (1 mg per day) for 3 months followed by letrozole (2.5 mg per day) for 3 months or the opposite sequence. Correlations between the estrogen suppression by each AI and BMI were assessed. Results Baseline values of estradiol and estrone sulfate were significantly correlated with BMI (r = 0.57; P < .001, and r = 0.38; P = .006, respectively). Levels of estrogen in patients receiving treatment were greater at higher levels of BMI with both AIs, but although this was significant with letrozole (r = 0.35; P = .013, and r = 0.30; P = .035 for estradiol and estrone sulfate, respectively), it was not with anastrozole. Suppression of both estrogen types was greater with letrozole across the full range of BMIs in this study. Conclusion The suppressed levels of plasma estradiol and estrone sulfate in postmenopausal women with early ER-positive breast cancer treated with the AIs anastrozole and letrozole are related to BMI.

Invasive lobular cancer (ILC) responds poorly to neoadjuvant chemotherapy but appears to respond well to endocrine therapy. We examined the effectiveness of neoadjuvant letrozole in postmenopausal women (PMW) with estrogen receptor (ER)-rich ILC. PMW were considered for treatment with neoadjuvant letrozole if they had ER-rich, large operable, or locally advanced cancers, or were unfit for surgical therapy. Tumor volume was estimated at diagnosis and at 3 months using calipers (clinical), ultrasound, and mammography. At 3 months, if physically fit, women were assessed for surgery. Responsive women with cancers too large for breast-conserving surgery continued with letrozole. Patients had surgery or were switched to alternative therapy if tumor volume was increasing. Sixty-one patients (mean age, 76.2 years) with 63 ILCs were treated with letrozole for ≥ 3 months. The mean reduction in tumor volume at 3 months was 66% (median, 76%) measured clinically, 61% (median, 73%) measured by ultrasound, and 54% (median, 60%) measured by mammography. Surgery was possible at 3 months in 24 cancers in 24 patients, and all but two of the remaining patients continued with letrozole therapy for a median duration of 9 months. At the time of this publication, 40 patients with a total of 41 cancers have undergone surgery. The rate of successful breast conservation was 81% (25/31). Twenty-one patients have continued with letrozole monotherapy, and 19 remain controlled on letrozole at a median of 2.8 years. There is a high rate of response to letrozole in PMW with ER-rich ILC.

Background: Around 70% of all breast cancers (BCs) are estrogen receptor positive (ER+), but some do not respond to endocrine therapy (ET) and many eventually develop resistance. ESR amplification (ESRA) linked to an increase in ESR1 gene expression is known to occur in some cancers that are endocrine resistant. However, the incidence of ESRA has been the object of debate and its clinical significance remains unclear. This study aimed to investigate the incidence of ESRA in BCs resistant to multiple sequential ETs and optimise a fluorescence in-situ hybridisation (FISH) methodology to robustly detect ESRA. Methods: Two unique cohorts have been studied: (A) 20 post-menopausal women with ER+ BC with acquired resistance to letrozole, subsequently treated with up to 4 different lines of ET. Serial RNA and DNA from 3-5 cancer samples per patient (58 samples from 20 patients) were analysed by Ribo0-RNAseq and DNA exome sequencing; (B) 18 post-menopausal women who developed ER+ BC recurrences on 1st line adjuvant letrozole, then on 2nd line tamoxifen and subsequently on 3rd line exemestane. Tissues were collected at the time of each surgery. We have optimised a FISH method to assess ESRA in these tissues. Results: In cohort A, 6/20 patients developed ESR1 gene amplification (ESRA) at some point during treatment. In 5 of these cases, ESRA was only found while on 2nd or 3rd line exemestane but was not present on acquired resistance to previous letrozole or tamoxifen. 1 patient had ESRA at the time of first recurrence on letrozole. The FISH method showed concordance with the genomic analysis. This suggests that ESRA may be associated with BCs that are treated with and then become resistant to exemestane. ESRA is also evident in samples from Cohort B, which includes 18 exemestane resistant cases. The complete analysis is ongoing. Conclusions: · ESRA can be seen in ER+ recurrent BCs. · ESRA may be associated with BCs treated with 2nd or 3rd line exemestane. · The frequency of ESRA in endocrine and exemestane resistance can now be ascertained using an optimised FISH-based method, which is more cost-effective than alternative genomic and biochemical methods. Citation Format: Turnbull AK, Martinez-Perez C, Mok S, Tanioka M, Fernando A, Renshaw L, Keys J, Wheless A, Garrett A, Parker J, He X, Sims AH, Carey LA, Perou CM, Dixon JM. Investigating the incidence of ESR1 gene amplification in breast cancers resistant to multiple endocrine agents [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 P5-04-27.

Background There are no data on the rate of progression of infirm or elderly postmenopausal patients unfit for surgery who have been treated with aromatase inhibiors alone. The aim was to determine the rate of disease control in a series of patients treated in the Edinburgh Breast Unit. Patients 79 patients with a median age of 81 years (range 57 to 98) with ER rich breast cancers (Allred score 6-8) have been treated with letrozole alone. The median follow up is 2.06 years, range 0.34 to 7.64. Of 71 patients with HER2 results, 59 (83%) were negative and 12 (17%) were positive. Results At two years, 65% were progression-free. By five years, this was 43%. The median time to first progression was 3.1 years (95% CI 1.7 to 4.5). By the Log Rank test, there was evidence of a significant difference between HER2+ve and-ve cancers (p=0.014). Median time to first progression for HER2-ve patients was 3.1 years (95% CI 1.9 to 4.2) and for HER2+ve patients was 1.8 years (95% CI 0.7 to 2.8). By two years, 67% of HER2-ve patients were progression-free, while 34% of the HER2+ve patients remained progression-free. At five years, 32% of the HER2-ve patients were progression free, however HER2+ve patients had insufficient follow up to estimate the 5 year progression-free rate. At two years, 77% of patients were still alive. By five years, that number was 33%. The median survival time was only 3.2 years (95% CI 2.4 to 4.1). There was no evidence to suggest a difference in mortality rates between HER2+ve and HER2-ve patients (p=0.79). Median survival time for HER2- ve patients was 2.6 years (95% CI 1.6 to 3.7) and for HER2+ve patients it was 3.0 years (95% CI 2.5 to 3.5). Conclusions In an elderly unfit population with ER rich breast cancer two thirds of these women will have their cancer controlled at 2 years by letrozole alone. HER2+ve cancers have a significantly shorter progression free survival than HER2-ve cancers. For older or infirm women unfit for surgegy with ER rich breast cancers, letrozole will control their cancer in the majority to the time of their death which was from other causes in all patients in this series. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-12-06.

Background Few data exist on local recurrence rates after breast conserving surgery in post menopausal patients following neoadjuvant endocrine therapy with letrozole. The aim was to investigate the outcome of a large series of patients treated in the Edinburgh Breast Unit. Patients 208 patients underwent breast conserving surgery after at least a 3 month course of neoadjuvant letrozole. The median age was 77 years (range 51 to 93). Of these patients 73% (151/208) received postoperative radiotherapy. Median follow up was 2.95 years (range 0.28 to 8.43) Results At two years 97% were local recurrence free and by five years 95% were recurrence free. By the Log Rank (Mantel-Cox) non-parametric method of analysis, there was a significant difference between patients treated by postoperative whole breast radiotherapy (RT) compared with no radiotherapy (p=0.02). There were very small numbers of local recurrences (five in no RT group, two in RT group by the end of five years). At two years 93% of patients with no RT were recurrence-free, compared with 98% of those with RT, at five years this is 86% and 98% respectively There was a borderline significant difference between HER2 positive and negative breast cancers, (p=0.055). At two years 97% of HER2 negative patients were recurrence-free, compared with 91% of HER2 positive patients. At five years, it was 96% and 85% respectively. Death from other causes was very common in this elderly age group and the median survival time was 7.2 years (95% CI 5.8 to 8.5). 90% of patients survived to the end of two years (95% CI: 86% to 94%), while only 68% survived to the end of years (60% to 76%). By the Log Rank test, there was a significant difference between patients treated by radiotherapy and those who did not receive radiotherapy (p=0.004). The median survival with no RT was 4.7 years (95% CI: 3.7 to 5.8) and for patients following RT was 8.1 years (95% CI: 6.2 to 10.0). The reason for this was selection of fitter patients for radiotherapy and avoiding radiotherapy in frail patients. Conclusions Local recurrence after breast conserving therapy following neoadjuvant endocrine therapy with letrozole is uncommon in this group of patients. Following radiotherapy local recurrence is only 2% at 5 years. Rates of recurrence are over 10% at 5 years without radiotherapy. HER2 positive patients have a higher local recurrence rate than HER2 negative patients. Breast Conserving Surgery followed by whole breast radiotherapy produces excellent rates of local control in postmenopausal women following neoadjuvant endocrine therapy with letrozole. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-12-05.

International audience; There is growing evidence that uncontrolled activation of the PI3K/Akt/mTOR pathway contributes to the development and progression of breast cancer. Inhibition of this pathway has antitumour effects in preclinical studies and efficacy in combination with other agents in breast cancer patients. The aim of this study is to characterise the effects of pre-operative everolimus treatment in primary breast cancer patients and to identify potential molecular predictors of response. Twenty-seven patients with oestrogen receptor (ER)-positive breast cancer completed 11–14 days of neoadjuvant treatment with 5-mg everolimus. Core biopsies were taken before and after treatment and analysed using Illumina HumanRef-8 v2 Expression BeadChips. Changes in proliferation (Ki67) and phospho-AKT were measured on diagnostic core biopsies/resection samples embedded in paraffin by immunohistochemistry to determine response to treatment. Patients that responded to everolimus treatment with significant reductions in proliferation (fall in % Ki67 positive cells) also had significant decreases in the expression of genes involved in cell cycle ( = 8.70E−09) and p53 signalling ( = 0.01) pathways. Highly proliferating tumours that have a poor prognosis exhibited dramatic reductions in the expression of cell cycle genes following everolimus treatment. The genes that most clearly separated responding from non-responding pre-treatment tumours were those involved with protein modification and dephosphorylation, including DYNLRB2, ERBB4, PTPN13, ULK2 and DUSP16. The majority of ER-positive breast tumours treated with everolimus showed a significant reduction in genes involved with proliferation, these may serve as markers of response and predict which patients will derive most benefit from mTOR inhibition.

Purpose: Tamoxifen reduces risk of recurrence after breast conservation surgery for ductal carcinoma in situ (DCIS), but no data exists on the effectiveness of aromatase inhibitors for DCIS. Cyclooxygenase-2 (COX-2) is overexpressed in DCIS, representing another potential therapeutic target. The aim of the study was to determine the effect of aromatase and/or COX-2 inhibition on epithelial proliferation and apoptosis in a presurgical study of estrogen receptor (ER)–positive DCIS. Methods: Postmenopausal women with ER-positive DCIS diagnosed by core biopsy were randomized to a 2 × 2 design of either 14 days of exemestane or placebo and celecoxib, or placebo immediately before surgery. Paired baseline and end point biopsies were analyzed for proliferation (Ki67), apoptosis, human epidermal growth factor receptor 2 (HER2), COX-2, and progesterone receptor (PR) expression by immunohistochemistry. The primary end point was a decrease in Ki67 between diagnosis and surgical excision. Results: Ninety women were randomized: all were ER positive, 49 (54%) had grade III tumors, and 29 (32%) were HER2 positive (3+). Exemestane reduced proliferation compared with placebo with a median reduction of 9% (95% confidence interval, 6-14; P < 0.001). Progesterone receptor was reduced by exemestane (mean decrease, 19%; 95% confidence interval, 9-28; P = 0.011). The effect of exemestane on proliferation was seen regardless of grade, HER2, or PR expression. Celecoxib had no effect on proliferation or apoptosis alone, or in combination with exemestane. Conclusions: Exemestane reduces proliferation in ER-positive DCIS. Aromatase inhibition is a potential alternative to tamoxifen in patients who have undergone breast conservation for ER-positive DCIS. Clin Cancer Res; 16(5); 1605–12

Purpose Endocrine agents, such as letrozole, are established in the treatment of hormone-dependent breast cancer. However, response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Thus there is a need to identify novel markers predicting for response and to understand molecular mechanisms of resistance. Patients and Methods Sequential tumor biopsies were taken before and after 10 to 14 days of neoadjuvant treatment with letrozole in patients with estrogen receptor–rich breast cancer. Expression profiles on high-density microarray chips were then related to clinical responses as assessed from tumor volume measurements after 3 months of treatment. Results Of 52 patients, 37 (71%) were classified as having a clinical response to letrozole and 15 being clinically resistant. Bioinformatic analysis identified 205 covariables (69 baseline expression, 45 day 14 expression, and 91 change in expression with treatment) which differentiated between clinical responders and nonresponders. Hierarchical clustering using the variables separated responders and nonresponders into two distinct groups. Ontological assessment indicated that discriminating genes were enriched toward cellular biosynthetic processes. In particular, functional gene assessment showed ribosomal protein probes to have higher baseline expression in tumors responsive to letrozole and increased expression with treatment in nonresponding cases. Conclusion To our knowledge, this is the first study to describe genetic covariables and molecular processes discriminating between tumors clinically responsive and resistant to an aromatase inhibitor. The understanding of such molecular phenotypes will be important in optimizing the clinical use of aromatase inhibitors, both in terms of identifying responsive breast cancers and developing new agents to target resistance pathways.

Introduction Changes in proliferation as measured by Ki67 occur within 14 days of starting treatment with an aromatase inhibitor and these changes have been shown to be predictors of long term outcome. This study aimed to compare changes in proliferation following 14 days of treatment with anastrozole and letrozole. Methods Two hundred and six women with 209 estrogen receptor (ER) positive operable breast cancers (three bilateral) were randomly allocated to receive either 14 days treatment with 2.5 mg of letrozole or 1 mg of anastrozole prior to surgery. Changes in expression of estrogen (ER) and progesterone receptors (PgR) as assessed by ALLRED scores and proliferation as assessed by Ki67 were analysed. The HER2 status of each tumour was also assessed using a combination of the Hercept test and FISH. Results Both letrozole and anastrozole reduced ER expression (ALLRED score) by a mean of 0.32 (0.20-0.44), P < 0.001 and PgR fell by a mean of 2.54 (2.20-2.89) P < 0.0001. Letrozole reduced proliferation from a geometric mean of 6.37% to 0.81%, P < 0.0001 and anastrozole reduced proliferation from 5.81% to 0.77%, P < 0.0001. There was no differences between drugs in the fall in ER, PgR or proliferation. Both letrozole and anastrozole produced significant falls in proliferation in both HER2 positive and HER2 negative cancers, all P < 0.001. Discussion 14 days of both letrozole and anastrozole reduces proliferation, ER and PgR expression. No significant difference between these two drugs was identified.

Purpose To compare the effects of anastrozole and letrozole on plasma estradiol (E2) and estrone sulfate (E1S) levels. Patients and Methods Fifty-four postmenopausal women with estrogen receptor–positive breast cancer receiving aromatase inhibitors (AIs) as part of their adjuvant therapy were randomly assigned to receive either 3 months of anastrozole (1 mg) followed by 3 months of letrozole (2.5 mg), both given orally once daily, or 3 months of the opposite sequence. Blood was taken at the same time and the same day of the week from each patient, before and after 3 months of each drug, and plasma levels of E2 and E1S were determined using highly sensitive radioimmunoassays. Results There were 27 patients in each group. The mean age of the patients was 63 years (range, 49 to 83 years). Baseline E2 levels ranged from 3 pmol/L to 91 pmol/L with a mean of 25.7pmol/L. Only one of 54 (2%) patients had an E2 value ≥ 3 pmol/L after receiving letrozole, versus 20 of 54 (37%) patients after receiving anastrozole (P < .001). Extrapolation revealed a mean E2 level after anastrozole treatment of 2.71 pmol/L (range, 2.38 to 3.08 pmol/L). Following letrozole, it was 1.56 pmol/L (range, 1.37 to 1.78 pmol/L). Mean residual E2 was 10.1% for anastrozole and 5.9% for letrozole. Residual E1S levels were 4.6% for anastrozole and 2.0% for letrozole (P = .001). Conclusion Letrozole reduces plasma E2 and E1S levels to a significantly greater extent than anastrozole in postmenopausal women taking AIs as part of their adjuvant therapy for hormone receptor–positive breast cancer.

// Carol Ward 1 , James Meehan 1 , Peter Mullen 2 , Claudiu Supuran 3 , J. Michael Dixon 4 , Jeremy S. Thomas 5 , Jean-Yves Winum 6 , Philippe Lambin 7 , Ludwig Dubois 7 , Nanda-Kumar Pavathaneni 6, 7 , Edward J. Jarman 1 , Lorna Renshaw 4 , InHwa Um 2 , Charlene Kay 1 , David J. Harrison 2 , Ian H. Kunkler 8 , Simon P. Langdon 1 1 Division of Pathology, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom 2 School of Medicine, University of St Andrews, North Haugh, St Andrews, United Kingdom 3 Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Sesto Fiorentino, Florence, Italy 4 Edinburgh Breast Unit, Western General Hospital, Edinburgh, United Kingdom 5 Department of Pathology, Western General Hospital, Edinburgh, United Kingdom 6 Institut des Biomolecules Max Mousseron (IBMM), UMR 5247, CNRS-UM1-UM2, Batiment de Recherche Max Mousseron, Ecole Nationale Superieure de Chimie de Montpellier, Montpellier, France 7 Department of Radiation Oncology (MaastRO), GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands 8 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom Correspondence to: Carol Ward, e-mail: drcward@hotmail.co.uk Keywords: carbonic anhydrase IX, breast cancer, tumour microenvironment, invasion, hypoxia Received: May 11, 2015 Accepted: June 22, 2015 Published: July 04, 2015 ABSTRACT Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

The management of the elderly patient with breast cancer is a challenge to the breast care team for a number of reasons. The higher rate of comorbidity in elderly patients increases the risk for complications and mortality following surgery and other adjuvant treatments such as chemotherapy and radiotherapy. The advent of using endocrine therapy in the neoadjuvant setting allows disease control and downstaging of tumors to allow less extensive surgery, with less morbidity compared with other available treatments. Tamoxifen has traditionally been the hormone therapy of choice for patients unable to undergo surgery, but development of resistance is a common feature. Newer third-generation aromatase inhibitors, in particular letrozole, are superior to tamoxifen in this setting with greater downstaging of tumor and disease control. The aromatase inhibitors are now the treatment of choice in elderly patients with estrogen receptor–positive breast cancer who are being considered for neoadjuvant therapy. These drugs are particularly suitable to the needs of an elderly population.

Sixty-three postmenopausal women with large primary breast cancers were treated with neoadjuvant letrozole (2.5 mg daily) for 3 months. Tumour samples were taken at diagnosis and after 10-14 days and 3 months treatment. Immunohistochemical staining for Ki67, oestrogen receptor (ER) and progesterone receptor (PgR) was performed and related to clinical (ClinR) and pathological responses (PathR) after 3 months treatment. ClinR was observed in 48 of 63 cases (76.2%) and PathR in 47 of 62 (75.8%). Pretreatment Ki67 scores were similar in responders (R) and non-responders (NR). Highly significant Ki67 decreases occurred in all tumour subgroups at 10-14 days (P0.005). A significant difference in Ki67 scores at 10-14 days (P0.007) was found between PathR and PathNR but not between ClinR and ClinNR. At 3 months, decreases from pretreatment Ki67 scores were highly significant in all tumour subgroups irrespective of response status. However, whereas Ki67 scores were significantly different between pathological R and NR (P = 0.009), the corresponding comparison of ClinR status was not. Significant decreases between 10-14 days and 3 months were found only in ClinR and PathR (P = 0.02 and 0.045, respectively). Treatment significantly reduced PgR expression at 14 days and 3 months (both P0.0001), but the level of changes was not different between response status groups. In summary, letrozole produces rapid and profound decreases in expression of Ki67 and PgR but changes do not always correlate with clinical and pathological responses.

Overexpression of erbB2 in breast tumours can predict resistance to tamoxifen therapy. We conducted a small trial to determine if erbB2 status correlates with tumour response and biochemical changes in postmenopausal women receiving neoadjuvant therapy with the aromatase inhibitor, anastrozole. Twenty-four postmenopausal women with oestrogen receptor (ER)-rich, large, operable breast tumours received three months of neoadjuvant anastrozole, 1 or 10 mg daily, then surgery, followed by another five years of anastrozole 1 mg daily. Response to the treatment was based on changes in clinical and ultrasound measurements of tumour volume and changes in tumour proliferation and progesterone receptor (PgR) status. After follow-up for a median duration of four years therapy, there was no apparent difference between erbB2 0/1+ and erbB2 3+ tumours in clinical response or changes in proliferation and PgR expression. In conclusion, anastrozole appears to be an effective endocrine option in this patient population, irrespective of the erbB2 status.

Background: Approximately 40% of ER+ breast cancer present with nodal metastasis. To date, there has been no comparison of the molecular response of primary cancers and metastases to ET. Recent evidence suggests that nodal metastases have different clones and subclones compared to the primary tumour. The aim of this study is to characterise the molecular response of primaries and nodal metastases to ET. Methods: A unique set of 7 post-menopausal women with ER-positive breast cancer had biopsies taken from the primary tumour and a positive lymph node at diagnosis and at surgery following 3-12 months of neoadjuvant letrozole. 14-day and 3-6 month on-treatment biopsies from the primary tumour and involved nodes were also taken from the same patients, giving a total of 75 samples. Lymph node FFPE blocks were stained for cytokeratin and macro-dissected to enrich for tumour tissue. RNA and DNA were extracted and Ribo0-RNAseq, DNA exome sequencing and somatic mutation detection using UNCeqR performed. Whole-transcriptome AmpliSeq targeted-sequencing has been analysed for 4 patients. Results: Multi-dimensional scaling and hierarchical clustering analysis based on all transcripts and the 500 most variably expressed genes revealed that primaries and nodal metastases are strongly associated at diagnosis but some nodes diverge during ET treatment. Analysis of estrogen-responsive proliferation-associated genes (n=60) in nodal metastasis revealed a reduction in expression of the majority of genes with ET. However, the expression levels of some remained high in the on-treatment node samples in all 4 patients analysed compared with the matched primary tumour on treatment. In particular, expression of genes involved in DNA replication and regulation of cell cycle including MCM6 and RRM2 (DNA replication), ASPM and CEP55 (mitosis) and CDKN3 (regulation of cell cycle) persisted at high levels in nodal metastases, but reduced in the primary cancers. Similarly, primary tumours had increased levels of ECM remodeling genes (n=60) as treatment continued, while levels in the nodal metastasis were heterogeneous on-treatment. Full genome sequencing results will be available by December 2017. Discussion · This is the first study to investigate genomic and transcriptomic changes with ET in both primary cancers and nodal metastases. · On-treatment changes in nodal disease are heterogeneous between patients and within the same patient. · Nodal metastases do respond to ET with reduced levels of proliferation-associated genes. · Some proliferation-associated genes appear to maintain higher expression in nodal disease. · Patterns of gene expression observed in some nodal metastases are consistent with profiles previously described by us for ET resistance and recurrent disease. · Nodal metastases may accumulate mutations during treatment with ET and on-going analysis will clarify this. Citation Format: Dixon JM, Turnbull AK, Tanioka M, Parker J, He X, Fernando A, Renshaw L, Keys J, Thomas JS, Sims AH, Carey LA, Perou CM. Characterising the effects of neoadjuvant endocrine therapy on primary cancers and nodal metastasis [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 P4-04-02.

Introduction Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. Methods Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n = 84), gene expression profiling (n = 47), matched pre- and post-AI aCGH (n = 19 pairs) and Ki67-based AI-response analysis (n = 39). Results Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). Conclusions These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0532-0) contains supplementary material, which is available to authorized users.

Invasive lobular carcinoma (ILC) accounts for approximately 10% to 15% of breast carcinomas, and although it responds poorly to neoadjuvant chemotherapy, it appears to respond well to endocrine therapy. Pre- and on-treatment (after 2 weeks and 3 months) biopsies and surgical samples were obtained from 14 postmenopausal women with estrogen receptor–positive (ER+) histologically confirmed ILC who responded to 3 months of neoadjuvant letrozole and were compared with a cohort of 14 responding invasive ductal carcinomas (IDC) matched on clinicopathologic features. RNA was extracted and processed for whole human genome expression microarray. Dynamic clinical response was assessed using periodic three-dimensional ultrasound measurements performed during treatment and defined as a reduction of >70% in tumor volume by 3 months. Pretreatment profiles of ILC and IDC tumors showed distinctive expression of genes associated with E-cadherin signaling, epithelial adhesion, and stromal rearrangement. The changes in gene expression in response to letrozole were highly similar between responding ILC and IDC tumors; genes involved in proliferation were downregulated and those involved with immune function and extracellular matrix remodeling were upregulated. However, molecular differences between the histologic subtypes were maintained upon treatment. This is the first study of molecular changes in ILC in response to endocrine therapy to date. The genes that change on letrozole are highly consistent between ILC and IDC. Differences in gene expression between ILC and IDC at diagnosis are maintained at each time point on treatment. Cancer Res; 74(19); 5371–6. ©2014 AACR.

555Background: Identifying breast cancer (BC) patients likely to recur on endocrine therapy (ET) is a major challenge. Many multigene classifiers are available for predicting prognosis. These are u...

International audience; mTOR plays a key role in tumor cell cycle control, proliferation, and survival. RAD001 (everolimus) is a novel macrolide that inhibits mTOR and thus downstream signaling pathways. 31 post-menopausal women with early breast cancer were given 5 mg RAD001 once daily for 14 days prior to surgery. Biopsies were taken at diagnosis and at surgery (post 14 days of treatment) and assessed for immunohistochemical changes in proliferation (Ki67), apoptosis (active caspase-3), p-AKT (s473), p-S6 (s235/236 and s240/244), p-mTOR (s2448), ER, and PR. Five patients did not complete the 2-week treatment period due to adverse events. All adverse events were grade 1 or 2 (NCIC-CTC scale). RAD001 treatment significantly decreased proliferation (geometric mean reduction 74% from baseline ( = 0.019)), particularly in HER-2 positive tumors. High Ki67 pre-treatment correlated with reduction in Ki67, an increase in apoptosis, a reduction in p-AKT (cytoplasmic) and reduction in p-mTOR following treatment. Nuclear expression of p-AKT was significantly reduced with treatment. Tumors that had a reduction in Ki67 with treatment exhibited a significant reduction in cytoplasmic p-AKT. p-S6 staining was significantly reduced independently of Ki67 (

International audience; Previous studies have demonstrated that both anastrozole and letrozole are well tolerated. Letrozole suppresses estrogen to a greater degree than anastrozole in the serum and breast tumor. Concerns have been raised that greater potency may adversely affect patients' quality of life (QOL). One hundred eighty-one postmenopausal women with invasive estrogen receptor-positive breast cancers were randomized to receive either 12 weeks of letrozole followed by 12 weeks of anastrozole or the reverse sequence. One hundred and six received immediate adjuvant aromatase inhibitors (AIs) following surgery, and 75 received extended adjuvant therapy. The Functional Assessment of Cancer Therapy Endocrine Subscale (FACT-B-ES) QOL questionnaires were completed to assess QOL on each drug. Additional side-effect profiles were collected. Each patient completed a patient preference form. Twenty-one patients withdrew before study end, 10/179 (5.6%) while taking letrozole and 4/173 (2.3%) while taking anastrozole ( = 0.12). Tamoxifen-naïve patients had a higher mean ES (endocrine symptoms subscale) score at entry versus those having extended therapy (66.0 vs. 61.9; = 0.001). There was no significant change in FACT-B-ES (overall) scores or ES scores while patients were taking anastrozole or letrozole and no significant differences between drugs. Nearly 80% of patients reported one or more side effects with either agent. No differences in frequency, grade, or range of side effects were seen between drugs. Of 160 patients, 49 (30.6%) preferred letrozole, 57 (35.6%) preferred anastrozole, and 54 (33.8%) had no preference ( = 0.26, Pearson's Chi-squared test). In conclusion, both AIs are equally well tolerated. There were no significant differences in QOL scores between the two drugs.

International audience; ALIQUOT (Anastrozole vs. Letrozole, an Investigation of Quality Of Life and Tolerability) was a prospective, open-label, randomized pharmacodynamic study designed to assess the effects of aromatase inhibitors (AIs) on bone turnover in healthy postmenopausal women with estrogen receptor-positive breast cancer. Ninety-four patients were randomized to receive either 12 weeks of letrozole (2.5 mg; = 42) followed by 12 weeks of anastrozole (1 mg), or 12 weeks of anastrozole (1 mg; = 42) followed by 12 weeks of letrozole (2.5 mg). After completion of the study period, patients in the immediate adjuvant group were either switched to tamoxifen ( = 38) or continued on anastrozole or letrozole. In the beginning of the study, 42 patients had taken tamoxifen within 3 months. Patients taking drugs likely to affect bone metabolism, including bisphosphonates, were excluded. Eighty-four patients had complete sample measurements and were included in the analysis. Prior tamoxifen therapy resulted in a significantly lower mean baseline procollagen type 1 N-terminal propeptide (PINP) compared with patients with no prior tamoxifen. There were no significant differences in bone markers between AIs at any time. By 6 months, significant increases were seen in PINP, C-terminal telopeptides (CTX), bone specific alkaline phosphatise (ALP), and urinary N-terminal telopeptides (NTX). Patients with prior tamoxifen had significantly greater increases than patients with no prior tamoxifen. Patients treated with 3 months of tamoxifen following 6 months of an AI showed a significant decrease in markers of bone resorption, serum CTX and urinary NTX. In conclusion, AI-induced bone turnover increases over time. Anastrozole and letrozole produce similar effects on bone metabolism and turnover. Stopping tamoxifen therapy and starting AIs results in a significantly greater increase in bone turnover compared with commencing AIs in tamoxifen-naïve patients. Patients given tamoxifen following AI therapy showed a decrease in markers of bone resorption.

In patients with hormone receptor positive DCIS tamoxifen reduces recurrence rates by almost 50%. Few data are available with aromatase inhibitors from randomised studies. In the ATAC study there were three DCIS lesions in the anastrozole arm and four in the tamoxifen arm in the women with ER positive invasive cancer. In the MA17 study which randomised patients to up to 5 years of letrozole or placebo there was only one DCIS event in the contralateral breast in patients taking letrozole and five on placebo. There were also four patients in this study who had DCIS in the conserved breast on placebo and none in the letrozole treated group. The few clinical data that are available therefore suggest the aromatase inhibitors are likely to be effective in DCIS. A histological review of a study of 206 postmenopausal women with invasive oestrogen receptor positive breast cancer who were randomised as part of a 14 day preoperative study to receive 2.5mg of letrozole or 1mg of anastrozole identified 27 patients with 28 pairs of tumours in whom there was sufficient ER positive DCIS in invasive cancer in the initial core biopsy and in the subsequent surgery specimen, to evaluate for PgR activity and proliferation. Within the DCIS both aromatase inhibitors significantly reduced PgR expression and both drugs also produced a significant fall in proliferation. There was a moderate degree of agreement between the fall in PgR in both the invasive cancer and DCIS (Kappa=0.5; p=0.0013) and between the fall in proliferation and between the invasive and in situ components (correlation coefficient=0.68; p

Fulvestrant (Faslodex) is a pure anti-oestrogen that reduces markers of hormone sensitivity and proliferation in postmenopausal women with oestrogen-receptor (ER)-positive breast cancer. This randomised trial compared the effects on the tumours of a single dose of 750mg fulvestrant to those of daily tamoxifen (20mg) taken 14-16 days prior to surgery in 60 premenopausal women with ER-positive primary breast cancer. There were statistically significant falls in the expression of ER and Ki67 levels compared to the baseline with both drugs. Both drugs caused a decrease in PgR expression from baseline but this was only statistically significant with fulvestrant. No statistically significant differences were seen between the two treatment groups. Fulvestrant caused an increase in circulating levels of oestradiol, irrespective of the stage of the menstrual cycle at which patients commenced treatment. No major changes were seen in LH, FSH and progesterone levels with either drug. The most common adverse events with fulvestrant were headaches, hot flushes, nausea and disturbance of menses. Contrary to previous studies with fulvestrant 250mg, these findings suggest that at a dose of 750mg fulvestrant is effective at reducing the effects of oestrogen on ER-positive breast cancer in premenopausal women.

Background: Axillary management in breast cancer is evolving. Recent ACSO guidelines have suggested that axillary node clearances (ANC) are not necessary in the positive axilla in early breast cancers with small nodal burdens, undergoing breast conserving surgery (BCT). According to ASCO guidelines, G1-2, T1-2 tumours with 1 or 2 SLN macrometastases, undergoing BCT should not undergo ANC. We compare current practice in a screening population against these guidelines. Methods: We retrospectively analysed all patients with invasive breast cancer under the regional screening programme (2011e2014), looking specifically at management of patients who would not be offered ANC under ASCO guidelines. Results: 687 screening patients were diagnosed in the time frame. 516 had normal axillary USS and 143 e abnormal. Of the 516 normal USS, 79 (15.3%) had malignant sentinel lymph nodes (SLNB), (45 (57%) were macrometastases of which, 93% had 1e2 positive nodes. 34 (43%) were micrometastases). 86 abnormal USS had SLNB, 16 of which were malignant, 12 macrometastases (100% 1e2 positive nodes). 36 of the malignantmacrometastatic SLNBmet ASCO guidelines. 24 of those received ANC (67%), 7 (30%) of which were positive, 2 with heavy nodal burdens (10/14 and 5/20). The remainder had 1 positive node. Conclusion: Under updated ASCO guidance on SLNB in early breast cancer, we are over-treating the majority of screening patients. However, had these guidelines been followed, 19% of our patients would have had a missed diagnosis of further axillary disease, and in 5% a significant nodal burden would have remained undetected. As clinicians, there is a responsibility to balance and articulate that trade off when discussing axillary management with patients.

PurposeTo investigate the impact of human epidermal growth factor receptor (HER) 1 and HER2 gene amplification on endocrine therapy responsiveness, a fluorescence in situ hybridization (FISH) study was conducted on tumor samples from 305 postmenopausal patients with stage II and III estrogen receptor (ER) –positive (ER ≥ 10%) breast cancers treated on two independent neoadjuvant endocrine therapy trials.Patients and MethodsFISH analysis focused on HER1 and/or HER2 immunohistochemistry (IHC) -positive patients and a random selection of HER1/2 IHC-negative patients. HER2 FISH status was correlated with response and changes in the proliferation marker Ki67.ResultsHER1 was rarely amplified (< 1%), and HER2 amplification was observed in 9.2% of patients. Letrozole response by clinical measurement (71% HER2 FISH positive v 71% HER2 FISH negative), mammogram (44% HER2 FISH positive v 47% HER2 FISH negative), or ultrasound (47% HER2 FISH positive v 54% HER2 FISH negative) was not impaired by HER2 FISH-positive status. In contrast, HER2 FISH-positive tumors showed higher histologic grade (P = .009), higher pretreatment Ki67 (P = .005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = .0001). Similar observations regarding Ki67 were made in a smaller cohort of tamoxifen-treated tumors.ConclusionNeoadjuvant letrozole is clinically effective in ER-positive HER2 FISH-positive tumors, indicating sensitivity to short-term estrogen deprivation. However, continued proliferation despite ongoing letrozole or tamoxifen treatment in the majority of ER-positive HER2 FISH-positive samples (88%) could imply therapeutic resistance that may manifest later in the clinical course of the disease. Discordance between clinical and biomarker findings in this study serves to emphasize the need for surrogate end point validation in neoadjuvant endocrine trials through correlation with information on long-term outcomes.

There are important surgical issues related to the use of the third generation aromatase inhibitors in both the neoadjuvant and adjuvant settings. Neoadjuvant hormone therapy is effective at downstaging tumours, particularly large tumours initially thought to be inoperable or requiring mastectomy. Randomised trials have shown that the newer aromatase inhibitors letrozole and anastrozole increase the numbers of women who are suitable for breast-conservation compared with tamoxifen, and that letrozole is superior to tamoxifen in terms of clinical response. Aromatase inhibitors are most effective in ER-rich tumours and are clinically and biologically effective in both HER2 positive and negative tumours, whereas HER2 positive tumours show a level of resistance to tamoxifen. In neoadjuvant studies comparing aromatase inhibitors with tamoxifen, the duration of use has been 3-4 months, by which time any response is usually evident but longer treatment periods produce continued shrinkage and response. The re-excision rate following breast conservation surgery after neoadjuvant hormone therapy is favourable compared with the rates following immediate wide local excision. Local recurrence rates are acceptable in patients undergoing neoadjuvant therapy and breast-conserving surgery providing post-operative radiotherapy is given. Adjuvant aromatase inhibitors, as well as having an effect on metastatic disease and survival, reduce local and regional recurrence.

INTRODUCTION Activation of the Phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K)/mTOR pathway is thought to play a role in the development of resistance to endocrine therapy. The aim was to investigate PIK3CA mutation and PTEN status in a series of patients with estrogen receptor positive alpha (ER+) breast cancer at diagnosis and on progression, recurrence or development of a new breast cancer during or following adjuvant endocrine treatment. METHODS Formalin fixed paraffin embedded tissue was available from a series of 120 patients at diagnosis and from; local recurrences (in breast tumour recurrence, or recurrence in mastectomy scars), metastatic recurrences (nodal, or distant metastatic disease), local progression (patients treated with neoadjuvant endocrine therapy who initially responded well and then progressed), and from second breast cancers (ipsilateral second breast cancers, or contralateral new primaries). Samples were profiled with a comprehensive qPCR based mutation assay covering 17 mutational hotspots in PIK3CA, as well as having immunohistochemistry for PTEN assessment. RESULTS PIK3CA mutation status was available from 88 paired-matched samples at both diagnosis and recurrence. 38 (43%) had at least one PIK3CA mutation at diagnosis, and 37 (42%) had a mutation present in tissue taken at the time of recurrence. The most common mutations in the 176 tissues analysed in order were: H1047R (35 samples), H1047L (11 samples), E545K (19 samples), E542K (6 samples), H1047R (3 samples), C420R (4 samples), 542K (1 sample). Three cancers had 2 mutations. PTEN protein status by immunohistochemistry at diagnosis was: null = 1, 1-100 = 7, 101-200 = 24, >200 = 14. At recurrence the PTEN status was null = 6, 1-100 = 5, 101-200 = 13, >200 = 32. The change in PIK3CA mutation status in different groups is outlined in Table 1. Table 1.EventNumberPIK3CA gainPIK3CA lossChange in PIK3CA mutation status%Local progression28011 (3.6%)Metastatic recurrences19112 (10.5%)Local recurrence30303 (10%)Contralateral new primary6235 (83%)Ipsilateral 2nd tumour5213 (60%)Total8814814 Few patients changed their PIK3CA mutation status on development of local recurrence, metastatic disease or progression on hormone treatment. Frequent changes in PIK3CA mutation status were seen in patients who developed a new cancer in the treated or in the contralateral breast. There was an increase in PTEN null cases (from 2.2% to 10.7%) and PTEN over-expression >200 (from 30.4% to 57%) from the first to the second samples. CONCLUSION • The acquisition of mutations in PIK3CA does not appear to be responsible for the development of endocrine resistance. • PIK3CA mutation status does not change in the majority of patients on progression or development of recurrence on or after endocrine therapy. PIK3CA mutation status at diagnosis can thus be used to determine whether a PI3-Kinase inhibitor might be suitable for treatment in patients with metastatic disease. • Patients who develop new breast cancers frequently develop cancers with a different PIK3CA mutation status. • These are the most comprehensive currently available data correlating PIK3CA status and endocrine resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-1.

Background: Older postmenopausal patients with large operable or locally advanced oestrogen receptor positive, invasive breast cancers are candidates for treatment with neoadjuvant letrozole. HER2 positivity is a potential marker for early endocrine therapy resistance. In this study we have evaluated the effects of HER2 amplification, gene and protein expression at diagnosis and following 14 and 90 days of treatment with neoadjuvant Letrozole. Methods: 70 postmenopausal women with large operable and locally advanced oestrogen receptor (ER) positive invasive breast cancers were treated with neoadjuvant letrozole. Response was assessed by 3D ultrasound. Sequential core biopsies were taken at 0, 14 days and 3 months of treatment. 12 patients were HER2 positive (either 3+ or 2+ FISH positive), 20 were HER2 2+ FISH negative and 38 were HER2 negative (0 or +). Results: 43 patients were responders to letrozole and 17 were non-responders. 7 of the 17 non-responders were HER2 positive. Analysing response in two groups (HER2 2+ and 3+ together versus 0 or +) there was a greater difference in response rate between these two groups than when splitting into conventional HER2 positive and negative groups (P < 0.0001). In non-responding patients HER2 gene expression levels from the microarrays taken at baseline were significantly higher than the HER2 expression in responding patients (p = 0.01). There was also an increase in HER2 gene expression seen during the first 14 days of treatment in non-responding but not responding patients (p = 0.08). Rank product analysis of gene expression identified 34 down-regulated genes and 7 up-regulated genes which were shared between the HER2 2+ FISH negative samples and samples which were HER2 3+ or HER2 2+ FISH positive. Conclusion: This large cohort of patients treated with neoadjuvant letrozole shows that: HER2 expression correlates with response to letrozole. A better cut off for prediction of response to letrozole is the split between 0 and + versus 2+ and 3+ rather than the traditional 3+. Patients with increased MRNA expression at diagnosis for HER2 have a significantly lower response rate to neoadjuvant letrozole. Endocrine therapy together with anti-HER2 therapies should be considered for patients having neoadjuvant endocrine therapy for cancers which over express HER2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-10.

Background:Aromatase inhibitors (AIs) reduce circulating oestrogen which may have a detrimental effect on lipid metabolism and subsequently ↑ cardiovascular risk. This study compares the effects of adjuvant A, L and E on lipid metabolism.Patients and Methods:This was an open, randomised pharmacodynamic study. Plasma lipids were measured in 120 postmenopausal women with invasive ER +ve breast cancer. As part of their adjuvant hormone therapy, patients were randomized to receive either:• 4 months of A or• 4 months of L or• 4 months of EFasting blood and urine samples were collected at the same time of day on each of (a) the day of entry, (b) after 3 months and (c) after 4 months. Patients were then switched to tamoxifen (T) and further samples collected after 12 months.• Triglycerides, cholesterol, ApoB and cLDL were measured.↑ levels are associated with ↑ cardiovascular risk.• ApoA-1 and HDL were also measured.↑ levels are associated with ↓ cardiovascular risk.Analyses were performed using repeated measures or analysis of covariance.ResultsDifferences between A vs L vs ETriglycerides E significantly ↓ triglycerides. There was a borderline significant difference between the drugs (p=0.055).Cholesterol E significantly ↓ cholesterol (p=0.03).ApoB No significant changes were detected.cLDL No significant changes were detected.ApoA-1 There were no significant differences detected between A or L although the fall with E was less but not significantly different to A or L (p=0.08).HDL E significantly ↓ HDL (p=0.008).Differences between steroidal vs non-steroidal AIsTriglyceride and ApoA-1 levels fell significantly more with E compared to the non-steroidal A+L (p=0.02, p=0.045 respectively).Effects of tamoxifenChanges from baseline after treatment on AIs and subsequent treatment with TFollowing T there was no evidence to suggest that the changes from baseline were influenced by the type of AI that patients received.Changes from end of treatment on AIs and subsequent treatment with TT increased HDL by a statistically significant greater amount following E than it did after A or L (p=0.009). From the end of treatment with AIs, T produced significant rises in triglyceride (p=0.0001), HDL (p=0.006) and ApoA1 (p=0.0003), and significant falls in cholesterol (p=0.03) and cLDL (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4086.