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Purpose Everolimus plus exemestane (EVE/EXE) is a registered treatment option for ER-positive, HER2-negative (ER +/HER2-) metastatic breast cancer (MBC), but resistance mechanisms limit efficacy. We aimed to find markers that might help select patients with a higher chance on benefit from EVE/EXE. Methods Immunohistochemistry (IHC) of PTEN, p-AKT(Thr308), p-AKT(Ser473), p-4EBP1, p-p70S6K, p-S6RP(Ser240/244), p-ERK1/2 and p-S6RP (Ser235/236) was performed on primary tumour tissue and on biopsies immediately taken from ER +/HER2- MBC patients before the start of standard EVE/EXE (Eudract 2013-004120-11). Unsupervised hierarchical clustering was executed to create heatmaps to distinguish subgroups of preferentially activated and less-activated PI3K/MAPK proteins. Uni- and multivariate Cox models were used for associations with PFS. Results Primary tumour tissue from 145 patients was retrieved. Median PFS was 5.4 months. Patients without (neo)adjuvant therapy (p = 0.03) or bone only disease (p = 0.04) had longer PFS on EVE/EXE. In primary tumours, neither single proteins nor PI3K/MAPK-associated heatmap subgroups were significantly associated with PFS. In 21 patients a non-osseous biopsy obtained before dosing was useful for continuous scoring, which demonstrated upregulation of several proteins as compared to readings in corresponding primary tumour tissues. These comparisons revealed that increased expression of p-4EBP1 was significantly associated with worse PFS (multivariate HR 3.69, p = 0.05). Conclusions IHC of single proteins or heatmap subgroups of the differentially activated PI3K/MAPK pathways was not able to discriminate patients on EVE/EXE with poor or better PFS. Upregulation of p-4EBP1 in pre-treatment biopsies as compared to levels in primary tumours pointed towards shorter PFS.

Background We previously showed that BRCA-like profiles can be used to preselect individuals with the highest risk of carrying BRCA mutations but could also indicate which patients would benefit from double-strand break inducing chemotherapy. A simple, robust, and reliable assay for clinical use that utilizes limited amounts of formalin-fixed, paraffin-embedded tumor tissue to assess BRCAness status in both ER-positive and ER-negative breast cancer (BC) is currently lacking. Methods A digital multiplex ligation-dependent probe amplification (digitalMLPA) assay was designed to detect copy number alterations required for the classification of BRCA1-like and BRCA2-like BC. The BRCA1-like classifier was trained on 71 tumors, enriched for triple-negative BC; the BRCA2-like classifier was trained on 55 tumors, enriched for luminal-type BC. A shrunken centroid-based classifier was developed and applied on an independent validation cohort. A total of 114 cases of a randomized controlled trial were analyzed, and the association of the classifier result with intensified platinum-based chemotherapy response was assessed. Results The digitalMLPA BRCA1-like classifier correctly classified 91% of the BRCA1-like samples and 82% of the BRCA2-like samples. Patients with a BRCA-like tumor derived significant benefit of high-dose chemotherapy (adjusted hazard ratio (HR) 0.12, 95% CI 0.04–0.44) which was not observed in non-BRCA-like patients (HR 0.9, 95% CI 0.37–2.18) (p = 0.01). Analysis stratified for ER status showed borderline significance. Conclusions The digitalMLPA is a reliable method to detect a BRCA1- and BRCA2-like pattern on clinical samples and predicts platinum-based chemotherapy benefit in both triple-negative and luminal-type BC.

Background: Recent data demonstrate that patients with early-stage triple negative breast cancer (TNBC) benefit from escalating adjuvant treatment with capecitabine. However, since a substantial proportion of patients does not benefit, predictive biomarkers to select those individuals upfront are needed. Over half of all TNBCs have a BRCA1-like DNA copy number signature similar to the profile found in germline BRCA1-mutated breast cancers and indicative for homologous recombination deficiency. We evaluate this signature as a predictive biomarker for capecitabine benefit in archived specimens of the randomized controlled FinXX trial. Additionally, we compared the concordance of our DNA-based BRCA1-like classifier with the RNA-based NanoString BRCAness signature. Methods: Early-stage TNBC patients were randomized between adjuvant capecitabine-containing chemotherapy (TX+CEX: capecitabine plus docetaxel, followed by cyclophosphamide, epirubicin and capecitabine) and conventional adjuvant chemotherapy (T+CEF: docetaxel, followed by cyclophosphamide, epirubicin, and fluorouracil). Breast tumor BRCA1-like status was determined on low coverage, whole genome next-generation sequencing data using an established DNA comparative genomic hybridization algorithm. We used interaction analysis in proportional hazards models to evaluate whether benefit of adjuvant capecitabine-containing versus conventional chemotherapy differs between BRCA1-like and non-BRCA1-like tumors in early-stage TNBC patients.Results: For 129 (63.9%) of the 202 TNBC patients the BRCA1-like status could be determined. Thirty-five recurrences and 32 deaths occurred during a median follow-up of 10.7 years. The capecitabine effect on recurrence-free survival did not significantly differ between the 68 patients (52.7%) with a BRCA1-like tumor (HR 0.66, 95% CI 0.24-1.81) and others (HR 0.23, 95% CI 0.08-0.70, P interaction = 0.17), also after adjustment for clinico-pathological variables. Conclusions: In the FinXX trial, the BRCA1-like status was not associated with a differential benefit from capecitabine-containing adjuvant chemotherapy compared to conventional chemotherapy in the TNBC subgroup. Based on this study, it is unlikely that the BRCA1-like classifier can be used to distinguish patients who do and do not benefit from capecitabine-enriched standard adjuvant chemotherapy.

Background Several (activated) proteins of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway have been analyzed for an association with adjuvant tamoxifen benefit in estrogen receptor-positive (ER+), HER2-negative (HER2-) breast cancer. Currently, no single protein has yielded convincing results allowing clinical implementation. Combining activated proteins could provide more robust predictive potential. We used unsupervised hierarchical clustering of 7 activated proteins downstream in these pathways, discriminated by expression profile, to analyze adjuvant tamoxifen benefit in ER+/HER2- postmenopausal breast cancer patients. Further, we developed a classification tool based on the generated heatmap groups by pathway activation status to categorize future patients into sensitive for or resistant to adjuvant tamoxifen. Methods Primary tumor blocks from 489 ER+/HER2- (stage I-III) postmenopausal patients previously randomized between tamoxifen (1 to 3 years) vs no adjuvant therapy (IKA trial 1982-1993; Beelen et al, Breast Cancer Res, 2014) were used for immunohistochemistry on tissue microarrays. Median follow-up of the patients without a recurrence event was 8.5 years. Scoring of PTEN, p-AKT(Thr308), p-AKT(Ser473) and p-p70S6K(Thr389) was by cytoplasmic intensity (0-3), p-4EBP1(Ser65) and pERK1/2(Thr202/204) by percentage of tumor cells with positive nuclear staining and p-S6RP(Ser235/236) by the percentage of tumor cells with positive membranous staining. Unsupervised hierarchical clustering was performed on tissue from 293 patients with informative data on all 7 proteins. Cox models were used to assess tamoxifen benefit (defined as the Hazard ratio (HR) of tamoxifen treatment vs no adjuvant therapy for recurrence-free interval) and to compare this benefit between subgroups generated by clustering as well as generated by the classification tool. Cox models included covariates (age, histological grade and subtype, tumor size, PR status) and were stratified for lymph node status. Results Two distinct groups were identified based on hierarchical clustering: one with preferentially activated pathway markers (A) and one with relatively no activation (N). Patients in group N derived significant benefit from tamoxifen (multivariable HR 0.28, 95% CI 0.14 − 0.56, p = 0.000324), while patients from group A had no benefit (multivariable HR 1.57, 95% CI 0.44 – 5.56, p = 0.482), p for interaction 0.018. Contradictory, patients in group A had a better prognosis compared to those in group N (multivariable HR 0.17, 95% CI 0.04 − 0.7, p = 0.014). The classification tool was also successful in differentiating ER+/HER2- patients with or without tamoxifen benefit (p for interaction = 0.016). Conclusions Hierarchical clustering of PI3K/MAPK pathway proteins is able to differentiate patients with adjuvant tamoxifen benefit and patients who would potentially need alternative treatment, for instance with inhibitors of the PI3K or MAPK pathway. The classification tool distinguishing patients with and without tamoxifen benefit should be validated in an independent cohort of postmenopausal patients with primary ER+/HER2- breast cancer. Citation Format: Kruger DT, Beelen K, Opdam M, Sanders J, van der Noort V, Boven E, Linn SC. Clustering of activated proteins of the PI3K and MAPK pathways distinguishes ER+/HER2- primary breast cancer patients with preferential tamoxifen benefit [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 PD4-12.

Background: Anthracycline-based adjuvant chemotherapy has substantially improved breast cancer survival. Both the addition of taxanes as well as using a dose dense treatment schedule can further ameliorate outcome, but inter-individual differences exist. Here we present the efficacy and toxicity of dose dense scheduled doxorubicin/cyclophosphamide (ddAC) versus docetaxel/doxorubicin/cyclophosphamide (TAC), which is, to our knowledge, the first direct comparison of these treatment regimens. Methods: In this Dutch, multicenter phase III trial (ISRCTN61893718), patients with pT1-3, pN0-3, M0 breast cancer were randomized between six cycles of either A60C600 every 2 weeks or T75A50C500 every 3 weeks. All patients received pegfilgrastim. Patients were evaluated for recurrence-free survival (RFS) and overall survival (OS). Survival was compared in a Cox regression analysis and adjusted for known prognostic factors. These factors include age, type of surgery, tumor size, histological grade, ER/PR status, HER2 status, and lymph node status. Adverse events were reported according to the common toxicity criteria (CTCAE version 3.0). Results: Between 2004 and 2012, 664 patients were enrolled of whom 531 (80%) had node positive disease. At a median follow up of 5 years, OS was 92% in the ddAC treated subgroup and 93% in the TAC treated subgroup (adjusted hazard ratio [HR] 0.75, 95% confidence interval [CI] 0.42-1.34, intention to treat population). Forty-two breast-cancer specific deaths were equally divided over both treatment arms. Similarly, no significant difference in RFS was observed between both treatment groups (adjusted HR 0.85, 95% CI 0.55-1.32). Molecular subtypes were defined by St. Gallen criteria: 548 patients (83%) had estrogen receptor positive disease and 102 patients (15%) triple negative disease. No heterogeneity regarding treatment efficacy was observed in these subtypes. In particular, there was no survival benefit for ddAC or TAC in the triple negative subtype. Both treatment regimens were well tolerated. Whereas anemia was more frequent in ddAC treated patients (19% vs 4.7%; p Conclusions: At a median follow up of 5 years no significant survival differences were observed between adjuvant ddAC and TAC, in all patients and in molecular subgroups, including triple negative. Our findings are in line with the Oxford overview, which reported no significant differences between taxane-based chemotherapy and more, non-taxane based chemotherapy given in a dose dense schedule. ddAC could be a reasonable alternative for patients with a contra-indication for TAC. Citation Format: van Rossum AGH, Oosterkamp HM, van Werkhoven E, Opdam M, Mandjes IAM, van Leeuwen-Stok E, van Tinteren H, Kok M, Imholz ALT, Portielje JEA, Bos MMEM, van Bochove A, Wesseling J, Rutgers EJ, Rodenhuis S, Linn SC. Adjuvant dose dense doxorubicin-cyclophosphamide (ddAC) or docetaxel-AC (TAC) for high-risk breast cancer: First results of the randomized MATADOR trial (BOOG-2004-04) [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 P5-14-03.

This abstract was withdrawn by the authors.

The prognostic value of tumour-infiltrating lymphocytes (TILs) differs by breast cancer (BC) subtype. The aim of this study was to evaluate TILs in stage III BC in the context of BRCA1/2-like phenotypes and association with outcome and benefit of intensified platinum-based chemotherapy.Patients participated in a randomised controlled trial of adjuvant intensified platinum-based chemotherapy versus conventional anthracycline-based chemotherapy carried out between 1993 and 1999 in stage III BC. Stromal TILs were scored according to International guidelines in these human epidermal growth factor receptor 2 (HER2)-negative tumours. BRCA-profiles were determined using Comparative Genomic Hybridization.TIL levels were evaluated in 248 BCs. High TILs were associated with Triple Negative BC (TNBC). BRCA-like tumours harboured higher TILs compared to non-BRCA-like tumours (median TILs of 20% versus 10%, p 0.01). TIL levels in BRCA1-like tumours were higher compared to BRCA2-like tumours (median TILs of 20% versus 10%, p 0.001). These correlations remained significant within the oestrogen (ER)-positive subgroup, however not within the TNBC subgroup. In this stage III BC cohort, high TIL level was associated with favourable outcome (TILs per 10% increment, recurrence-free survival (RFS): multivariate hazard ratio (HR) 0.82, 95% confidence interval (CI) 0.71-0.94, p = 0.01; overall survival (OS): multivariate HR 0.80, 95% CI 0.68-0.94, p = 0.01). There was no significant interaction between TILs and benefit of intensified platinum-based chemotherapy.In this high-risk breast cancer cohort, high TILs were associated with TNBC and BRCA1-like status. Within the ER-positive subgroup, TIL levels were higher in BRCA1-like compared to BRCA2-like tumours. When adjusted for clinical characteristics, TILs were significantly associated with a more favourable outcome in stage III BC patients.

Purpose The prognostic and predictive values of the MAPK/AKT/ERα phosphorylation axis (pT202/T204MAPK, pT308AKT, pS473AKT, pS118ERα and pS167ERα) in primary tumours were assessed to determine whether these markers can differentiate between patient responses for switching adjuvant endocrine therapy after 2–3 years from tamoxifen to exemestane and continued tamoxifen monotherapy in the Intergroup Exemestane Study (IES). Methods Of the 4724 patients in IES, 1506 were managed in a subset of centres (N = 89) participating in PathIES. These centres recruited 1282 (85%, 1282/1506) women into PathIES of whom 1036 had phospho-marker data. All phospho-markers were analysed by immunohistochemistry staining. Multivariable Cox proportional hazards models of the phospho-markers for disease-free survival (DFS) and overall survival (OS) were adjusted for clinicopathological factors. Treatment effects on the biomarker expression were determined by interaction tests. Benjamini–Hochberg adjustment for multiple testing with a false discovery rate of 10% was applied (pBH). Results Phospho-T202/T204MAPK, pS118ERα and pS167ERα were all found to be correlated (pBH = 0.0002). These markers were not associated with either DFS or OS when controlling for the established clinicopathological factors. Interaction terms between the phospho-markers and treatment strategies for either DFS or OS were not statistically significant (pBH > 0.05 for all). Conclusions This PathIES study confirmed previously described associations between the phosphorylation site markers of AKT, MAPK and ERα activity in postmenopausal breast cancer patients. No prognostic correlations between the phosphorylation markers and clinical outcome were found, nor were they predictive for clinical outcomes among patients who switched therapy over those treated with tamoxifen alone. Electronic supplementary material The online version of this article (10.1007/s10549-018-05110-x) contains supplementary material, which is available to authorized users.

Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERα) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERα transcriptional action in breast and endometrial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERα binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERα binding sites proximal to genes involved in classical ERα target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERα and FOXA1 together with the enhancer-enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERα expression was associated with a longer interval between breast cancer and the emergence of endometrial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERα are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773–84. ©2016 AACR.

We recently reported on the development of a 4-protein-based classifier (PDCD4, CGN, G3BP2, and OCIAD1) capable of predicting outcome to tamoxifen treatment in recurrent, estrogen-receptor-positive breast cancer based on high-resolution MS data. A precise and high-throughput assay to measure these proteins in a multiplexed, targeted fashion would be favorable to measure large numbers of patient samples to move these findings toward a clinical setting. By coupling immunoprecipitation to multiple reaction monitoring (MRM) MS and stable isotope dilution, we developed a high-precision assay to measure the 4-protein signature in 38 primary breast cancer whole tissue lysates (WTLs). Furthermore, we evaluated the presence and patient stratification capabilities of our signature in an independent set of 24 matched (pre- and post-therapy) sera. We compared the performance of immuno-MRM (iMRM) with direct MRM in the absence of fractionation and shotgun proteomics in combination with label-free quantification (LFQ) on both WTL and laser capture microdissected (LCM) tissues. Measurement of the 4-proteins by iMRM showed not only higher accuracy in measuring proteotypic peptides (Spearman r: 0.74 to 0.93) when compared with MRM (Spearman r: 0.0 to 0.76) but also significantly discriminated patient groups based on treatment outcome (hazard ratio [HR]: 10.96; 95% confidence interval [CI]: 4.33 to 27.76; Log-rank P < 0.001) when compared with LCM (HR: 2.85; 95% CI: 1.24 to 6.54; Log-rank P = 0.013) and WTL (HR: 1.16; 95% CI: 0.57 to 2.33; Log-rank P = 0.680) LFQ-based predictors. Serum sample analysis by iMRM confirmed the detection of the four proteins in these samples. We hereby report that iMRM outperformed regular MRM, confirmed our previous high-resolution MS results in tumor tissues, and has shown that the 4-protein signature is measurable in serum samples.

Background Dose-dense administration of chemotherapy and the addition of taxanes to anthracycline-based adjuvant chemotherapy have improved breast cancer survival substantially. However, clinical trials directly comparing the additive value of taxanes with dose-dense anthracycline-based chemotherapy are lacking. Patients and methods In the multicentre, randomised, biomarker discovery Microarray Analysis in breast cancer to Tailor Adjuvant Drugs Or Regimens (MATADOR) trial, patients with pT1-3, pN0-3 breast cancer were randomised (1:1) between six adjuvant cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 2 weeks (ddAC) and six cycles of docetaxel 75 mg/m2, doxorubicin 50 mg/m2 and cyclophosphamide 500 mg/m2 every 3 weeks (TAC). The primary objective was to discover a predictive gene expression profile for ddAC and TAC benefit. Here we report the preplanned secondary end-point recurrence-free survival (RFS) and overall survival (OS). Results Between 2004 and 2012, 664 patients were randomised. At 5 years, RFS was 87% (95% confidence interval [CI] 83%–91%) in the ddAC-treated patients and 88% (84–92%) in the TAC-treated subgroup (hazard ratio [HR] 0.89, 95% CI 0.62–1.28, P = 0.53). OS at 5 years was 93% (90%–96%) in the ddAC-treated and 94% (91%–97%) in the TAC-treated patients (HR 0.89, 95% CI 0.57–1.39, P = 0.61). Anaemia was more frequent in ddAC-treated patients (62/327 patients [18.9%] versus 15/319 patients [4.7%], P Conclusions With a median follow-up of 7 years, no significant differences in RFS and OS were observed between six adjuvant cycles of ddAC and TAC in high-risk breast cancer patients. Trial registration numbers ISRCTN61893718 and BOOG 2004-04.

Background Controversy exists for the use of Ki67 protein expression as a predictive marker to select patients who do or do not derive benefit from adjuvant endocrine therapy. Whether other proliferation markers, like Cyclin D1, and mitotic count can also be used to identify those estrogen receptor α (ERα) positive breast cancer patients that derive benefit from tamoxifen is not well established. We tested the predictive value of these markers for tamoxifen benefit in ERα positive postmenopausal breast cancer patients. Methods We collected primary tumor blocks from 563 ERα positive patients who had been randomized between tamoxifen (1 to 3 years) vs. no adjuvant therapy (IKA trial) with a median follow-up of 7.8 years. Mitotic count, Ki67 and Cyclin D1 protein expression were centrally assessed by immunohistochemistry on tissue microarrays. In addition, we tested the predictive value of CCND1 gene copy number variation using MLPA technology. Multivariate Cox proportional hazard models including interaction between marker and treatment were used to test the predictive value of these markers. Results Patients with high Ki67 (≥5%) as well as low (

Background: The phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways are frequently activated in breast cancer which can result in antioestrogen resistance. Single protein markers failed to be introduced into clinical practice. We, therefore, aimed to find a better read-out of activation of the PI3K and MAPK pathways in ER+/HER2− breast cancer. Assessment of seven PI3K/MAPK proteins might better reflect pathway activation and distinguish patients without adjuvant tamoxifen benefit. Methods: Tumour blocks were recollected from 293 primary postmenopausal ER+/HER2− breast cancer patients randomised between tamoxifen and no adjuvant therapy. PTEN, p-AKT(Thr308), p-AKT(Ser473), p-p70S6K, p-4EBP1, p-ERK1/2 and p-S6RP expression was assessed by immunohistochemistry followed by unsupervised hierarchical clustering. The primary endpoint was recurrence-free interval. Multivariate Cox models were used to assess tamoxifen benefit. A classification tool was developed based on protein expression profile. Results: Subgroups were identified with preferentially activated (A) and preferentially not activated (N) proteins. Patients in group N derived significant benefit from tamoxifen (multivariate hazard ratio (HR) = 0.23, p = 0.000101), while patients from group A did not (multivariate HR = 1.37, p = 0.64), p for interaction 0.020. Our generated classification tool confirmed these results (p for interaction 0.024). Conclusions: Hierarchical clustering of seven PI3K/MAPK proteins reflects pathway activation and can guide treatment decisions in primary ER+/HER2− postmenopausal breast cancer patients.

IntroductionCurrently used tools for breast cancer prognostication and prediction may not adequately reflect a young patient’s prognosis or likely treatment benefit because they were not adequately validated in young patients. Since breast cancers diagnosed at a young age are considered prognostically unfavourable, many treatment guidelines recommend adjuvant systemic treatment for all young patients. Patients cured by locoregional treatment alone are, therefore, overtreated. Lack of prognosticators for young breast cancer patients represents an unmet medical need and has led to the initiation of the PAtients with bReAst cancer DIaGnosed preMenopausally (PARADIGM) initiative. Our aim is to reduce overtreatment of women diagnosed with breast cancer aged≤40 years.Methods and analysisAll young, adjuvant systemic treatment naive breast cancer patients, who had no prior malignancy and were diagnosed between 1989 and 2000, were identified using the population based Netherlands Cancer Registry (n=3525). Archival tumour tissues were retrieved through linkage with the Dutch nationwide pathology registry. Tissue slides will be digitalised and placed on an online image database platform for clinicopathological revision by an international team of breast pathologists. Immunohistochemical subtype will be assessed using tissue microarrays. Tumour RNA will be isolated and subjected to next-generation sequencing. Differences in gene expression found between patients with a favourable and those with a less favourable prognosis will be used to establish a prognostic classifier, using the triple negative patients as proof of principle.Ethics and disseminationObservational data from the Netherlands Cancer Registry and left over archival patient material are used. Therefore, the Dutch law on Research Involving Human Subjects Act (WMO) is not applicable. The PARADIGM study received a ‘non-WMO’ declaration from the Medical Ethics Committee of the Netherlands Cancer Institute - Antoni van Leeuwenhoek hospital, waiving individual patient consent. All data and material used are stored in a coded way. Study results will be presented at international (breast cancer) conferences and published in peer-reviewed, open-access journals.

Both preclinical and clinical data suggest that activation of the PI3K/AKT/mTOR pathway in response to hormonal therapy results in acquired endocrine therapy resistance. We evaluated differences in activation of the PI3K/AKT/mTOR pathway in estrogen receptor α (ERα) positive primary and corresponding metastatic breast cancer tissues using immunohistochemistry for downstream activated proteins, like phosphorylated mTOR (p-mTOR), phosphorylated 4E Binding Protein 1 (p-4EBP1) and phosphorylated p70S6K (p-p70S6K). For p-mTOR and p-4EBP1, the proportion of immunostained tumor cells (0–100%) was scored. Cytoplasmic intensity (0–3) was assessed for p-p70S6K. The difference between expression of these activated PI3K/AKT/mTOR proteins- in primary and metastatic tumor was calculated and tested for an association with adjuvant endocrine therapy. In patients who had received endocrine therapy (N = 34), p-mTOR expression increased in metastatic tumor lesions compared to the primary tumor (median difference 45%), while in patients who had not received adjuvant endocrine therapy (N = 37), no difference was found. Similar results were observed for p-4EBP1 and p-p70S6K expression. In multivariate analyses, adjuvant endocrine therapy was significantly associated with an increase in p-mTOR (p = 0.01), p-4EBP1 (p = 0.03) and p-p70S6K (p = 0.001), indicating that compensatory activation of the PI3K/AKT/mTOR pathway might indeed be a clinically relevant resistance mechanism resulting in acquired endocrine therapy resistance. What's new? Inhibitors of the PI3K/AKT/mTOR pathway can overcome the resistance to estrogen-depletion therapy that often develops in metastatic breast cancer. In this study, the authors compared primary and metastatic tumors; their results suggest that activation of the PI3K/AKT/mTOR pathway in patients who receive adjuvant endocrine therapy is a clinically relevant mechanism of acquired hormone resistance. For identification of companion diagnostics for PI3K/AKT/mTOR inhibitors, the authors conclude that analyzing primary tumor tissue may often fail to predict treatment response in metastatic breast cancer.

Background The prognostic value of tumour-infiltrating lymphocytes (TILs) differs by breast cancer (BC) subtype. The aim of this study was to evaluate TILs in stage III BC in the context of BRCA1/2-like phenotypes and their association with outcome and benefit of intensified platinum-based chemotherapy. Methods Patients in this study participated in a randomized controlled trial of adjuvant intensified platinum-based chemotherapy versus conventional anthracycline-based chemotherapy carried out between 1993-1999 in stage III BC. Stromal TILs were scored according to International guidelines in these HER2-negative tumours. BRCA-profiles were determined using array-based Comparative Genomic Hybridization (aCGH) data. Results TIL levels were evaluated in 248 stage III breast tumours. High TILs are associated with TNBC. Tumours were classified as non-BRCA-like (n = 167), BRCA1-like (n = 30), BRCA2-like (n = 39) or BRCA1/2-like (n = 12). BRCA-like tumours harboured higher TILs compared to non-BRCA-like tumours (median TILs of 20% vs 10%, respectively, p Conclusions In this high-risk breast cancer cohort, high TILs were associated with TNBC and BRCA1-like status. Within the ER-positive subgroup, TIL levels were higher in BRCA1-like compared to non-BRCA-like tumours, but this was not seen within the TNBC subgroup. When adjusted for clinical characteristics, TIL levels were significantly associated with a more favourable outcome in stage III BC patients. Legal entity responsible for the study The authors. Funding Dutch Cancer Society. Disclosure A. Cimino-Mathews: Research grant / Funding (self): BMS. S.C. Linn: Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: Cergentis; Advisory / Consultancy: Novartis; Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Sanofi; Research grant / Funding (institution): Adienne; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Genentch; Research grant / Funding (institution): Tesaro. M. Kok: Advisory / Consultancy, Research grant / Funding (institution): BMS; Research grant / Funding (institution): Roche. All other authors have declared no conflicts of interest.

Background In the 1990s, the use of adjuvant systemic therapy was based on nodal involvement but not on tumor size, as was recommended in the Dutch breast cancer guidelines. Tumor size is an important prognostic variable in breast cancer, although this may not hold true for all molecular subtypes or patient ages. Here, we studied the prognostic value of tumor size for young (≤40 years at diagnosis), systemically untreated, node-negative (N0), breast cancer patients, and assessed whether for any given T-stage survival exceeds 90% at 20 years. Methods All female, breast cancer patients, ≤40 years at time of diagnosis (between 1989 and 2000), were selected from the Netherlands Cancer Registry. All lymph node-positive and systemically treated patients were excluded, resulting in a cohort of 2302 women. Clinico-pathological and follow-up data were collected from national databases, patient records, and the original pathology reports. Multivariate cox regression for overall survival was performed using T-stage (TNM 8.0), molecular subtype, and tumor grade (according to Nottingham Histologic Score) as covariates. Results Survival rates at 20-years are shown in Table. Median follow-up was 17 years. Only 1% had a T3 tumor and these patients were excluded from analysis. The 20-yr OS for the 96 (4%) T1a tumors was 81% (not shown). Women with T2 tumors had a significantly lower OS compared to women with T1a (HR 1.9 P = 0.024), T1b (HR 1.7 P = > 0.001) and T1c tumors (HR 1.31 P = 0.002), adjusted for tumor grade and molecular subtype. No OS data is shown for subgroups with less than 35 patients. Table . 179PD Overall survival (OS; %) at 20 years by T-stage, molecular subtype and tumor grade (Gr) T1b T1c T2 All n OS n OS n OS n OS 359 73 1114 69 611 60 2302 68 ER+HER2- Gr 1 + 2 Gr 3 172 82 464 77 165 56 973 73 43 68 130 55 83 49 269 54 All 79 72 55 69 ER-HER2- Gr 1 + 2 Gr 3 6 - 31 - 27 - 70 65 31 - 205 73 158 66 417 68 All 61 71 63 68 HER2+ Gr 1 + 2 Gr 3 31 - 74 54 36 54 159 57 22 - 81 53 54 61 169 56 All 70 54 57 58 Conclusions T-stage is associated with OS in this patient group. At 20 years follow-up, we found no T-stage with OS rates >90% in N0, systemically untreated patients, meaning that patients regardless of T-stage may benefit from some form of systemic treatment. Legal entity responsible for the study The PARADIGM study group. Funding The Netherlands Organization for Health Research and Development (ZonMW); A Sister’s Hope; De Vrienden van UMC Utrecht. Disclosure S.C. Linn: Research grant / Funding (self): Amgen; Research grant / Funding (self), unrestricted research grant SUBITO study (NCT02810743): Eurocept-pharmaceuticals; Research grant / Funding (institution), research grant NCT02810743 & olaparib;fulvestrant NCT00738777;advisory board olaparib breast cancer: AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Roche; Research grant / Funding (institution), unrestricted research grant for NCT02285179 and study drug (taselisib): Genentech; Advisory / Consultancy, Member of the Scientific Advisory Board of Cergentis (unpaid): Cergentis; Advisory / Consultancy, scientific advisory board for Watson for Oncology (paid to institution): IBM; Research grant / Funding (institution), unrestricted research grant and study drug NCT03283384; research support biomarker study NCT02109913: Novartis; Advisory / Consultancy, Research grant / Funding (institution), scientific advisory board palbociclib; TEAM 2b through unrestricted research grant from Pfizer: Pfizer; Research grant / Funding (institution), Unrestricted research grant paid to institution for ABC study and study drug: Tesaro; Honoraria (institution), Fee for teaching paid to institution: Bayer. All other authors have declared no conflicts of interest.

Background: Peripheral neuropathy is a common and potentially debilitating side-effect of microtubule-stabilizing agents like docetaxel and is characterized by sensory symptoms like numbness, tingling and burning pain. Docetaxel induced neuropathy may occur in up to 37% of patients and can be dose limiting, thus potentially reducing its chemotherapeutic efficacy. Doxorubicine has occasionally been described to be associated with neuropathy. With this background we investigated the role of various single nucleotide polymorphisms (SNPs) to predict chemotherapy-induced neuropathy. The exploration of SNPs and an association with incidence or severity of neuropathy is important, because it can result in identifying individuals being at higher risk of neurotoxicity. Methods: The MATADOR study is a prospective, non-blinded randomized phase II/III trial for patients with invasive breast cancer, comparing TAC (docetaxel 75 mg/m2 as 1 hour i.v. infusion q 3 weeks, in combination with doxorubicin and cyclophosphamide for 6 cycles) with AC dose dense (doxorubicin and cyclophosphamide q 2 weeks)(ISRCTN61893718). In total 180 interesting SNPs were selected that are involved in metabolism, distribution and excretion of drugs and tested on DNA that was isolated from tumor or from tumor surrounding tissue. We selected 20 SNPs for further analyses because of reported associations with chemotherapy-induced neuropathy (see table). In the first analysis DNA of 45 patients was analyzed as a pilot, comparing DNA of 23 neuropathy patients with that of 22 controls. The primary hypothesis was to identify SNPs associated with NCI-CTC grade ≥2 neuropathy. Results: Interim toxicity data from the MATADOR study demonstrated that 30 of 580 patients had grade ≥2 neuropathy. GSTP1 114Ala/114Val genotype was associated with grade ≥2 neuropathy. Twelve out of 14 (86%) patients with this genotype had chemotherapy-induced neuropathy compared to 11 out of 31 (35%) without this genotype (chi-square test p = 0.002). Conclusion: We found a correlation between GSTP1 114Ala/114Val genotype, involved in detoxification, and development of grade ≥2 neuropathy during treatment with chemotherapy. Updated results will be presented at the meeting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-15-05.

Estrogen receptor (ER) positive tumors represent the majority of breast malignancies, and are effectively treated with hormonal therapies, such as tamoxifen. However, in the recurrent disease resistance to tamoxifen therapy is common and a major cause of death. In recent years, in‐depth proteome analyses have enabled identification of clinically useful biomarkers, particularly, when heterogeneity in complex tumor tissue was reduced using laser capture microdissection (LCM). In the current study, we performed high resolution proteomic analysis on two cohorts of ER positive breast tumors derived from patients who either manifested good or poor outcome to tamoxifen treatment upon recurrence. A total of 112 fresh frozen tumors were collected from multiple medical centers and divided into two sets: an in‐house training and a multi‐center test set. Epithelial tumor cells were enriched with LCM and analyzed by nano‐LC Orbitrap mass spectrometry (MS), which yielded >3000 and >4000 quantified proteins in the training and test sets, respectively. Raw data are available via ProteomeXchange with identifiers PXD000484 and PXD000485. Statistical analysis showed differential abundance of 99 proteins, of which a subset of 4 proteins was selected through a multivariate step‐down to develop a predictor for tamoxifen treatment outcome. The 4‐protein signature significantly predicted poor outcome patients in the test set, independent of predictive histopathological characteristics (hazard ratio [HR] = 2.17; 95% confidence interval [CI] = 1.15 to 4.17; multivariate Cox regression p value = 0.017). Immunohistochemical (IHC) staining of PDCD4, one of the signature proteins, on an independent set of formalin‐fixed paraffin‐embedded tumor tissues provided and independent technical validation (HR = 0.72; 95% CI = 0.57 to 0.92; multivariate Cox regression p value = 0.009). We hereby report the first validated protein predictor for tamoxifen treatment outcome in recurrent ER‐positive breast cancer. IHC further showed that PDCD4 is an independent marker., Highlights Analysis of hormonal naïve cohorts of patients with recurrent breast cancer.Laser microdissection based enrichment of epithelial breast tumors.High resolution mass spectrometric analysis of epithelial breast tumors.Development and validation of a protein signature predicting tamoxifen outcome.Validation by immunohistochemistry in independent cohort.

BACKGROUND: Activation of the PI3K/MAPK pathways results in anti-estrogen resistance in vitro, however a biomarker that can predict clinical resistance has not yet been identified. Common drivers of these pathways are PIK3CA mutations, loss of PTEN and over-expression of HER2 and IGF-1R. We aimed to test the prognostic and predictive value of PI3K/MAPK pathway drivers as well as downstream activated proteins in postmenopausal breast cancer patients. METHODS: We collected primary tumor tissue from 563 ERα positive breast cancer patients who were randomized between tamoxifen (1–3 years) versus no adjuvant systemic therapy. PIK3CA hotspot mutations were assessed by Sequenom Mass Spectrometry. Immunohistochemistry was performed for expression of PTEN, IGF-1R and the downstream markers p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interaction between these markers and treatment. RESULTS: No significant interaction between any of the tested PI3K/MAPK pathways drivers and tamoxifen was found. [/bold]However, interactions were identified between tamoxifen and the downstream activated proteins (p-AKT(Thr308), p- mTOR, p-p70S6K and p-ERK1/2). After correcting for multiple testing, p-p70S6K remained significantly associated with tamoxifen resistance. Patients whose tumor did not express p-p70S6K did derive significant benefit from tamoxifen (HR 0.24, 95 % CI= 0.12–0.47, p < 0.0001), while patients whose tumor did express p-p70S6K did not (multivariate HR=1.02, 95% CI=0.48–2.21, p = 0.95), p for interaction 0.003. CONCLUSION: We conclude that the downstream marker of PI3K/MAPK activation p-p70S6K predicts tamoxifen resistance in ERα positive postmenopausal breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-03.

Introduction: Therapy resistance to adjuvant tamoxifen in estrogen receptor positive (ER+) breast cancer (BC) is related to activation of downstream proteins in the PI3K/AKT/mTOR pathway. However, clinical efficacy of mTOR inhibitors has so far been modest. Growing evidence shows that genomic make-up of cancer cells plays a crucial role in an anti-cancer immune response and this is reflected in the presence of tumor infiltrating immune cells. This begs the need for understanding the relationship between tumor-related immune activity, PIK3CA hot-spot mutations (PIK3CAm) and PI3K/AKT/mTOR pathway activation in ER+BC. Material and methods: The IKA trial recruited stage I to III postmenopausal BC patients (1982 till 1994), who were randomized to tamoxifen or no adjuvant therapy. Sequenom mass spectrometry-based genotyping was used for PIK3CAm assessment. Immune markers and phosphorylation status of proteins in the PI3K/AKT/mTOR pathway [p-AKT (Thr308 and 473), p-mTOR, p-ERK1/2, p-p-70S6K and p-4EBP1] were assessed by immunohistochemistry and scored by two pathologists. Expression of CD4, CD8 and FOXP3 was evaluated using automatic scoring by image-analysis software (SlidePath® or AxioVision®) and compared with manual scoring by two pathologists. Associations were assessed using multivariable logistic regression models, including as co-variables: age, tumor grade, lymph node status, tumor size, and progesterone receptor and HER2 status. Results: We included 563 ER+BC cases. PIK3CAm were found in 159 (32%) of the 486 tumors genotyped. On average, PI3K/AKT/mTOR downstream proteins and immune markers were scored in 409 tumors (range: 366 to 438). Stromal CD8 expression, but not CD4 or FOXP3, was significantly higher in PIK3CA mutated tumors (OR=1.11; 95%CI: 1.02-1.22). Stromal FOXP3 expression, but not CD4 or CD8, was significantly increased in tumors with activated proteins from the PI3K/AKT/mTOR pathway (except p-mTOR). The largest association was with p-4EBP1 (OR=1.34; 95%CI: 1.21-1.48) and smallest with p-70S6K (OR=1.15; 95%CI: 1.08-1.22). Conclusion: In our dataset of ER+BC, PIK3CAm are associated with higher level of CD8+ T cells. Tumors with activation of the PI3K/Akt/mTOR pathway tend to have more FOXP3+ T cells. Together, our results suggest that PIK3CA mutation/activation harbor an immune-related tumor microenvironment. Citation Format: Marcelo Sobral-Leite, Izhar Salomon, Mark Opdam, Karin Beelen, Ronald L. van Vlierberghe, Erik J. Blok, Hugo M. Horlings, Joyce Sanders, Koen Van de Vijver, Peter J. Kuppen, Sabine Linn, Marjanka K. Schmidt, Marleen Kok. Cancer-immune interactions in luminal breast cancers: PI3KCA mutations, PI3K/AKT/mTOR activation and tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5612. doi:10.1158/1538-7445.AM2017-5612

535 Background: Young age at breast cancer diagnosis is considered a poor prognostic factor. As a result, many treatment guidelines advice adjuvant systemic treatment for young patients. Answering prognostic questions on young patients has therefore become a challenge. The PARADIGM (PAtients with bReast cAncer DIaGnosed preMenopausally) project aims to assess the long-term outcome of women diagnosed with breast cancer ≤40 years in the absence of adjuvant systemic therapy, using real world data from the nationwide Netherlands Cancer Registry (NCR) coupled with tissue biobanking. Methods: All women ≤40 years, diagnosed in the Netherlands between 1989-2000 with a primary invasive, histologically proven, TanyN0M0 breast cancer, without adjuvant systemic treatment were identified through the NCR. Back then N0 patients were considered low risk and did not receive adjuvant systemic treatment. Tissue specimens were revised by a team of dedicated breast pathologists. Cox regression was performed to estimate hazard ratios for recurrence-free (RFS) and overall survival (OS) according to immunohistochemical (IHC) subtype. Analyses were adjusted for grade, pathological T-stage, histological subtype and radiotherapy. Results: We included 2310 patients with a mean follow-up of 15.4 years (range 0-25 years). OS for the whole cohort was 68% and RFS 58.4% at 25 years. In total 740 deaths and 1043 recurrences were observed. Hormone receptor (HR)+/HER2+ patients had a significantly worse OS when compared to HR-HER2+ patients (adjusted Hazard Ratio 1.58; 95% confidence interval 1.05-2.38; p=0.029). No difference was observed between HR-HER2+ and the triple negative and HR+/HER2- subgroups at 25-years. RFS was similar for all IHC subtypes. Conclusions: In this large cohort of non-adjuvant systemically treated young breast cancer patients with long-term follow-up HR+/HER2+ patients have a significantly worse survival when compared to triple negative, HR-/HER2+ and HR+/HER2- patients. The latter three subtypes have similar OS at 25 years. Future molecular studies have been planned to distinguish the favorable from the unfavorable prognostic patients.

// Annelot G.J. van Rossum 1 , Marleen Kok 2, 3 , Danielle McCool 1 , Mark Opdam 1 , Nienke C. Miltenburg 4 , Ingrid A.M. Mandjes 5 , Elise van Leeuwen-Stok 6 , Alex L.T. Imholz 7 , Johanneke E.A. Portielje 8 , Monique M.E.M. Bos 9 , Aart van Bochove 10 , Erik van Werkhoven 11 , Marjanka K. Schmidt 1 , Hendrika M. Oosterkamp 12, * and Sabine C. Linn 1, 3, 13, * 1 Division of Molecular Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands 2 Division of Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands 3 Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands 4 Department of Neurology, Medical Center Slotervaart, Amsterdam, The Netherlands 5 Data Center, Netherlands Cancer Institute, Amsterdam, The Netherlands 6 Dutch Breast Cancer Research Group, BOOG Study Center, Amsterdam, The Netherlands 7 Department of Medical Oncology, Deventer Ziekenhuis, Deventer, The Netherlands 8 Department of Medical Oncology, HagaZiekenhuis, The Hague, The Netherlands 9 Department of Medical Oncology, Reinier de Graaf Groep, Delft, The Netherlands 10 Department of Medical Oncology, Zaans Medisch Centrum, Zaandam, The Netherlands 11 Biometrics Division, Netherlands Cancer Institute, Amsterdam, The Netherlands 12 Department of Medical Oncology, Haaglanden Medisch Centrum, The Hague, The Netherlands 13 Department of Pathology, University Medical Center, Utrecht, The Netherlands * These authors contributed equally to this work Correspondence to: Sabine C. Linn, email: s.linn@nki.nl Keywords: replication; single nucleotide polymorphisms; association; toxicity; chemotherapy Received: April 13, 2017 Accepted: October 27, 2017 Published: November 27, 2017 ABSTRACT Introduction: Although pharmacogenomics has evolved substantially, a predictive test for chemotherapy toxicity is still lacking. We compared the toxicity of adjuvant dose-dense doxorubicin-cyclophosphamide (ddAC) and docetaxel-doxorubicin-cyclophosphamide (TAC) in a randomized multicenter phase III trial and replicated previously reported associations between genotypes and toxicity. Results: 646 patients (97%) were evaluable for toxicity (grade 2 and higher). Whereas AN was more frequent after ddAC ( P < 0.001), TAC treated patients more often had PNP ( P < 0.001). We could replicate 2 previously reported associations: TECTA (rs1829; OR 4.18, 95% CI 1.84-9.51, P = 0.001) with PNP, and GSTP1 (rs1138272; OR 2.04, 95% CI 1.13-3.68, P = 0.018) with PNP. Materials and methods: Patients with pT1-3, pN0-3 breast cancer were randomized between six cycles A60C600 every 2 weeks or T75A50C500 every 3 weeks. Associations of 13 previously reported single nucleotide polymorphisms (SNPs) with the most frequent toxicities: anemia (AN), febrile neutropenia (FN) and peripheral neuropathy (PNP) were analyzed using logistic regression models. Conclusions: In this independent replication, we could replicate an association between 2 out of 13 SNPs and chemotherapy toxicities. These results warrant further validation in order to enable tailored treatment for breast cancer patients.

Contains fulltext : 136517.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Activation of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways results in anti-estrogen resistance in vitro, but a biomarker with clinical validity to predict intrinsic resistance has not been identified. In metastatic breast cancer patients with previous exposure to endocrine therapy, the addition of a mammalian target of rapamycine (mTOR) inhibitor has been shown to be beneficial. Whether or not patients on adjuvant endocrine treatment might benefit from these drugs is currently unclear. A biomarker that predicts intrinsic resistance could potentially be used as companion diagnostic in this setting. We tested the clinical validity of different downstream-activated proteins in the PI3K and/or MAPK pathways to predict intrinsic tamoxifen resistance in postmenopausal primary breast cancer patients. METHODS: We recollected primary tumor tissue from patients who participated in a randomized trial of adjuvant tamoxifen (1-3 years) versus observation. After constructing a tissue micro-array, cores from 563 estrogen receptor alpha positive were immunostained for p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interactions between these markers and tamoxifen treatment efficacy. RESULTS: Interactions were identified between tamoxifen and p-AKT(Thr308), p-mTOR, p-p70S6K and p-ERK1/2. Applying a conservative level of significance, p-p70S6K remained significantly associated with tamoxifen resistance. Patients with p-p70S6K negative tumors derived significant benefit from tamoxifen (HR 0.24, P < 0.0001), while patients whose tumor did express p-p70S6K did not (HR = 1.02, P =0.95), P for interaction 0.004. In systemically untreated breast cancer patients, p-p70S6K was associated with a decreased risk for recurrence. CONCLUSIONS: Patients whose tumor expresses p-p70S6K, as a marker of downstream PI3K and/or MAPK pathway activation, have a favorable prognosis, but do not benefit from adjuvant tamoxifen. A potential benefit from inhibitors of the PI3K/Akt/mTOR pathway in these patients needs to be further explored.

Estrogen catabolism is a major function of CYP2C19. The effect of CYP2C19 polymorphisms on tamoxifen sensitivity may therefore not only be mediated by a variation in tamoxifen metabolite levels but also by an effect on breast cancer risk and molecular subtype due to variation in lifelong exposure to estrogens. We determined the association between these polymorphisms and tamoxifen sensitivity in the context of a randomized trial, which allows for the discernment of prognosis from prediction. We isolated primary tumor DNA from 535 estrogen receptor-positive, stages I–III, postmenopausal breast cancer patients who had been randomized to tamoxifen (1–3 years) or no adjuvant therapy. Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of CYP2C19*2 and CYP2C19*17. Hazard ratios and interaction terms were calculated using multivariate Cox proportional hazard models, stratified for nodal status. Tamoxifen benefit was not significantly affected by CYP2C19*17. Patients with at least one CYP2C19*2 allele derived significantly more benefit from tamoxifen (HR 0.26; p = 0.001) than patients without a CYP2C19*2 allele (HR 0.68; p = 0.18) (p for interaction 0.04). In control patients, CYP2C19*2 was an adverse prognostic factor. In conclusion, breast cancer patients carrying at least one CYP2C19*2 allele have an adverse prognosis in the absence of adjuvant systemic treatment, which can be substantially improved by adjuvant tamoxifen treatment. Electronic supplementary material The online version of this article (doi:10.1007/s10549-013-2568-0) contains supplementary material, which is available to authorized users.

In previous studies, high expression of XIST and low expression of 53BP1 were respectively associated with poor systemic therapy outcome in patients and therapy resistance in BRCA1-deficient mouse tumor models, but have not been evaluated in BRCA1-deficient patients. Previously, we demonstrated that classifying breast cancer copy number profiles as BRCA1-like or non–BRCA1-like identified patients enriched for defects in BRCA1 that benefit from high-dose (HD) alkylating chemotherapy compared with a conventional standard regimen. We investigated whether XIST and 53BP1 expression predicted poor outcome of HD chemotherapy within 28 BRCA1-like patients from a trial randomizing between HD [4 cycles 5-fluorouracil, epirubicin, cyclophosphamide (FEC) followed by 1 cycle HD carboplatin, thiotepa, cyclophosphamide] or conventional chemotherapy (5 cycles FEC), for which both XIST and 53BP1 statuses were available. High RNA expression of XIST (n = 5) and low protein expression of 53BP1 (n = 3) expression did not coincide. Patients with either one had poor outcome after treatment with HD chemotherapy, whereas patients with low expression of XIST and high expression of 53BP1 derived substantial benefit of this regimen on recurrence-free survival, disease-free survival, and overall survival, corroborating preclinical findings. XIST and 53BP1 may be predictive biomarkers in BRCA1-like breast cancer. Mol Cancer Ther; 15(1); 190–8. ©2015 AACR.

BACKGROUND: Activation of the PI3K pathway as an adaptive change in response to estrogen depletion results in acquired endocrine therapy resistance in vitro. Metastatic breast cancer patients with previous exposure to endocrine therapy do derive substantial benefit from the addition of an mTOR inhibitor to endocrine therapy compared to endocrine therapy alone. This suggests that compensatory PI3K pathway activation might play a role in the development of clinical resistance to endocrine therapy. We evaluated the activation of the downstream PI3K pathway protein mTOR in primary breast tumors and matched metastatic lesions. METHODS: From a previously described series of breast cancer patients from whom both primary tumor tissue as well as metastatic tumor tissue was collected, we selected estrogen receptor α (ERα) positive patients who had received endocrine therapy (N = 34) and who did not receive endocrine therapy (N = 37). The difference in expression of phosphorylated mTOR (p-mTOR) between primary and metastatic tumor was assessed by immunohistochemistry, which was scored by using the proportion of tumor cells with sub-membranous p-mTOR expression. We assessed whether this p-mTOR difference between primary and metastatic tumor was associated with clinico-pathological factors (location of metastasis, lymph node status, T-stage, grade, progesterone receptor status) or varied between patients who did and did not receive endocrine therapy, using Mann-Whitney tests. In addition we performed a linear regression model including the same clinico-pathological factors. RESULTS: In patients who had received endocrine therapy we observed an increase in p-mTOR expression in metastatic tumor lesions compared to the primary tumor (median difference 45%). This was significantly different from the mTOR changes observed in patients who did not receive endocrine therapy (median difference between metastatic and primary lesion 0 %) (p = 0.003). The difference remained significant in the multivariate regression model (p = 0.001). None of the other factors was significantly associated with a differential p-mTOR change. CONCLUSION: In patients who received previous adjuvant endocrine therapy, the increase in p-mTOR expression in metastatic tumor lesions compared to the primary tumor is significantly higher than in patients who did not receive endocrine therapy. Compensatory activation of the PI3K pathway might therefore be a clinically relevant resistance mechanism resulting in secondary endocrine therapy resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-02.

Tamoxifen is one of the most frequently used drugs in the treatment of estrogen receptor positive breast cancer. Although tamoxifen significantly increases patient survival in the adjuvant setting, a major factor that decreases its efficacy in the recurrent setting is drug resistance. The aim of our study is to identify a protein signature predicting outcome of tamoxifen treatment using high resolution mass spectrometry. A total of 112 snap frozen breast tumor tissues were gathered from patients that had received tamoxifen as first line treatment upon recurrence. Samples were divided into a discovery (n= 56; Erasmus Medical Center) and validation sets (n=56; multi-center cohort). Based on progression of disease from start of tamoxifen therapy, patients were categorized having either Good (time-to-progression; ttp>6 months) or Poor (ttp A total of 2741 proteins were identified in both datasets. Comparative proteome analysis showed differential expression of 99 proteins in the discovery set. A subset of 4 proteins (PDCD4, OCIAD1, G3BP2, and CGN) derived from the multivariate model was selected to develop a predictor for tamoxifen outcome. The 4 protein abundances in the discovery samples were used to build a receiver operating characteristic curve, in which Youden J was selected as grouping cutoff. Group prediction was tested in the validation set through log rank test, which resulted in a significant ttp difference (p=0.004; HR=2.33, 95% CI [1.30-4.16]) between Good and Poor outcome. Our signature significantly predicts Poor outcome patients with 86.7% sensitivity, 41.46% specificity, 35.1% PPV, and 89.5% NPV in the multi-center cohort. OCIAD1, PDCD4, and CGN were significantly enriched in patients with Good outcome, while G3BP2 was more abundant in patients with Poor outcome. We are currently validating these results by immunohistochemistry in a large independent cohort. In addition, we are developing a targeted, multiplexed, quantitative MS-based assay for larger scale clinical validation. Our 4-protein signature significantly predicts outcome to tamoxifen treatment for recurrent breast cancer. Large scale clinical validation will enable more tailored treatment of breast cancer patients with advanced ER+ disease. In addition, our findings may help elucidate the mechanisms underlying endocrine therapy resistance in recurrent breast cancer. Citation Format: Tommaso De Marchi, NingQing Liu, Mila Tjoa, Christoph Stingl, Marcel Smid, Maxime P. Look, Mieke A. Timmermans, Rene BH Braakman, Mark Opdam, Sabine Linn, Paul Span, Fred CGJ Sweep, John WM Martens, Theo M. Luider, John A. Foekens, Arzu Umar. 4-protein signature predicts outcome to tamoxifen treatment in estrogen receptor positive recurrent breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2014-1611

544 Background: Elevated insuline-like growth factor 1 receptor (IGF-1R) signaling activates the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways and result...