ER Pathway Activity Research Articles

Hormone Receptor Expression and Activity for Different Tumour Locations in Patients with Advanced and Recurrent Endometrial Carcinoma.

Response to hormonal therapy in advanced and recurrent endometrial cancer (EC) can be predicted by oestrogen and progesterone receptor immunohistochemical (ER/PR-IHC) expression, with response rates of 60% in PR-IHC > 50% cases. ER/PR-IHC can vary by tumour location and is frequently lost with tumour progression. Therefore, we explored the relationship between ER/PR-IHC expression and tumour location in EC. Pre-treatment tumour biopsies from 6 different sites of 80 cases treated with hormonal therapy were analysed for ER/PR-IHC expression and classified into categories 0-10%, 10-50%, and >50%. The ER pathway activity score (ERPAS) was determined based on mRNA levels of ER-related target genes, reflecting the actual activity of the ER receptor. There was a trend towards lower PR-IHC (33% had PR > 50%) and ERPAS (27% had ERPAS > 15) in lymphogenic metastases compared to other locations (p = 0.074). Hematogenous and intra-abdominal metastases appeared to have high ER/PR-IHC and ERPAS (85% and 89% ER-IHC > 50%; 64% and 78% PR-IHC > 50%; 60% and 71% ERPAS > 15, not significant). Tumour grade and previous radiotherapy did not affect ER/PR-IHC or ERPAS. A trend towards lower PR-IHC and ERPAS was observed in lymphogenic sites. Verification in larger cohorts is needed to confirm these findings, which may have implications for the use of hormonal therapy in the future.

Abstract PO5-21-06: Pathway profiling for prediction of response to neoadjuvant Letrozole therapy in ER positive postmenopausal breast cancer: gaining new insights for targeted treatment

Abstract Introduction: The NEOLBC (NCT03283384) study included postmenopausal patients with hormone receptor positive (ER≥50%, PR any), HER2 negative, stage II/III breast cancer. A baseline biopsy was taken prior to start letrozole treatment and after two weeks of treatment. Based on the two week Ki67-IHC, patients were randomized (Ki67 ≥1%) to receive either Letrozole + Ribociclib or standard chemotherapy until surgery, or continued (Ki67 < 1%) Letrozole mono therapy. Here we describe the signal transduction pathway profiles to better understand the molecular mechanisms of response to neoadjuvant letrozole. Methods: Signal transduction pathway activities for the ER, AR, MAPK, PI3K, HH, TGFβ and Notch pathways were measured on baseline and two weeks samples using the OncoSIGNal qPCR test (InnoSIGN). High pathway activity in a tumor sample was defined as pathway activity value above the 95th percentile of reference normal epithelial breast tissue. The pathway activity patterns of the Ki67 < 1% (n=30) and Ki67 > 1% (n=63) groups were compared between baseline and two weeks of treatment. Results: Although all measured baseline samples (n=93) were ER IHC staining positive (≥50%), there is a large range in baseline ER signal transduction pathway activity (32 to 78 on a scale from 0 – 100) and 20% of the samples show an ER-activity range (32 - 45) that normally falls in the range of ER IHC stained negative breast tissue. The group with Ki67 > 1% after 2 weeks Letrozole treatment showed non elevated ER pathway activity at baseline more often (25%) compared to the Ki67 < 1% group (10%). 92% of the patients showed a decrease in ER pathway (11.6 ± 8.6) activity after 2 week letrozole treatment compared to baseline (p=8.5e-13). Interestingly, baseline AR pathway activity has a good differentiating predictive value for the two week response to letrozole treatment and the overall AR pathway activity score was higher in the low Ki67 group (51.4 ± 7.4) than in the Ki67 ≥ 1% group (46.7 ± 6.4; p=0.0053) and more often classified as high in 30% of the low Ki67 group vs. 13% of the Ki67 ≥ 1% group. The Ki67 > 1% group showed activation of non-hormonal pathways (PI3K, MAPK and/or HH) more frequent (38%) at baseline compared to the < 1% Ki67 group (19%). Conclusion: Positive ER staining does not always relate to a high ER pathway activity, possibly explaining that not all patients respond equally well to ER inhibition therapy. Activation of hormonal pathways (ER, but also AR) appear to be predictive for early (2 weeks) response towards Letrozole as assessed by Ki67 staining. Involvement of non-hormonal pathways is associated with less effective response towards Letrozole alone. Pathway activity patterns could improve insight to underlying hormonal treatment escape mechanisms which could lead to alternative treatment options in patients with ER-positive breast cancer. Citation Format: Nadia de Gruil, Anne Florine de Groot, Yvonne Wesseling-Rozendaal, Diederick Keizer, Sigi Neerken, Judith Kroep. Pathway profiling for prediction of response to neoadjuvant Letrozole therapy in ER positive postmenopausal breast cancer: gaining new insights for targeted treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-21-06.

Abstract PS17-01: Key drivers of therapeutic response and resistance to giredestrant from GO39932: a Phase Ia/b study in patients with estrogen receptor-positive, HER2-negative, locally advanced or metastatic breast cancer

Abstract BACKGROUND Several investigational oral selective estrogen receptor antagonists and degraders (SERDs) are under investigation. These were partly developed to benefit patients (pts) with breast cancer (BC) who acquired resistance to standard-of-care (SoC) endocrine therapies (ET); e.g., through gain of ESR1 mutations that enable estrogen-independent ER activity. One such SERD, giredestrant (G), demonstrates activity in pts with ESR1-wild type (WT) or -mutant (m) tumors, and in pts who progressed on other ETs. Results from Phase I/III trials in ER-positive, HER2-negative, locally advanced/metastatic BC (ER+, HER2– LA/mBC) showed that next-generation SERDs had increased activity in ESR1m tumors vs SoC ETs. However, enthusiasm was hindered by heterogeneous responses, driven in part by study design differences. Across acelERA BC (NCT04576455), EMERALD (NCT03778931), SERENA-2 (NCT04214288), and AMEERA-3 (NCT04059484), 30–50% of pts progressed rapidly (< 2 months) when treated with SoC ETs or next-generation SERDs, while others benefited for ≥ 1 year. We present an exploratory biomarker analysis aimed at understanding the mechanistic basis for heterogeneous responses to SERDs in ER+, HER2– LA/mBC, by assessing a Phase Ia/b G cohort (NCT03332797) with paired baseline and on-treatment (tx) biopsies. METHODS Pts had ≤ 2 prior therapies for LA/mBC; disease recurrence/progression while on adjuvant ET for ≥ 24 months and/or ET for ER+, HER2– LA/mBC; and tumor response/stable disease for ≥ 6 months. Single-agent dose-escalation stage: 10, 30, 90, or 250 mg G once daily (QD) on Days (D) 1–28 of 28-D cycles (C). Dose-expansion stage: 30, 100, or 250 mg G QD. 100 mg G + 125 mg palbociclib on a 21-D on/7-D off schedule was also explored. Pre-/perimenopausal pts received LHRH agonists. Paired pre- and on-tx (C2D8) tumor biopsies (n = 29) were immunolabeled for ER, progesterone receptor, and Ki67, and assessed via bulk RNA-sequencing. Pre-tx liquid biopsies (n = 85) were evaluated by FoundationOne Liquid CDx assay. To validate our clinical observations, we generated a SERD-resistant MCF7 cell line and tested its sensitivity to other therapies. RESULTS We compared fast-progressing pts (FP; progression-free survival [PFS] < 2 months) with those experiencing long-term benefit (LTB; PFS > 12 months). LTB was significantly associated with high tumor baseline ER pathway activity. Although the benefit of G was of larger magnitude among pts with ESR1m tumors compared with SoC ET in acelERA BC, here pts with predominantly ESR1WT tumors received LTB on G tx. At the molecular level, G acted on LTB tumors by suppressing cell cycle- and ER-associated genes. In contrast, G had no effect on the transcriptome of FP tumors, which had lower baseline ER activity than LTB tumors. FP tumors were instead enriched for multiple cancer-associated pathways, e.g., RAS/MAPK and PI3K, which may drive resistance to G and thus enable fast progression. By liquid biopsy, most FP pts (> 90%) did not harbor a mutation in the associated pathway (e.g., NF1, KRAS), and thus could not be identified by genomic profiling alone. FP tumors were instead enriched for multiple cancer-associated pathways by gene expression. We explored these mechanisms further in the SERD-resistant cell line, which was similarly enriched for EGFR and MAPK activation vs standard MCF7 cells. Although cells became resistant to ER-targeted drugs, they acquired sensitivity to EGFR/MAPK inhibitors e.g., gefitinib, cobimetinib. CONCLUSIONS We identified molecular features associated with LTB to G and revealed a set of oncogenic pathways associated with FP pts on tx; demonstrating that these pathways represent acquired dependencies and potential therapeutic targets for SERD-resistant tumors and FP pts. Citation Format: Jackson Liang, Christy Ong, Jennifer Giltnane, Junko Aimi, Ching-Wei Chang, Mary Gates, Jennifer Eng-Wong, Pablo Perez-Moreno, Komal Jhaveri, Elgene Lim, Nicholas Turner, Heather Moore. Key drivers of therapeutic response and resistance to giredestrant from GO39932: a Phase Ia/b study in patients with estrogen receptor-positive, HER2-negative, locally advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-01.

Increased PI3K pathway activity is associated with recurrent breast cancer in patients with low and intermediate 21-gene recurrence score

AimsWe investigated key signalling pathways’ activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence.MethodsThis is...

Exploratory biomarker analysis of acelERA breast cancer (BC): Phase II study of giredestrant vs. physician’s choice of endocrine therapy (PCET) for previously treated, estrogen receptor-positive, HER2-negative advanced BC (ER+, HER2– aBC).

1069 Background: ET is the mainstay treatment of ER+ aBC. Giredestrant is a highly potent, nonsteroidal, oral selective ER antagonist and degrader (PO SERD) that achieves robust ER occupancy. acelERA BC (NCT04576455) is a Phase II, randomized, open-label study that evaluated giredestrant vs. PCET in the second- or third-line ER+, HER2– aBC setting. The study did not reach statistical significance for the primary endpoint of investigator-assessed progression-free survival, although giredestrant demonstrated a more pronounced effect in patients (pts) with ESR1-mutated ( ESR1m) tumors. We present an exploratory biomarker analysis. Methods: Pts (N = 303) were post- or pre-/perimenopausal women, and men, with ER+, HER2– aBC who had received 1–2 prior lines of systemic therapy for aBC. Randomization was 1:1 to giredestrant (30 mg PO daily) or PCET (fulvestrant/aromatase inhibitor). Baseline plasma circulating tumor (ct)DNA (n = 232) was evaluated with the FoundationOne Liquid CDx (n = 229) or PredicineCARE (n = 3) assays; ESR1 and other gene mutations in ctDNA were defined as variants with known or likely impact on protein function. Gene expression and PAM50 molecular subtype were assessed by RNAseq from tumor tissue (n = 184) which, based on collection date relative to aBC, was defined as representing early BC (eBC, n = 67), first line (1L, n = 64), or post-1L (n = 46). ER pathway activity was quantified by expression of a predefined set of 38 ER-induced or -repressed genes. Results: The majority of tumors were of the Luminal A (51%) and Luminal B (43%) PAM50 subtypes. Post-1L ESR1m tumors (n = 17) were significantly enriched for Luminal B (64%) compared with eBC (39%), 1L (44%), and post-1L tumors with no ESR1m detected ( ESR1nmd; n = 29; 45%) (p < 0.001). Post-1L ESR1nmd tumors, but not ESR1m tumors, had significantly lower ER pathway activity compared with eBC or 1L tumors (p = 0.0001, 0.001). Within the post-1L subgroup, those with ESR1m had a distinct ctDNA mutational profile with more GATA3 (n = 29) and fewer CHEK2 mutations (n = 0) than ESR1nmd (n = 3 and n = 11, respectively). Tumor ER pathway activity and ESR1 expression in tumor tissue, as well as pre-treatment ctDNA composite tumor fraction and ESR1 variant allele frequency, were significantly correlated with best overall response to single-agent ET (partial response vs. progressive disease; p < 0.001, p = 0.022, p = 0.001, and 0.045, respectively). Conclusions: These data show that tumor response to single-agent ET is correlated with baseline ctDNA features and with tumor ER pathway activity. Interestingly, ER pathway activity was significantly lower in late-line tumors, except in ESR1m tumors. Overall, these results support the continued investigation of improved and personalized treatment options for pts with ER+, HER2– aBC. Clinical trial information: NCT04576455 .

Abstract PD13-02: PD13-02 Exploratory gene expression analysis of coopERA Breast Cancer (BC): a study evaluating neoadjuvant giredestrant versus anastrozole alone and in combination with palbociclib in ER-positive, HER2-negative untreated early BC

Abstract Background: Endocrine therapy remains the mainstay treatment for ER+ BC. CDK4/6 inhibitors induce cell cycle arrest and decrease tumor cell proliferation, as measured by the biomarker Ki67, when used in combination with aromatase inhibitors (AI) such as anastrozole (A). Giredestrant is an oral, well-tolerated, and highly potent selective ER degrader (SERD) that achieves robust ER suppression and has demonstrated antitumor activity in the metastatic setting either as monotherapy or in combination with the CDK4/6 inhibitor palbociclib. The randomized, phase 2 coopERA Breast Cancer study (NCT04436744) evaluated giredestrant in postmenopausal women with untreated ER+/HER2- early BC and met its primary endpoint, demonstrating superior Ki67 suppression with giredestrant vs A after two weeks of single agent treatment. This suppression was maintained at surgery where giredestrant vs A was evaluated in combination with palbociclib. Here, we present gene expression analysis and associations with Ki67 response. Methods: 221 eligible patients with measurable ER+/HER2– untreated early BC and baseline Ki67 ≥ 5% were randomized 1:1 to receive 30 mg oral daily (PO QD) giredestrant or 1 mg PO QD A on Days 1–14 of a neoadjuvant window-of-opportunity phase, followed by four 28-day cycles of PO QD giredestrant or A with 125 mg PO QD palbociclib on Days 1–21 before surgery. FFPE specimens were collected at baseline, week 2 and surgery; and RNA-sequencing (seq) was performed. Gene expression analysis included ER pathway activity, PAM50 intrinsic subtypes, and other pathway analyses, assessed by Ki67 response. Results: 112 and 92 patients had paired tumor samples at baseline/week 2 and baseline/surgery, respectively, that were evaluable for RNA-seq and Ki67. The trend for greater Ki67 protein suppression by giredestrant vs A from baseline to week 2 was maintained in the RNA-seq evaluable subset. Interestingly, the same subset revealed similar suppression of both proliferation gene signatures and ER pathway activity between A and giredestrant. PAM50 subtyping showed that 69% of tumors were luminal (Lum) A and 29% were LumB at baseline. Less than 1% were classified as basal or HER2. Interestingly, giredestrant (G) showed greater suppression of both Ki67 and ER pathway activity vs A in LumB tumors (Ki67: -82% [G] vs -62% [A]; ER activity: -0.83 [G] vs -0.66 [A]) compared to LumA at week 2 (Ki67: -74% [G] vs -71% [A]; ER activity: -0.60 [G] vs -0.70 [A]). Moreover, at week 2, 83% (13/18) of LumB tumors at baseline transitioned into a LumA subtype after giredestrant treatment compared to 46% (5/11) of A-treated tumors. Giredestrant-treated tumors also achieved lower mean ER pathway activity compared to those treated with A at surgery (p=0.023). Gene set enrichment analysis showed downregulation of cell-cycle and ER-related pathways at week 2 and surgery in both treatment arms. A subset of cytokine signaling and immune response pathways were increased at week 2 compared to baseline after treatment with A but not with giredestrant. These pathways were also associated with Ki67 resistance (Ki67 ≥ 7.4%) in A-treated tumors. This was consistent with differential expression analysis of samples collected at week 2, in which cytokine signaling pathways were enriched in A compared to giredestrant. Notably, IL12 signaling was enriched in tumors resistant to A but not giredestrant. Conclusions: Giredestrant has a greater effect on Ki67 protein suppression in ER+/HER2- early BC compared to A, which is more pronounced in LumB tumors. This benefit may involve differential regulation of cytokine and immune responses. These exploratory findings reveal novel mechanisms that may differentiate the activity of SERDs vs AIs, which warrant further validation. Citation Format: Alejandro M. Chibly, Tharu M. Fernando, Ciara Metcalfe, Marc Hafner, Gilbert Owusu-Manu, Sara Hurvitz, Aditya Bardia, Peter A. Fasching, Yeon H. Park, Vanesa Quiroga, Jutta Steinseifer, Pablo Perez-Moreno, Heather M. Moore. PD13-02 Exploratory gene expression analysis of coopERA Breast Cancer (BC): a study evaluating neoadjuvant giredestrant versus anastrozole alone and in combination with palbociclib in ER-positive, HER2-negative untreated early BC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD13-02.

Abstract P3-07-07: The use of ribociclib/letrozole combination as an alternative for neoadjuvant chemotherapy in selected patients with early luminal breast cancer

Abstract Introduction In hormone receptor-positive, HER2-negative early-stage breast cancer (BC), cyclin-dependent kinases 4 and 6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) could represent an alternative to neoadjuvant chemotherapy (NAC). Methods NEOLBC is a randomized phase II trial that tailored neoadjuvant therapy in postmenopausal patients with early, luminal (ER >50%, PR any), HER2-negative, stage II/III BC based on the percentage of Ki67 positive cancer cells after a window of opportunity of two weeks letrozole. Patients with a Ki67 >= 1% after 2 weeks were randomized between ribociclib plus letrozole (RL) and chemotherapy (CT; AC-T regimen). The primary objective was to determine if RL gives a doubling in complete cell cycle arrest (CCCA; Ki67 < 1% on IHC) as compared to CT in the surgical specimen (70% vs. 35% of patients, respectively). Secondary endpoints included pathological response, toxicity and ER pathway activity (measured by the OncoSIGNal qPCR test). Results Out of 161 registered patients, 70 patients were randomized and 66 patients started treatment; 34 RL and 32 CT. Patient characteristics were equally distributed between the two groups, except for the PR status (RL 23.5% negative vs. 50.0% negative in the CT group). In the intention to treat analysis, the CCCA in the surgical specimen was similar for both groups: 35.3% in the RL vs. 31.3% in the CT group (p = 0.73). The pathological complete response (pCR) in the breast was not significantly different between the two groups (11.8% vs. 3.1%, p = 0.36) nor was the pCR rate in breast plus lymph nodes (8.8% vs. 3.1%, p = 0.61) for the RL vs. CT group, respectively. An explorative analysis on the difference in Ki67% (decline, no change, increase) from baseline to surgery showed a decline in 73.5% vs. 50.0%, no change in 17.6% vs. 31.3% and an increase in 8.8% vs. 18.8% of patients (p = 0.06) for the RL vs. CT group, respectively. In the RL group eight patients (23.5%) discontinued ribociclib early due to toxicity (two SAE’s were observed) vs. 10 patients (31.3%) discontinuing treatment in the CT group (one SAE was observed). Secondary endpoints, including the ER pathway activity analysis, will be presented in-depth during the meeting. Conclusion Although the primary endpoint was not met, the NEOLBC trial showed a similar CCCA and pathological response at surgery for RL vs. CT. Therefore, RL as an alternative for NAC merits further investigation in follow-up studies. ClinicalTrials.gov: NCT03283384 Citation Format: Anne Florine de Groot, Danielle Cohen, Joan B. Heijns, Caroline Mandigers, Diederick M. Keizer, Hein Putter, Elma Meershoek-Klein Kranenbarg, Marjolijn Duijm-de Carpentier, Kyra Dijkstra, Elise van Leeuwen-Stok, Gerrit-Jan Liefers, Sabine Linn, Judith Kroep. The use of ribociclib/letrozole combination as an alternative for neoadjuvant chemotherapy in selected patients with early luminal breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-07.

Abstract PD10-06: PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer

Abstract Background: Endocrine therapy (ET) is the mainstay of ER+ BC treatment. However; up to 20% of ER+ BC tumors progress into metastatic disease and develop ET resistance, underscoring the need for combination therapies. Preclinical data suggest that combining phosphatidylinositol 3-kinase (PI3K) inhibitors with ET may overcome resistance. OPPORTUNE, a preoperative phase II window trial, evaluated whether the combination of the PI3K inhibitor pictilisib with anastrozole (PIC+ANA) can increase the antitumor effect of ANA in newly diagnosed operable ER+ BC. Early results showed greater suppression of tumor Ki67 in patients treated with PIC+ANA versus ANA alone. Here, we present gene expression analysis from tumors collected pre- and post-treatment, and their associations with Ki67 outcomes. Methods: Postmenopausal women with newly diagnosed operable ER+/HER2-negative BC were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with ANA 1 mg once per day (n = 47) or the combination of ANA 1 mg with PIC 260 mg once per day (n = 89). The primary end point was inhibition of tumor cell proliferation measured by change in Ki67 protein expression via IHC between tumor samples taken pre- and post-treatment. Samples were analyzed by RNA-sequencing—ER pathway activity, PAM50 intrinsic subtypes, and pathway analyses were assessed by Ki67 outcomes. Elastic net regression analysis and transcription factor activity inference with Dorothea were performed to identify features strongly associated with Ki67 outcomes. Results: 124 patients (ANA, n=43; PIC+ANA, n=81) had paired tumor samples at baseline/week 2 that were evaluable for both Ki67 and RNA-seq. PIC+ANA showed improved suppression of Ki67 compared to ANA alone (-83.78% vs -73.85%, p=0.012) and a greater proportion of patients achieved complete cell cycle arrest (CCCA, as defined by Ki67< 2.7%) in the PIC+ANA arm (45.68% vs 36.59%). ER pathway activity suppression was comparable between treatments. PAM50 classification based on RNA-seq showed that 83.0% of tumors were luminal (Lum) A at baseline and 12.9% were LumB, with the remainder being classified as Normal-like. PIC+ANA showed greater suppression of Ki67 in LumB tumors compared to LumA (-92.29% vs -81.62%). This effect was much greater in the PIC+ANA arm compared to LumB tumors treated with ANA alone (-92.29% vs -37.87%); however, given the low number of LumB tumors in the ANA arm (n=3), we could not determine statistical significance. Bioinformatic analysis of RNA-seq from baseline specimens showed that MYBL2 activity was associated with resistance to ANA (as defined by Ki67≥7.5% at week2). Tumors in the top quartile of MYBL2 activity at baseline showed improved Ki67 outcomes in the PIC+ANA arm compared to ANA alone (-91.71% vs -51.44%), while no differences were observed between treatments for tumors with lower MYBL2 activity (-79.16% vs -76.72%). Single-nucleus (sn)-RNA-seq from untreated ER+ BC tumors showed that MYBL2 was enriched in a single cluster of tumor cells, which also had the highest expression of a subset of actionable targets (including CDK1, CDK2, CDK4, EZH2, and AKT1), as compared to other tumor cells. Expression data from DepMap and drug-sensitivity data from ER+ BC cell lines show a positive trend between high MYBL2 expression and sensitivity to the ER degrader, giredestrant. Conclusions: PIC exhibited greater antiproliferative effects in combination with ANA in ER+/HER2- early BC compared to ANA alone, particularly in LumB and MYBL2-high tumors. Furthermore, the transcriptional profile and in vitro response of tumor cells with high MYBL2 expression suggest potential sensitivity to other combination therapies. Citation Format: Alejandro Martinez Chibly, Alice Shia, Radia Johnson, Michael S. Hwang, Marc Hafner, Ciara Metcalfe, Kalpit Shah, Michal Slyper, Chris Bolen, Sarah E. Pinder, Alastair M. Thompson, Steven Gendreau, Peter Schmid. PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-06.

Abstract 3015: Serine starvation silences estrogen receptor signaling through histone hypoacetylation

Abstract Estrogen receptor (ER) plays important roles in regulating normal development and female reproductive system function. Loss of ER pathway activity is a hallmark of breast cancer progression, associated with accelerated tumor proliferation and resistance to endocrine therapy. How ER loss occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in solid tumors, downregulates estrogen receptor alpha (ERα) expression, represses transcriptional targets such as progesterone receptor (PR), and reduces sensitivity to antiestrogens, suggesting a transition of ER-positive (ER+) breast cancer cells to an ER/PR-negative (ER-/PR-) state. ER downregulation under serine starvation is accompanied by a global loss of histone acetylation. These chromatin changes are driven by metabolic reprogramming triggered by serine starvation, particularly lower glucose flux through glycolysis and the TCA cycle, leading to reduced acetyl-CoA levels and histone hypoacetylation. Supplementation with acetate or glycerol triacetate (GTA), precursors of acetyl-CoA, restores H3K27 acetylation and ERα expression under serine starvation. Therefore, a major consequence of serine starvation in breast cancer could be global chromatin changes that influence lineage-specific gene expression. Citation Format: Albert M. Li, Yang Li, Bo He, Haowen Jiang, Chao Lu, Joshua J. Gruber, Erinn B. Rankin, Jiangbin Ye. Serine starvation silences estrogen receptor signaling through histone hypoacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3015.

Abstract PD13-07: Activity and biomarker analyses from a phase Ia/b study of giredestrant (GDC-9545; G) with or without palbociclib (palbo) in patients with estrogen receptor-positive, HER2-negative locally advanced/metastatic breast cancer (ER+/HER2- LA/mBC)

Abstract Background A mainstay of ER+ BC treatment is to target ER activity and/or estrogen synthesis; however, during or after endocrine therapy (ET), many patients either relapse or develop resistance to such treatments due to several mechanisms, including ESR1 mutations which can be drivers of estrogen-independent transcription and proliferation. The majority of tumors remain dependent on ER signaling, and it is possible that patients will be responsive to second- or third-line ET after they have progressed on previous therapies. The highly potent, nonsteroidal oral selective ER antagonist and degrader G achieves robust ER occupancy and is active in patients with either ESR1-wildtype or -mutant tumors and in patients who had previously been treated with ET. G was well tolerated as a single agent and in combination with palbo, with antitumor activity shown in the nonrandomized, open-label, dose-escalation and -expansion, phase Ia/b GO39932 study (NCT03332797). We present updated activity and biomarker analyses in patients treated with 30 mg G monotherapy and those treated with 100 mg G + 125 mg palbo ± luteinizing hormone-releasing hormone (LHRH) agonists (clinical cutoff: Apr 16, 2021); 30 mg has since been selected as the dose in both the single agent and combination settings. Methods Eligible patients had ≤2 prior therapies for LA/mBC, disease recurrence, or progression while on adjuvant ET for ≥24 months and/or ET for LA/mBC, and tumor response or stable disease ≥6 months. All were postmenopausal (premenopausal women were allowed co-administered LHRH agonists with 100 mg G). Oral G was given daily on Days (D)1-28 of each 28-day cycle (C); 125 mg palbo, on a 21-day on/7-day off schedule. Modulation of ER signaling and proliferation of paired pre- and on-treatment (C2D8) tumor biopsies were assessed with immunohistochemistry for ER, progesterone receptor (PgR), and the proliferation marker Ki67, as well as with gene expression analysis of a predefined set of 38 ER target genes using the Illumina TruSeq RNA Access method. Plasma samples collected pre- and on-treatment (C1D15 and/or C2D1) were assessed for circulating tumor (ct)DNA using a digital PCR BEAMing assay detecting a total of 22 mutations across ESR1, AKT1, and PIK3CA. Results Objective response rates in patients with measurable disease at baseline were 20.0% in the 30 mg G group (6/30 patients) and 47.7% in the 100 mg G + palbo ± LHRH (combination) group (21/44). Clinical benefit rates were 55.0% (22/40 patients) and 81.3% (39/48), respectively. Overall, 21 pre- and on-treatment-paired tumor biopsies were collected in the 30 mg (n=13) and combination (n=8) groups. Consistent downregulation of ER, PgR, Ki67, and ER pathway activity was observed at C2D8, regardless of clinical benefit or baseline ESR1 mutation, demonstrating consistent on-target activity of G. Stronger Ki67, ER, and PgR suppression was seen with the addition of palbo to G. Of the 36 patients across both cohorts with a detectable ctDNA baseline ESR1 mutation, 16 had >1 baseline ESR1 mutation. Of the eight unique ESR1 mutations detected, none showed an association with response. At C2D1, 34 patients had a decrease in ESR1 ctDNA levels from baseline, of which 27 became undetectable for ESR1 ctDNA. Early changes in PIK3CA and AKT1 ctDNA levels, but not ESR1, trended with response. Conclusions Encouraging clinical activity was observed with single agent G at 30 mg and with G at 100 mg in combination with palbo. Biomarker analyses demonstrated consistent biologic activity of G, and largely consistent decreases of ESR1 ctDNA levels. Further updated activity and biomarker data, including PAM50 subtype classifications, will be presented. Citation Format: Nicolas C Turner, Sherene Loi, Heather M Moore, Ching-Wei Chang, Jennifer Eng-Wong, Aditya Bardia, Valentina Boni, Joohyuk Sohn, Komal L Jhaveri, Elgene Lim. Activity and biomarker analyses from a phase Ia/b study of giredestrant (GDC-9545; G) with or without palbociclib (palbo) in patients with estrogen receptor-positive, HER2-negative locally advanced/metastatic breast cancer (ER+/HER2- LA/mBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD13-07.

Abstract 740: A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology

Abstract Introduction: OncoSignal test was developed to determine and quantify functional signal transduction pathway activity levels of 4 key oncogenic signaling pathways (ER, AR, PI3K and MAPK), based on the quantitative measurement of mRNA expression levels of the direct target genes. Here we present data of a 4 pathway qPCR test, that can be used in external labs and is able to measure simultaneously the 4 pathway activities in tumor tissue samples possibly providing important information for optimal therapy selection for treatment of breast cancer. Materials and methods: OncoSignal 4 pathway test was compared between 2 reference laboratories; the Philips service lab in The Netherlands and at Protean BioDiagnostics, a US CAP-certified CLIA service lab. mRNA was isolated from unstained FFPE tissue sections of well characterized breast tissues obtained from 65 patients (retrospective). Annotated areas with at least 50% tumor cells were used (total volume about 0.25mm3). The OncoSignal test was used to measure the signal transduction pathway activity of AR, ER, PI3K and MAPK pathways. IHC staining for ER, PR, Ki67 and HER2 was also available for all samples enabling tumor categorization into conventional breast cancer subtypes. Results: Excellent inter-lab concordance was found with correlation coefficients of 0.99, 0.99, 0.98 and 0.99 for ER, AR, PI3K and MAPK, respectively. Average absolute difference between pathway activities was 3.6 activity points on a 0-100 activity score scale. All 32 cases passed the criteria set for reproducibility. Of the 65 tested breast cancer samples, 58 could be subtyped based on IHC. Five of the 58 samples failed QC. Of interest, ER IHC positive luminal A (41%) and B (28%) breast cancer showed high variation in ER pathway activity between cases, suggesting that the ER signaling pathway is not the tumor driving pathway in a significant subset of IHC ER positive tumors. As expected the Her2-enriched (17%) and TNBC (17%) sub-groups predominantly had a very low ER pathway activity score. AR pathway activity was most prominent in the HER2 enriched tumor groups, while in TNBC the MAPK pathway was most active. Conclusion: The OncoSignal 4 pathway test is robust and can be implemented in external (local) labs as shown by the excellent correlation results. The results of the signal pathway activity analysis correlate well with breast subtype classification, however, the assay has the potential to uncover specific pathway activation independent of subtype which may have significant value for precision oncology and targeted therapy selection. The test has potential for wide utilization as it can be performed on conventional FFPE prepared breast specimens, and may be complementary to conventional mutational and IHC analysis for classifying breast cancer and selecting appropriate therapy. Citation Format: Eveline den Biezen, Saskia Vermeer, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Hannah Park, Anthony Magliocco. A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 740.

Abstract 4261: Development of quantitative multi-platform tests for easy readout of AR, ER, PI3K and MAPK pathway activity to unravel pathophysiology across cancer and tissue types

Abstract Introduction: Currently, the selection of targeted therapy is commonly based on genetic data, such as well-known gene mutations, but these mutations do not provide conclusive evidence for the functional activation of the affected pathway. For this reason, we have developed computational models to assess the molecular phenotype of individual tumors, determining the functional activity of oncogenic signaling pathways based on mRNA expression levels of the direct target genes of the respective pathway transcription factors (Verhaegh et al., Cancer Res 2014; Van Ooijen et al., Am J Pathol 2018; Van de Stolpe et al., Sci Rep 2019). For a broader application of these models, we translated them to RT-qPCR, RNA sequencing and microarray tests, for a quantitative and reproducible assessment of signaling pathway activity. Materials and methods: Focusing on the AR, ER, PI3K-FOXO and MAPK-AP1 pathways, we developed PCR primers and probes to assess mRNA expression levels of the pathways' direct target genes. For model parameter calibration, we performed controlled cell line experiments in which the respective pathways were inactivated and activated, to have calibration samples with known pathway activity status. Per pathway, the same set of calibration samples and the same set of target genes were used to develop RT-qPCR, RNA sequencing and microarray tests, to maximize similarity. The resulting OncoSignal tests can be used for a quantitative assessment of pathway activity on individual samples, giving a score on a 0-100 scale. We verified these tests on independent samples with known pathway status. We ran repeat experiments to assess the reproducibility within each platform, and tested the same samples on all three platforms to assess cross-platform concordance. Results: Using the OncoSignal score, low activity test samples were clearly separated from highly active ones. For example, AR-PCR scores went from 19±3 on androgen-deprived LNCaP cell lines to 70±2 after stimulation with DHT, and went down again to 33±1 and 23±1 when subsequently treated with bicalutamide and enzalutamide, respectively. PI3K-FOXO-microarray scores went from 12±1 on control MCF7 experiments to 50±4 after alpelisib treatment. Measurements on 100+ samples on all three platforms showed Pearson correlations between 0.93 and 0.99 in OncoSignal scores, with absolute differences typically below 10. Conclusion: We developed broadly applicable tests to assess AR, ER, PI3K-FOXO and MAPK-AP1 pathway activity in a quantitative manner on individual samples, with good reproducibility on repeat experiments and good concordance between PCR, RNA sequencing and microarray measurements. These tests can be used to unravel disease pathophysiology in (pre-)clinical samples. The added value of OncoSignal for therapy response prediction is currently being evaluated in different studies. Citation Format: Martijn Akse, Henk van Ooijen, Anke Pierik, Sandra van den Bosch, Hans van Zon, Laurent Holtzer, Anne van Brussel, Márcia Inda, Yvonne Wesseling-Rozendaal, Wim Verhaegh, Eveline den Biezen-Timmermans. Development of quantitative multi-platform tests for easy readout of AR, ER, PI3K and MAPK pathway activity to unravel pathophysiology across cancer and tissue types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4261.

Oestrogen receptor pathway activity is associated with outcome in endometrial cancer

BackgroundOestrogen receptor (ER) expression is a prognostic biomarker in endometrial cancer (EC). However, expression does not provide information about the functional activity of the ER pathway. We evaluated a model to quantify ER pathway activity in EC, and determined the prognostic relevance of ER pathway activity.MethodsER pathway activity was measured in two publicly available datasets with endometrial and EC tissue, and one clinical cohort with 107 samples from proliferative and hyperplastic endometrium and endometrioid-type EC (EEC) and uterine serous cancer (USC). ER pathway activity scores were inferred from ER target gene mRNA levels from Affymetrix microarray data (public datasets), or measured by qPCR on formalin-fixed paraffin-embedded samples (clinical cohort) and related to ER expression and outcome.ResultsER pathway activity scores differed significantly throughout the menstrual cycle supporting the validity of the pathway test. The highest ER pathway scores were found in proliferative and hyperplastic endometrium and stage I EEC, whereas stage II–IV EEC and USCs had significantly lower levels. Low ER pathway activity was associated with recurrent disease, and added prognostic value in patients with low ER expression.ConclusionThe ER pathway test reflects activity of the ER pathway, and may improve prediction of outcome in EC patients.

ER and PI3K Pathway Activity in Primary ER Positive Breast Cancer Is Associated with Progression-Free Survival of Metastatic Patients under First-Line Tamoxifen.

Estrogen receptor positive (ER+) breast cancer patients are eligible for hormonal treatment, but only around half respond. A test with higher specificity for prediction of endocrine therapy response is needed to avoid hormonal overtreatment and to enable selection of alternative treatments. A novel testing method was reported before that enables measurement of functional signal transduction pathway activity in individual cancer tissue samples, using mRNA levels of target genes of the respective pathway-specific transcription factor. Using this method, 130 primary breast cancer samples were analyzed from non-metastatic ER+ patients, treated with surgery without adjuvant hormonal therapy, who subsequently developed metastatic disease that was treated with first-line tamoxifen. Quantitative activity levels were measured of androgen and estrogen receptor (AR and ER), PI3K-FOXO, Hedgehog (HH), NFκB, TGFβ, and Wnt pathways. Based on samples with known pathway activity, thresholds were set to distinguish low from high activity. Subsequently, pathway activity levels were correlated with the tamoxifen treatment response and progression-free survival. High ER pathway activity was measured in 41% of the primary tumors and was associated with longer time to progression (PFS) of metastases during first-line tamoxifen treatment. In contrast, high PI3K, HH, and androgen receptor pathway activity was associated with shorter PFS, and high PI3K and TGFβ pathway activity with worse treatment response. Potential clinical utility of assessment of ER pathway activity lies in predicting response to hormonal therapy, while activity of PI3K, HH, TGFβ, and AR pathways may indicate failure to respond, but also opens new avenues for alternative or complementary targeted treatments.

Abstract P5-02-14: The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance

Abstract Background: Based on preclinical research, the MAPK growth factor pathway is thought to be an oncogenic pathway driving breast cancer, potentially in association with the ER pathway. In patients it is unclear how functional activity of the MAPK pathway activity relates to activity of the ER pathway to drive cancer growth. We have reported before on the development of assays, e.g. for the ER pathway, that enable quantitative measurement of activity of signal transduction pathways based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945). We now present (1) development of the MAPK-AP1 pathway model, (2) MAPK-AP1 pathway activity and its relation to ER pathway activity in breast cancer; (3) emergence of MAPK pathway activity associated with hormonal therapy resistance. Methods: The MAPK-AP1 pathway model was developed and biologically validated on Affymetrix mRNA data from samples (different cell types) with a known MAPK pathway activity. MAPK-AP1 and ER pathway activity scores were measured on Affymetrix expression data of primary untreated breast cancer samples (GSE45827, n=141) and of fulvestrant sensitive and resistant MCF7 cell line clones (GSE74391). Results: In clinical breast cancer samples, average MAPK pathway activity was increased in all subtypes compared to normal breast tissue, with a large dynamic range indicating that in each subtype there are samples with high and with low pathway activity; ER pathway activity in Luminal A and B subtypes ranged from high to low, and was low in all other subtypes. MAPK-AP1 pathway activity did not correlate with ER pathway activity. Fulvestrant-sensitive MCF7 cells had high ER and low MAPK pathway activity scores, while resistant MCF7 clones exhibited increased MAPK pathway activity and complete loss of ER pathway activity, explaining therapy resistance. Discussion: The MAPK-AP1 pathway is frequently activated in all breast cancer subtypes, independent of ER pathway activity. We hypothesize that in case of resistance to hormonal therapy measurement of both pathway activities may be informative as to the underlying resistance mechanism, i.e. loss of ER pathway activity and/or gain of MAPK pathway activity. Future clinical studies are needed to confirm the role of these signaling pathways in hormonal resistance. Citation Format: Anja van de Stolpe, Laurent Holtzer, Yvonne Wesseling-Rozendaal, Wim Verhaegh. The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance [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 P5-02-14.

Abstract P5-02-08: Androgen receptor pathway activity and the ratio between androgen and estrogen receptor pathway activity in breast cancer subtypes

Abstract Background: Androgen receptor (AR) immunohistochemistry staining in breast cancer has revealed frequent AR expression in all breast cancer subtypes. Activity of the AR signaling pathway can be either tumor suppressive or tumor promoting, depending on tumor context (Pharmacol Ther., In press, https://doi.org/10.1016/j.pharmthera.2019.05.005). For this reason, in an individual patient, AR phenotypic activity needs to be defined in order to choose the proper AR targeted therapy. The AR/ER protein ratio has been suggested as a biomarker to define this AR function and enable the appropriate therapy choice, i.e. androgen therapy in case of a low AR/ER protein expression ratio, and anti-androgen therapy in case of a high ratio. However, AR and ER expression are not necessarily associated with an active signaling pathway. Therefore we investigated the ratio between functional AR and ER pathway activities as this may have an advantage in guiding therapy choice. Methods: AR and ER pathway activity and AR/ER pathway activity ratio were determined on public Affymetrix HG-U133 Plus 2.0 gene expression microarray data of clinical breast cancer studies (available in the GEO database: GSE12276, GSE21653, GSE20685, GSE42568, GSE6532, GSE9195, GSE45827, GSE7307, GSE10780, GSE17907, GSE21422, GSE5764, GSE31192, GSE54002 and Array Express: E-MTAB-365; total n=2090). For this, we used previously described tests that enable quantification of functional pathway activity, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER and AR transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945; Sci Rep., 2019, vol. 9, no. 1, p. 1603). Intrinsic subtyping of all samples has been done using the same methodology as described elsewhere (Am J Pathol, 2018, vol. 188, no. 9, pp 1956-1972).Results: AR pathway activity was increased in all breast cancer subtypes compared to normal breast tissue (p<0.0001); highest AR activity scores were observed in the HER2-enriched subtype. As shown before, ER activity scores were highest in luminal A and B subtypes. The AR/ER pathway activity ratio was low in luminal A and B, and comparable with normal tissue, but high in HER2 and basal breast cancer subtypes (p<0.0001). Discussion: Results are compatible with currently available evidence and show that the AR pathway can be activated in all breast cancer subtypes. We hypothesize that its function depends on the level of co-existent ER pathway activity, i.e. a tumor suppressive function in ER pathway active breast cancer, and a tumor promoting function in ER pathway inactive breast cancer. Upon future clinical conformation, the AR/ER pathway activity ratio as determined on an individual patient tissue sample may be an improved biomarker to guide the choice of (complementary) AR pathway targeted therapy in breast cancer, i.e. androgen receptor pathway inhibitors in case of a high ratio and potential use of androgen receptor modulators in case of a low ratio. Citation Format: Anja van de Stolpe, Yvonne Wesseling-Rozendaal, Marcia Alves de Inda, Henk van Ooijen, Wim Verhaegh. Androgen receptor pathway activity and the ratio between androgen and estrogen receptor pathway activity in breast cancer subtypes [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 P5-02-08.

Abstract P1-18-07: Can some ER+/HER2+ patients be safely spared from treatment with chemotherapy plus herceptin?

Abstract 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.

Are contralateral parenchymal enhancement on dynamic contrast-enhanced MRI and genomic ER-pathway activity in ER-positive/HER2-negative breast cancer related?

PurposeTo retrospectively explore the relation between parenchymal enhancement of the healthy contralateral breast on dynamic contrast-enhanced magnetic resonance imaging (MRI) and genomic tests for estrogen receptor (ER)-pathway activity in patients with ER-positive/HER2-negative cancer. MethodsA subset of 227 consecutively included patients with unilateral invasive ER-positive/HER2-negative breast cancer underwent dynamic contrast-enhanced MRI prior to breast-conserving therapy between 2000 and 2008. Perfusion of the parenchyma in the healthy breast was assessed using a previously reported measure of contralateral parenchymal enhancement (CPE), consisting of the mean of the top-10% late enhancement. ER-pathway activity was assessed from the surgical resection specimen by the previously reported sensitivity to endocrine therapy (SET)-index and ER-factor. The SET-index is a genetic test to estimate survival benefit from endocrine therapy, consisting of genes related to the ESR1 gene. The ER-factor examines other factors as well including protein expression. The relation between CPE and ER-pathway activity was modeled using linear regression. ResultsPatients had a median age of 59 years. CPE was not significantly associated with the SET-index (R-squared = 0.005) nor the ER-factor (R-squared = 0.0002). The only variable significantly different between low and high CPE was age at diagnosis (P < 0.001). ConclusionsContralateral parenchymal enhancement on dynamic contrast-enhanced MRI was not associated with tumor-derived estrogen receptor pathway activity.

284P - Changes in ER pathway activity score during neoadjuvant letrozole to assess therapy response and predict disease free survival (DFS) in ER positive breast cancer patients

BackgroundDespite ER positive IHC staining, some patients do not respond to neoadjuvant endocrine therapy, suggesting that ER staining lacks specificity to predict response. We developed a method to infer a quantitative signal transduction pathway activity score (PAS) from mRNA levels (microarray, qPCR) of pathway-associated transcription factor target genes. Initial studies suggest that ER PAS may have higher specificity than ER IHC in predicting endocrine therapy response. In this study, we correlated pre-treatment ER PAS and changes in ER PAS during neoadjuvant letrozole treatment to therapy response and DFS. MethodsWe collected fresh frozen RNA from tumor samples of 30 ER IHC positive post-menopausal patients with primary localized breast cancer, treated with neoadjuvant letrozole at Edinburgh Western General. In total, 30 pre, 25 mid (median 27 days), and 29 post-treatment (median 136 days) samples were analysed. Clinical outcome was assessed (RECIST, n=29) at circa 3 months treatment by 3D ultrasound, with 1 complete (CR), 21 partial responses (PR), 2 stable (SD), and 5 progressive diseases (PD). Using RT-qPCR, target gene expression was measured for ER, androgen receptor, PI3K, Hedgehog, TGFβ and Wnt pathways. PAS were expressed on a normalized scale (0 to 100). ResultsPre-treatment ER PAS was significantly higher in responders (CR/PR) than non-responders (SD/PD), PAS=45 vs 24, respectively, T-test p=0.01. Pre-treatment ER PAS correlated with decrease in ER PAS during treatment (cor=0.87 and 0.7, mid and-post treatment, respectively). At mid-treatment, ER PAS of responders had decreased to PAS of non-responders (20 vs 19, respectively), remaining low during further treatment. Decrease in ER PAS was significantly higher in responders (-30) than non-responders (-6), p=0.01. Higher ER PAS after treatment correlated to shorter DFS (COX proportional hazards p=0.02). Baseline PAS of other pathways did not correlate with response, but changed significantly during treatment. ConclusionsThis study confirms that ER PAS in ER-positive patients, measured before and after neoadjuvant endocrine therapy, has potential to predict and assess therapy response, and predict DFS. Legal entity responsible for the studyPhilips Electronics Nederland B.V., acting through its HealthWorks Molecular Pathway Dx. FundingHas not received any funding. DisclosureM. A. Inda: Full / Part-time employment: Philips Reseach. A. van de Stolpe: Full / Part-time employment, has Philips stocks: Philips Research. D. Keizer: Full / Part-time employment: Philips. D. Clout: Full / Part-time employment: Philips Reasearch. H. van Zon: Full / Part-time employment: Philips Reasearch. M. Akse: Full / Part-time employment: Philips. All other authors have declared no conflicts of interest.

50P - ER pathway activity score as a predictive biomarker to improve stratification for neoadjuvant endocrine therapy

50P - ER pathway activity score as a predictive biomarker to improve stratification for neoadjuvant endocrine therapy