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Background:Increased body mass index (BMI) and metabolic syndrome are associated with an increased risk of breast cancer recurrence. The mechanism(s) responsible for this increased risk are not known, though multiple mechanisms including resistance to endocrine therapy have been proposed. We sought to determine whether obesity and/or metabolic syndrome were associated with intrinsic tumor risk as determined by the Oncotype recurrence score (RS), or with resistance to neoadjuvant endocrine therapy, as determined by Ki67 response after short-term presurgical treatment. Methods:To investigate whether BMI was associated with increased intrinsic recurrence risk, we analyzed a cohort of 779 patients treated at Vanderbilt for early stage breast cancer and who had Oncotype test results. Medical records were reviewed to record BMI at the time of breast cancer diagnosis. We compared the Oncotype RS between patients with BMI greater or less than 27, and in normal, overweight, and obese ranges. To investigate whether the increase in breast cancer recurrence risk associated with obesity could be identified by a surrogate marker of short-term response to endocrine therapy, we analyzed data from a presurgical trial of endocrine therapy in early stage breast cancer patients previously completed at Vanderbilt. In this study patients were treated with 1-2 weeks of letrozole prior to surgery, then the surgical specimen was analyzed for Ki67. The original goal of the study was to compare endocrine sensitive tumors with endocrine resistant tumors as a platform for discovery of resistance mechanisms to endocrine therapy. We retrospectively analyzed medical records for 143 patients in that study who had post-treatment Ki67 values available, and for whom BMI at the time of surgery could be obtained. We also reviewed medical records for components of the metabolic syndrome (hypertension, hyperglycemia or anti-hyperglycemic medications, low HDL, high triglycerides or statin use, and BMI > 30 as a surrogate for waist circumference). Metabolic syndrome was defined as 3 or more of the 5 components. For endocrine therapy response, a cutoff of 1 for the natural log Ki67 expression was defined as sensitive (approximately 2.5%) and tumors with natural log of Ki67 expression between 1 and 2 (approximately 7.5%) were categorized as intermediate sensitivity. Results:We did not find any significant association between BMI and recurrence score and the distribution of risk scores was similar across all BMI groups. We did not observe any correlation between RS and BMI. However we did observe a difference in the proportion of endocrine sensitive and resistant tumors in overweight and obese groups (only a minority of patients in this cohort, 18%, had normal weight). In patients with overweight, 70% of the tumors had a post-treatment Ki67 level classified as sensitive, whereas only 40% of the tumors in patients with obesity were endocrine sensitive (chi square = 0.0518). Similarly, 72% of patients without metabolic syndrome had sensitive tumors, but only 51% of patients with metabolic syndrome had sensitive tumors (relative risk 1.408, chi square = 0.0154). Conclusions:Our findings suggest that the association between obesity and/or metabolic syndrome and increased recurrence risk is not reflected in tumor-intrinsic properties present at the time of diagnosis. Rather, the effect of obesity and metabolic syndrome appears to be in response to treatment, at least as measured by a short-term surrogate marker of long-term endocrine sensitivity. Importantly, this suggests that interventions to reverse obesity and/or metabolic syndrome may be effective in improving outcomes for breast cancer recurrence, at least in part by improving sensitivity to endocrine therapy. Citation Format: Riley Bergman, Yvonne Berko, Brent Rexer. Association of factors of the obesity phenotype with breast cancer recurrence risk measured by oncotype recurrence score and short-term response to neoadjuvant endocrine therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-18.

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy [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 P3-08-15.

BackgroundCyclin-dependent kinases (CDK) 4 and 6 regulate G1 to S cell cycle progression and are often altered in cancers. Abemaciclib is a selective inhibitor of CDK4 and CDK6 approved for administration on a continuous dosing schedule as monotherapy or as combination therapy with an aromatase inhibitor or fulvestrant in patients with advanced or metastatic breast cancer. This Phase 1b study evaluated the safety and tolerability, pharmacokinetics, and antitumor activity of abemaciclib in combination with endocrine therapy for metastatic breast cancer (MBC), including aromatase inhibitors (letrozole, anastrozole, or exemestane) or tamoxifen.Patients and MethodsWomen ≥18 years old with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) MBC were eligible for enrollment. Eligibility included measurable disease or non-measurable but evaluable bone disease by Response Evaluation Criteria in Solid Tumours (RECIST) v1.1, Eastern Cooperative Oncology Group performance status 0–1, and no prior chemotherapy for metastatic disease. Adverse events were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 and tumor response were assessed by RECIST v1.1.ResultsSixty-seven patients were enrolled and received abemaciclib 200 mg every 12 hours in combination with letrozole (Part A, n=20), anastrozole (Part B, n=16), tamoxifen (Part C, n=16), or exemestane (Part D, n=15). The most common treatment-emergent adverse events (TEAE) were diarrhea, fatigue, nausea, and abdominal pain. Grade 4 TEAEs were reported in five patients (one each with hyperglycemia, hypertension, neutropenia, procedural hemorrhage, and sepsis). There was no effect of abemaciclib or endocrine therapy on the pharmacokinetics of any combination study drug. Across all treated patients, the median progression-free survival was 25.4 months (95% confidence interval: 18.0, 35.8). The objective response rate was 38.9% in 36 patients with measurable disease.ConclusionsAbemaciclib in combination with multiple endocrine therapy options exhibited manageable safety and promising antitumor activity in patients with HR+, HER2- MBC.Clinical Trial Registrationhttps://clinicaltrials.gov/, identifier NCT02057133

Background: Somatic alterations in the FGFR pathway (mainly FGFR1, amplified in about 15% of ER+ BC) have been implicated in resistance to endocrine therapy (ET), and more recently, to CDK4/6i as well. Based on our preclinical data showing that the FGFR pan-inhibitor erdafitinib when added to fulvestrant/palbociclib resulted in marked PDX regressions, we initiated a phase Ib trial combining erdafitinib with fulvestrant/palbociclib in patients with ER+/HER2-/FGFR-amplified MBC (NCT03238196) to determine safety, tolerability and anti-tumor activity of this combination. Methods: Patients with evaluable ER+/HER2- MBC with FGFR1-4 amplification (detected on tumor next generation sequencing or plasma ctDNA) exposed to at least one ET regimen (but no more than 2 lines of chemotherapy) in the metastatic setting, were treated with fulvestrant, palbociclib (standard of care dosing/ schedule) and oral erdafitinib, tested in 4 doses ranging from 4 mg to 8 mg daily. Once MTD reached, we planned to enroll 20 patients in the expansion portion of the trial. Tumor blocks were collected for FGFR1 FISH amplification analysis, and plasma ctDNA was collected at baseline, 4 weeks and at treatment discontinuation. Tumor assessments were performed every 8 weeks. Results: Since August 2017, 26 eligible patients with evaluable ER+/HER2-/FGFR-amplified MBC were enrolled across 4 institutions. Patient characteristics are summarized in Table 1. Main grade 1 and 2 adverse events (AE) were consistent with on-target toxicities of erdafitinib and/or palbociclib: mucositis (67%), hyperphosphatemia (61%), dysgeusia (52%), diarrhea (48%), fatigue (48%), neutropenia (47%), hand-foot syndrome (38%), anemia (29%), and onycholysis (14%). Febrile neutropenia occurred in 5% patients, no cases of central serous retinopathy were seen. Serious AE were rare: one grade 4 elevation of transaminases (DLT; attributed to fulvestrant), one grade 3 colitis (attributed to erdafitinib), and one thromboembolic event (attributed to palbociclib). In combination with fulvestrant/ palbociclib, the MTD of erdafitinib was 6 mg. No drug-drug interaction was seen. 8 patients were deemed non-evaluable for anti-tumor effect as treatment discontinuation (mainly due to AE) occurred prior to first tumor assessment. Of the 18 evaluable patients: 7 had disease progression, 8 had stable disease (4 of which discontinued treatment due to AE), 3 have not completed their first tumor assessment, 4 are still on treatment; median PFS was 3 months and CBR at 6 months was 28%. However, higher PFS (6 months) was seen in 6/8 patients with high levels of FGFR1 amplification (FISH FGFR1:CEP8 ratio >5; gene copy number >10) and in both patients with FGFR3 amplification. Conclusion: To our knowledge, this is the first time an FGFR inhibitor has been tested in combination with ET and CDK4/6i in patients with MBC harboring FGFR alterations. Erdafitinib-related side effects appeared to be on target, leading to treatment discontinuation in several patients despite optimal medical treatment. Clinical activity was seen in heavily pre-treated patients with molecular evidence of high FGFR amplification despite 100% prior exposure to ET and CDK4/6i. Full clinical and correlative work will be presented at the meeting, and a future phase II trial is being planned. Table 126 / 35 patients accrued13 in escalation, 13 in expansion(ongoing)Median age53 (35 - 75)Race/ ethnicityWhite 22Black 1Asian 2Hispanic 1Median number of lines of treatment in the metastatic setting4 (1 - 5)Prior lines of treatment in the metastatic settingEndocrine therapy 100%Fulvestrant 28%CDK4/6i 100%PI3K pathway inhibitor 80%1 line chemo 65%2 lines chemo 45%FGFR1 amplification23FGFR3 amplification2FGFR4 amplification1 Citation Format: Ingrid A. Mayer, Barbara B. Haley, Vandana G. Abramson, Adam Brufsky, Brent Rexer, Erica Stringer-Reasor, Komal L. Jhaveri, Melinda Sanders, Paula I. Ericsson-Gonzalez, Fei Ye, Carlos L. Arteaga. A phase Ib trial of fulvestrant + CDK4/6 inhibitor (CDK4/6i) palbociclib + pan-FGFR tyrosine kinase inhibitor (TKI) erdafitinib in FGFR-amplified/ ER+/ HER2-negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-03.

Background Identification of HER2-positive breast cancers with high anti-HER2 sensitivity could help de-escalate chemotherapy. Here, we tested a clinically applicable RNA-based assay that combines ERBB2 and the HER2-enriched (HER2-E) intrinsic subtype in HER2-positive disease treated with dual HER2-blockade without chemotherapy. Methods A research-based PAM50 assay was applied in 422 HER2-positive tumors from five II–III clinical trials (SOLTI-PAMELA, TBCRC023, TBCRC006, PER-ELISA, EGF104090). In SOLTI-PAMELA, TBCRC023, TBCRC006, and PER-ELISA, all patients had early disease and were treated with neoadjuvant lapatinib or pertuzumab plus trastuzumab for 12–24 weeks. Primary outcome was pathological complete response (pCR). In EGF104900, 296 women with advanced disease were randomized to receive either lapatinib alone or lapatinib plus trastuzumab. Progression-free survival (PFS), overall response rate (ORR), and overall survival (OS) were evaluated. Results A total of 305 patients with early and 117 patients with advanced HER2-positive disease were analyzed. In early disease, HER2-E represented 83.8% and 44.7% of ERBB2-high and ERBB2-low tumors, respectively. Following lapatinib and trastuzumab, the HER2-E and ERBB2 (HER2-E/ERBB2)-high group showed a higher pCR rate compared to the rest (44.5%, 95% confidence interval [CI] = 35.4% to 53.9% vs 11.6%, 95% CI = 6.9% to 18.0%; adjusted odds ratio [OR] = 6.05, 95% CI = 3.10 to 11.80, P Conclusions Combining HER2-E subtype and ERBB2 mRNA into a single assay identifies tumors with high responsiveness to HER2-targeted therapy. This biomarker could help de-escalate chemotherapy in approximately 40% of patients with HER2-positive breast cancer.

Background In a randomized trial (CREATE-X) patients who had residual disease following standard neoadjuvant chemotherapy had improved survival with the addition of adjuvant capecitabine. For patients who required radiation (RT), capecitabine was given sequentially. Concurrent capecitabine-RT may be more efficacious. We hypothesized that the safety, feasibility, and toxicity of adjuvant capecitabine-RT is not significantly different compared to adjuvant RT alone. Patient and Methods We retrospectively studied patients with stage I-III invasive mammary carcinoma. Patients treated with capecitabine-RT were matched 1:3 with control patients treated with RT alone. Logistic regression evaluated predictors of radiation dermatitis. Results A total of 64 patients were enrolled including 16 patients treated by capecitabine-RT and 48 treated by RT alone. Cohorts were balanced with regard to clinicopathologic factors. No treatment in either cohort resulted in hospitalization, short-term disability, or fatality. Most toxicities of capecitabine-RT were related to radiation dermatitis. Radiation dermatitis was not significantly different between the capecitabine-RT and RT cohort at either the grade 2 level (OR 1.36, 95% CI 0.38-4.93, p=0.63) or grade 3 level (OR 3.00, 95% CI 0.85-10.63, p=0.09) or after multivariable analysis. However, patients treated with capecitabine-RT were more likely to require modifications in the radiation schedule, including treatment breaks or cancelled fractions (44% versus 17%, OR 3.89, 95% CI 1.12-13.52, p=0.03). Conclusion Capecitabine-RT appears to be safe in the adjuvant treatment of breast cancer with comparable toxicity to RT alone. It may lead to more treatment adjustments. Prospective studies are needed to evaluate the safety and tolerability of this combination.

Background: HER2-positive breast cancer consists of 4 intrinsic molecular subtypes (Luminal A, Luminal B, HER2-enriched [HER2-E], and Basal-like) and a Normal-like subtype, with the HER2-E subtype having the highest activation of the EGFR-HER2 pathway. Concordant with this, the HER2-E subtype was significantly associated with pathological complete response (pCR) following lapatinib and trastuzumab without chemotherapy on the PAMELA phase II neoadjuvant trial (Lancet Oncol 2017). Here, we aimed to further validate this observation in a different cohort using tumor samples from the TBCRC023 trial. Methods: TBCRC023 (NCT00999804) was a randomized phase II trial combining a Simon Phase 2 design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Ninety-seven women with HER2+ breast cancer measuring 2 cm or larger (median = 5 cm) were randomized in a 1:2 ratio to 12 vs. 24 weeks of lapatinib and trastuzumab. Letrozole (along with ovarian suppression if premenopausal) was added in patients whose tumors were also estrogen receptor (ER)-positive. All evaluable patients were assessed for pCR, defined as no residual invasive carcinoma in the breast. Intrinsic molecular subtypes from tumor biopsy formalin-fixed, paraffin-embedded samples taken at baseline (day 0) were determined with the nCounter-based PAM50 predictor. Gene expression PAM50 data was performed at Hospital Clínic in Barcelona blinded from clinical data. The primary outcome was the association of the HER2-E subtype (vs. non-HER2-E) with pCR. A logistic regression model adjusted for tumor size, ER status, nodal status and treatment arm was performed. Results: A total of 85 of the 97 (87.6%) baseline tumor samples were available. Most patients had the HER2-E subtype (51 [60.0%]), followed by Normal-like (12 [14.1%]), Basal-like (11 [13.0%]), Luminal B (7 [8.2%]) and Luminal A (4 [4.7%]). The proportion of patients with HER2-E tumors within ER+ and ER-negative disease was 54.9% and 67.7%, respectively. At the time of surgery, 17 of 85 patients (20.0%; 95% confidence interval [CI] 0.13-0.30) had a pCR in the breast. Fourteen of 51 patients with the HER2-E subtype (27.5%; 95% CI 0.17 to 0.41) and 3 of 34 patients with non-HER2-E subtypes (8.8%; 95% CI 0.03 to 0.23) achieved a pCR at the time of surgery (adjusted odds ratio 4.33; 95% CI 1.08-17.41; P=0.039). No other clinical-pathological variable was found significantly associated with pCR in the multivariable model. Conclusions: In an independent validation study performed while blinded to clinical outcomes, HER2-E subtype confirms its ability to identify patients with HER2-positive breast cancer who are likely to benefit from dual HER2 blockade therapies without chemotherapy. Further studies should be performed to prospectively validate this biomarker, alone or in combination with other biomarkers. Citation Format: Prat A, De Angelis C, Pascual T, Gutierrez C, Wang T, Paré L, Rexer B, Cortes J, Forero A, Llombart A, Wolff AC, Krop I, Galván P, Pavlick AC, Villagrasa P, Hilsenbeck SG, Schiff R, Osborne CK, Rimawi MF. Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-12.

Background: FGFR1 amplification (amp) occurs in ˜15% of breast cancers (BC) and associates with poor prognosis and resistance to endocrine therapy. CCND1 regulates cell cycle progression and is amplified in 15-20% of BC. Co-amp of FGFR1 occurs in 30-40% of CCND1amp tumors, suggesting the possibility of oncogene cooperativity. CDK4/6 inhibitors, which block the action of cyclin D1/CDK4 complexes at the G1-to-S transition, are approved for treatment of ER+ BC and FGFR inhibitors are in early phase clinical trials. Results: Between 11/2013 and 03/2017, 191 BCs from 188 patients with metastatic (M) or refractory locoregional recurrent (RLRR) BC at Vanderbilt (VICC) were profiled by targeted next gen sequencing (Foundation OneTM). These are included within the 2131 publicly available BC sequencing results in GENIE (Foundation OneTM and MSK-IMPACT). Among the GENIE cohort, rates were: FGFR1amp 7% (n=156), CCND1amp 12% (n=261) and CCND1/FGFR1co-amp 3% (n=58). Additional cases showed FGFR1 missense mutations (n=16) and deep deletions (n=5). When the analysis was limited to the VICC cohort allowing restriction to ER+ BC, FGFR1amp (16%) and CCND1amp (23%) rates are similar to rates in primary BC in TCGA (13% FGFR1 [p = 0.44] and 19% CCND1 [p = 0.24]). In GENIE, the most frequent co-mutations in FGFR1amp tumors were TP53 (31%), PIK3CA (21%), GATA3 (13%), CDH1 (11%) and MAP3K1 (10%). However, TP53 and PIK3CA mutations were less common among FGFR1amp tumors than FGFR1non-amp cases (p 0.016). Histopathologic correlation on tumors from our institution show a majority of FGFR1 and/or CCND1 amp BC (64%) were ER+/HER2–; 33% of ER+/FGFR1amp tumors were PR–. Distinctive histologic features associated with FGFR1 and/or CCND1 amp were lobular histology (17%) and neuroendocrine differentiation (14%), 0-10%TILs (94%) and high proliferative rate (46%). Conclusion: FGFR1amp and CCND1amp rates in TCGA are similar to those seen in MBC/LRRBC (GENIE) suggesting FGFR1 can function as both a driver mutation and de novo mechanism of endocrine resistance early in tumorigenesis. Frequent co-amp with CCND1 and lower rates of TP53 and PIK3CAmut also support a driver role for FGFR1amp and FGFR1/CCND1co-amp. The observation of neuroendocrine features in a subset of these tumors suggests lineage plasticity. This may be a consequence of genomic alterations promoting anti-estrogen resistance and is consistent with recently published BC outcome data associating neuroendocrine differentiation with higher grade ER+ tumors, frequent 8p amp, which includes FGFR1, and worse disease-free and overall survival. The frequency of FGFR1amp suggests genotype specific trials with FGFR inhibitors would be highly feasible. Whether FGFR1/CCND1 co-amplified tumors are candidates for treatment with a combination of FGFR and CDK4/6 inhibitors requires further investigation. Citation Format: Gonzalez-Ericsson PI, Estrada MV, Formisano L, Jansen VM, Mayer IA, Rexer BN, Abramson VG, Levy M, Balko JM, Stricker TP, Arteaga CL, Sanders ME. Genenomic landscape of breast cancers with FGFR1 amplification and FGFR1/CCND1 co-amplification revealed by targeted capture next generation sequencing [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 PD4-07.

Purpose: Prior neoadjuvant trials with 12 weeks of dual anti-HER2 therapy without chemotherapy demonstrated a meaningful pathologic complete response (pCR) in patients with HER2-positive breast cancer. In this trial, we sought to determine whether longer treatment would increase the rate of pCR. Patients and Methods: TBCRC023 (NCT00999804) is a randomized phase II trial combining a Simon phase II design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Women with HER2-positive breast tumors measuring ≥2 cm (median = 5 cm) were randomized in a 1:2 ratio to 12 versus 24 weeks of lapatinib and trastuzumab. Letrozole (along with ovarian suppression if premenopausal) was administered in patients whose tumors were also estrogen receptor (ER) positive. All evaluable patients were assessed for in-breast pCR. Results: Ninety-seven patients were enrolled (33 in 12-week arm and 64 in 24-week arm), of whom 94 were evaluable. Median age was 51 years, and 55% were postmenopausal. Median tumor size was 5 cm, and 65% were ER-positive. The rate of pCR in the 24-week arm was 28% and numerically superior to the 12-week arm (12%). This was driven by increased pCR in the ER-positive subgroup (33% vs. 9%). Study treatment was well tolerated, with grade 1–2 diarrhea and acneiform rash being the most common toxicities. Conclusions: Treatment with dual anti-HER2 therapy for 24 weeks led to a numeric increase in pCR rate in women with HER2-positive breast cancer, without using chemotherapy. If validated, this approach may help identify patients who may benefit from deescalation of therapy.

HER2 overexpression drives Akt signaling and cell survival and HER2-enriched breast tumors have a poor outcome when Akt is upregulated. Akt is activated by phosphorylation at T308 via PI3K and S473 via mTORC2. The importance of PI3K-activated Akt signaling is well documented in HER2-amplified breast cancer models, but the significance of mTORC2-activated Akt signaling in this setting remains uncertain. We report here that the mTORC2 obligate cofactor Rictor is enriched in HER2-amplified samples, correlating with increased phosphorylation at S473 on Akt. In invasive breast cancer specimens, Rictor expression was upregulated significantly compared with nonmalignant tissues. In a HER2/Neu mouse model of breast cancer, genetic ablation of Rictor decreased cell survival and phosphorylation at S473 on Akt, delaying tumor latency, penetrance, and burden. In HER2-amplified cells, exposure to an mTORC1/2 dual kinase inhibitor decreased Akt-dependent cell survival, including in cells resistant to lapatinib, where cytotoxicity could be restored. We replicated these findings by silencing Rictor in breast cancer cell lines, but not silencing the mTORC1 cofactor Raptor (RPTOR). Taken together, our findings establish that Rictor/mTORC2 signaling drives Akt-dependent tumor progression in HER2-amplified breast cancers, rationalizing clinical investigation of dual mTORC1/2 kinase inhibitors and developing mTORC2-specific inhibitors for use in this setting. Cancer Res; 76(16); 4752–64. ©2016 AACR.

Background: Abemaciclib, a small molecule inhibitor of CDK4 and CDK6, induces G1 cell cycle arrest in Rb-proficient human cancers.1 The clinical safety profile of abemaciclib enables continuous oral dosing to achieve sustained target inhibition, resulting in single-agent antitumor activity against multiple human cancers. The drug also reaches relevant concentrations in the central nervous system and, in patients taking the drug orally, can be detected in the cerebrospinal fluid.2 For women with previously treated hormone receptor positive (HR+) metastatic breast cancer (MBC), abemaciclib as a single agent achieved a six-month clinical benefit rate of 61.1% and an objective response rate of 33.3%.3 Clinical trials investigating abemaciclib combined with fulvestrant4 or aromatase inhibitors5 have led to randomized Phase 3 studies for women with HR+ breast cancer.6,7 Methods: This Phase 1b study (NCT02057133) with multiple cohorts evaluates safety and tolerability of abemaciclib combined with endocrine or HER2-targeted therapies for MBC. Secondary objectives include pharmacokinetics (PK) and antitumor activity of abemaciclib when given in combination with other therapies. Cohorts were opened to enrollment sequentially. Patients with HR+ HER2 negative MBC received abemaciclib orally every 12 hours (Q12H) in combination with the following standard therapies daily until progression: letrozole (Part A), anastrozole (Part B), tamoxifen (Part C), exemestane (Part D), or exemestane plus everolimus (Part E). Patients with HER2 positive MBC received abemaciclib orally Q12H in combination with trastuzumab every 21 days until progression (Part F). Adverse events (AEs) were graded by NCI CTCAE v4.0 and tumor response was assessed radiographically using RECIST v1.1. Results: Abemaciclib has been combined with multiple targeted therapies for the treatment of women with MBC. We previously reported safety and early efficacy results for the combinations of abemaciclib with letrozole, anastrozole, tamoxifen, exemestane, and exemestane plus everolimus.5 Due to limited follow-up at that time, the efficacy results were not mature. Safety, PK, and efficacy results with approximately 6 months of additional follow-up will be reported across all parts of the study. The most common treatment-emergent AEs include effects on the gastrointestinal and hematopoietic systems. Consistent with previously reported results for both single-agent abemaciclib and the combination of abemaciclib with fulvestrant, tumor responses have been observed among women receiving abemaciclib in combination with targeted therapies for MBC. Conclusions: This study for women with MBC demonstrates the potential for abemaciclib to be combined with therapies targeting specific signaling pathways. References: 1Gelbert et al. Invest New Drugs. 2014;32(5):825-37. 2Shapiro et al. J Clin Oncol 31, 2013 (suppl; abstr 2500). 3Tolaney et al. SABCS 2014: Abstract 763. 4Patnaik et al. J Clin Oncol 32:5s, 2014 (suppl; abstr 534). 5Tolaney et al. J Clin Oncol 33, 2015 (suppl; abstr 522). 6Llombart et al. SABCS 2014: OT1-1-07 (MONARCH 2, NCT02107703). 7Goetz et al. J Clin Oncol 33, 2015 (suppl; abstr TPS624) (MONARCH 3, NCT02246621). Citation Format: Goetz MP, Beeram M, Beck T, Conlin AK, Dees EC, Dickler MN, Helsten TL, Conkling PR, Edenfield WJ, Richards DA, Turner PK, Cai N, Chan EM, Pant S, Becerra CH, Kalinsky K, Puhalla SL, Rexer BN, Burris HA, Tolaney SM. Abemaciclib, an inhibitor of CDK4 and CDK6, combined with endocrine and HER2-targeted therapies for women with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-25.

1011 Background: Several trials have shown 25-30% pathologic complete response (pCR) rates in patients with HER2+ BC treated with LT therapy (+ endocrine therapy if ER+), but no chemotherapy (CTX). We hypothesize that a multiparameter classifier, comprised of HER2 gene and protein levels, intratumor heterogeneity (ITH), HER2-enriched (E) subtype, and PIK3CA mutation status can identify patients whose tumors are “addicted” to HER2 signaling and are likely to achieve pCR from a CTX-sparing de-escalation strategy. Methods: Baseline specimens from 2 trials (TBCRC023 [NCT00999804] and PAMELA [NCT01973660]) of neoadjuvant CTX-sparing LT (+ endocrine therapy if ER+) in HER2+ BC were used. HER2 protein and ITH (scored for % 3+ by IHC), and gene amplification (HER2:CEP17 ratio and copy number (CN) by CISH) were measured on the same slide by the dual gene protein assay (GPA). HER2-E and PIK3CA mutation status were assessed by research-based PAM50 and MSK-IMPACT platforms, respectively. A decision tree algorithm was used to determine the GPA cutoffs and to construct the classifier of response (by pCR) in TBCRC023, which was then validated in PAMELA. Results: Of the evaluable patients from TBCRC023 (N = 130) and PAMELA (N = 151), GPA data were available for 121 and 94 cases, respectively. Both cohorts exhibited similar distributions for HER2 ratio, CN, and % 3+, and a strong correlation between HER2 ratio and CN (R > 0.92). In TBCRC023, 73 cases had data from GPA, PAM50, and IMPACT, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio > 4.6 and % 3+ > 97.5% in both the GPA data cohort (N = 121) and complete data cohort (N = 73). With PAM50 and IMPACT data, the model added HER2-E and PIK3CA wild-type (wt). For practical reasons, the classifier was locked as HER2 ratio ≥ 4.5 AND % 3+ ≥ 90% AND PIK3CA-wt AND HER2-E, which yielded a PPV of 55% and NPV of 94%. Validation in PAMELA using 45 cases with data for all 3 assays yielded PPV of 44% and NPV of 82%. 29 TBCRC023 cases without IMPACT data could be predicted to be non-pCR, of which 26 were correct (NPV = 89%). In PAMELA, 66 additional cases could be predicted to be non-pCR, of which 54 were correct (NPV = 81%). Conclusions: We have constructed a multiparameter classifier that can predict pCR with targeted therapy alone that compare to pCR rates of CTX + dual anti-HER2 in unselected patients. Prospective validation in a clinical trial is warranted. [Table: see text]

Background: Current evidence indicates that patients with Human Epidermal Growth Factor Receptor 2 (HER2)-positive Breast Cancer (BC) benefit from HER2-targeted therapies. Accurate determination of HER2 status is critical to ensure that the correct patients are offered targeted treatment while patients with HER2-negative tumors–who are unlikely to benefit from anti-HER2 therapy– are spared from the adverse effects of these costly drugs. Guidelines for performance and reporting of HER2 testing, first published in 2007 by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP), were updated in October 2013. The new reporting criteria are based on a combination of HER2:CEP17 ratio and average HER2 copy number. Methods: We retrospectively reviewed HER2 dual probe FISH test results sequentially performed by the Cytogenetics Laboratory at Vanderbilt University from January to May 2014 since implementation of the updated guidelines. The cases were then rescored using the 2007 guidelines. The clinicopathologic features of cases with a change in Her2 status after scoring with the 2013 guidelines were examined for statistical significance. Results: If the 266 performed HER2 FISH tests had been scored using the 2007 guidelines the results would have been the following: amplified (n = 44, 16%), equivocal (n = 28, 10%) and not amplified (n = 194, 70%). However, using the 2013 guidelines the cases were actually reported as follows: amplified (n=57, 21%), equivocal (n=22, 8.3%) and not amplified (n=187, 68%). Additionally, 18 cases demonstrated genetic heterogeneity in >25% of cells. The updated guidelines resulted in a change in Her2 status in 12% of tests (32/266; p less than 0.0001); 13 changed from equivocal to amplified, 13 cases changed from not amplified to equivocal and 6 cases changed from equivocal to not amplified. Cases with a change in Her2 status following implementation of the new guidelines were more likely to demonstrate genetic heterogeneity (28% [9/32] vs. 4% [9/234]; p less than 0.0001). Furthermore, hormone receptor (HR)-negative tumors scored as not amplified by the 2007 guidelines were more likely to undergo an upgrade in HER2 status under the 2013 guidelines than HR-positive tumors (26% [11/41] vs. 7% [11/153], p less than 0.0001). Conclusions: The 2013 reporting criteria, based on a combination of HER2:CEP17 ratio and average HER2 copy number, increase the number of patients eligible for HER2-targeted therapies while decreasing equivocal results. Tumors that demonstrate genetic heterogeneity for Her2 or are HR-negative account for a significant percentage of these newly eligible cases. Correlation with clinical response is required to confirm the proposed improved analytical validity of the updated guidelines. Clinical trials to evaluate the benefit of anti-Her2 therapy in patients with genetic heterogeneity are in the planning phase. Citation Format: Monica V Estrada, Jena M Giltnane, Ferrin C Wheeler, Ashwini Yenamandra, Vandana Abramson, Ingrid A Mayer, Julie Means, Brent Rexer, Ingrid M Meszoely, Melinda E Sanders. Genetic heterogeneity for Her2 accounts for a significant percentage of breast cancers changing Her2 status following implementation of the 2013 CAP/ASCO HER2 reporting guidelines [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-32.

BACKGROUND: Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti–human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance. METHODS: HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided. RESULTS: Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm(3), SD = 924 mm(3), vs Pan-HER mean = 565 mm(3), SD = 499 mm(3), P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts. CONCLUSIONS: These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

The combination of a PI3K inhibitor with trastuzumab has been shown to be effective at overcoming trastuzumab resistance in models of HER2+ breast cancer by inhibiting HER2–PI3K–FOXO–survivin signaling. In this review the potential clinical implications of these findings are discussed. Cancer Res; 73(13); 3817–20. ©2013 AACR.

The optical redox ratio (fluorescence intensity of NADH divided by that of FAD), was acquired for a panel of breast cancer cell lines to investigate how overexpression of human epidermal growth factor receptor 2 (HER2) affects tumor cell metabolism, and how tumor metabolism may be altered in response to clinically used HER2-targeted therapies. Confocal fluorescence microscopy was used to acquire NADH and FAD auto-fluorescent images. The optical redox ratio was highest in cells overexpressing HER2 and lowest in triple negative breast cancer (TNBC) cells, which lack HER2, progesterone receptor, and estrogen receptor (ER). The redox ratio in ER-positive/HER2-negative cells was higher than what was seen in TNBC cells, but lower than that in HER2 overexpressing cells. Importantly, inhibition of HER2 using trastuzumab significantly reduced the redox ratio in HER2 overexpressing cells. Furthermore, the combinatorial inhibition of HER2 and ER decreased the redox ratio in ER+/HER2+ breast cancer cells to a greater extent than inhibition of either receptor alone. Interestingly, trastuzumab had little impact upon the redox ratio in a cell line selected for acquired resistance to trastuzumab. Taken together, these data indicate that the optical redox ratio measures changes in tumor metabolism that reflect the oncogenic effects of HER2 activity within the cell, as well as the response of the cell to therapeutic inhibition of HER2. Therefore, optical redox imaging holds the promise of measuring response and resistance to receptor-targeted breast cancer therapies in real time, which could potentially impact clinical decisions and improve patient outcome.

Phosphatiditylinositide-3-kinase (PI3K) is activated in some cancers by direct mutation, but it is activated more commonly in cancer by mutation of upstream acting receptor tyrosine kinases (TK). At present, there is no systematic method to determine which TK signaling cascades activate PI3K in certain cancers, despite the likely utility of such information to help guide selection of tyrosine kinase inhibitor (TKI) drug strategies for personalized therapy. Here, we present a quantitative liquid chromatography tandem mass spectrometry approach that identifies upstream activators of PI3K both in vitro and in vivo. Using non–small cell lung carcinoma to illustrate this approach, we show a correct identification of the mechanism of PI3K activation in several models, thereby identifying the most appropriate TKI to downregulate PI3K signaling. This approach also determined the molecular mechanisms and adaptors required for PI3K activation following inhibition of the mTOR kinase TORC1. We further validated the approach in breast cancer cells with mutational activation of PIK3CA, where tandem mass spectrometry detected and quantitatively measured the abundance of a helical domain mutant (E545K) of PIK3CA connected to PI3K activation. Overall, our findings establish a mass spectrometric approach to identify functional interactions that govern PI3K regulation in cancer cells. Using this technique to define the pathways that activate PI3K signaling in a given tumor could help inform clinical decision making by helping guide personalized therapeutic strategies for different patients. Cancer Res; 71(18); 5965–75. ©2011 AACR.

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable, whereas active phosphoinositide-3 kinase (PI3K)-Akt and mitogen-activated protein kinase (MAPK) were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFKs) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.

Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K, but not E545K PI3K, markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors.

509Background: HER2-Enriched (HER2-E) intrinsic subtype within HER2-positive breast cancer is characterized by high expression of ERBB2 and related genes. Here we retrospectively evaluated the value of the HER2-E subtype and ERBB2 mRNA expression alone to predict pathological complete response (pCR) in tumor samples from PAMELA and TBCRC 006/023 trials. Methods: All patients had HER2-positive early breast cancer and were treated with neoadjuvant lapatinib and trastuzumab. Patients with hormone receptor-positive tumors were also treated with letrozole or tamoxifen. In PAMELA (NCT01973660), 151 patients were treated for 18 weeks. TBCRC 006 (NCT00548184) treated 66 patients for 12 weeks and TBCRC 023 (NCT00999804) randomized 97 patients to 12 vs. 24 weeks of treatment. pCR was defined as no residual invasive carcinoma in the breast. Baseline intrinsic subtypes and ERBB2 mRNA expression were determined using the nCounter-based PAM50 predictor. ERBB2 expression was dichotomized as low (lowest 1/3) vs high (hig...

Background: Molecular tumor profiling is increasingly important in the management of oncologic patients. Targeted next-generation sequencing (T-NGS), using formalin fixed paraffin embedded (FFPE) clinical samples, allows for molecular characterization of genes with known or potential therapeutic and prognostic importance in cancer. Actionable alterations include those with on- or off-label therapeutic implications, those that might be biomarkers of response to clinical trial agents, or those which are non-indicators for response. Design: We correlated information on patients with metastatic or refractory locoregional recurrence of breast cancer (BC) with potentially actionable genetic alterations detected by a commercially available T-NGS assay, which sequences the coding regions of 315 genes and introns of 28 genes involved in rearrangements selected for their demonstrated role in solid malignancy. We developed an informatics pipeline to capture test results in real-time and store them for subsequent research analysis. Results were analyzed by clinical subtype (estrogen receptor positive [ER+]; HER-2 amplified [HER2+]; triple-negative [TN]) for actionable alterations, most frequently altered genes/pathways and variants of standard biomarkers not detected by routine studies. Results: Between 11/2013 and 4/2015, 141 FFPE samples from 139 patients were tested by T-NGS. At least one potentially actionable genetic alteration was identified in 98% of patients (median 5.5 alterations/tumor [range 0-18]). 64% had alterations predicting sensitivity to approved BC therapies and 10% to approved therapies in other tumor types. An additional 1231 variants of uncertain significance (VUS) (median 9 per tumor [range 0-28]) were identified. The most frequently altered genes were TP53 (64%), PIK3CA (37%) and MYC (24%). Genes involved in cell cycle, DNA damage and PIK3CA/mTOR pathways were highly altered among all receptor subtypes. The RAS/MAPK pathway was more commonly altered in ER+ (28%) vs. HER2+ (13%) and TN (17%). CCND1 amplifications were found in 16% (57% ER+, 30% HER2+, 13% TN) and FGFR1 amplifications in 13% (61% ER+, 22% HER2, 17% TN). Co-amplification of 8p and 11q (including FGFR1/ZNF703 and CCND1/FGF3/FGF4/FGF19) was found in 28% of patients (ER+ 21%, HER2+ 25%, TN 7.5%). The combination of PIK3CA mutation and MAP3K1/MAP2K4 alteration occurred in 12% of patients (82% ER+, 18% TN). 4 ESR1 mutations and 2 amplifications (all in ER+) as well as 4 HER2 mutations (1 ER+ and 3 TN) were also identified. The number of patients receiving genotype-directed treatments informed by T-NGS results and patient outcome after genotype-directed treatment will be subsequently presented. Conclusion: Mutation profiling using T-NGS identified potentially actionable alterations in a majority of advanced BC patients, providing novel yet rational therapeutic options and facilitating clinical trial enrollment. T-NGS results will be used to guide therapy in increasing numbers of BC patients. Citation Format: Estrada MV, Warner J, Rioth M, Balko JM, Rexer B, Sanders ME. Targeted next generation sequencing of advanced breast cancers identifies potentially actionable alterations and variants of standard biomarkers in the majority of patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-02.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

A small molecule inhibitor of MAP/ERK kinase (MEK) was effective against human breast cancer cells with a basal-like gene expression signature. Antitumor activity was limited by both feedback upregulation of phosphatidylinositol-3 kinase (PI3K)/AKT upon inhibition of MEK as well as loss of the phosphatase PTEN. Therefore, MEK inhibitors should preferably be investigated in combination with PI3K inhibitors in basal-like breast cancers.(Clin Cancer Res 2009;15(14) July 2009).

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K), have been shown to transform mammary epithelial cells (MEC). Studies suggest this transforming activity requires binding of mutant p110α via p85 to phosphorylated YXXM motifs in activated receptor tyrosine kinases (RTK) or adaptors. Using transgenic mice, we examined if ErbB3, a potent activator of PI3K, is required for mutant PIK3CA-mediated transformation of MECs. Conditional loss of ErbB3 in mammary epithelium resulted in a delay of PIK3CAH1047R-dependent mammary gland hyperplasia, but tumor latency, gene expression, and PI3K signaling were unaffected. In ErbB3-deficient tumors, mutant PI3K remained associated with several tyrosyl phosphoproteins, potentially explaining the dispensability of ErbB3 for tumorigenicity and PI3K activity. Similarly, inhibition of ErbB RTKs with lapatinib did not affect PI3K signaling in PIK3CAH1047R-expressing tumors. However, the p110α-specific inhibitor BYL719 in combination with lapatinib impaired mammary tumor growth and PI3K signaling more potently than BYL719 alone. Furthermore, coinhibition of p110α and ErbB3 potently suppressed proliferation and PI3K signaling in human breast cancer cells harboring PIK3CAH1047R. These data suggest that PIK3CAH1047R-driven tumor growth and PI3K signaling can occur independently of ErbB RTKs. However, simultaneous blockade of p110α and ErbB RTKs results in superior inhibition of PI3K and mammary tumor growth, suggesting a rational therapeutic combination against breast cancers harboring PIK3CA activating mutations. Cancer Res; 73(13); 4075–85. ©2013 AACR.

HER2 amplification and activating mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, often co-occur in breast cancer. We generated a transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. In these mice, PIK3CAH1047R accelerates HER2-mediated tumor formation and promotes resistance to HER2 inhibitors (Hanker et al. PNAS 2013). HER2+/PIK3CA tumor growth was inhibited by treatment with the HER2 antibodies trastuzumab and pertuzumab in combination with the pan-PI3K inhibitor BKM120 (TPB). We sought to discover mechanisms of acquired resistance to the triple therapy by long-term treatment of established HER2+/PIK3CA tumors. Tumor transplants derived from a transgenic HER2+/PIK3CA tumor were initially growth inhibited by TPB. After several weeks, a subset of transplants (3/11) resumed growth in the presence of continuous TPB therapy. TPB-resistant tumors were cross-resistant to the combination of T + P + BYL719 (a p110α-specific inhibitor). Whole exome sequencing did not identify acquired somatic alterations in TPB-resistant tumors, including in HER2. However, RNA-seq revealed significant transcriptional upregulation of extracellular matrix (ECM) genes and genes involved in cell adhesion, including collagens, tenascins, and thrombospondins. Likewise, trichrome staining revealed a significant increase in collagen fibers and IHC analysis confirmed increased Tenascin expression in the TPB-resistant tumor stroma. In addition, western blot analysis revealed increased expression of an activated form of integrin β1, a substrate for ECM ligands such as collagen, as well as P-SrcY416 (activated by integrins/focal adhesion). We also found that transcription of many of these genes is induced by short-term TPB treatment in human breast cancer cell lines by qRT-PCR. Interestingly, primary tumor cells derived from TPB-resistant tumors no longer displayed resistance when grown in vitro. These cells regained TPB resistance when re-introduced into mice. Plating primary tumor cells on growth factor-reduced Matrigel or on Collagen I-coated plates restored resistance, suggesting that the ECM directly promotes TPB resistance. We are currently investigating whether inhibition of Integrin/Src signaling reverses TPB resistance. We are also exploring whether components of the ECM are altered in residual disease specimens from HER2+ breast cancer patients treated with neoadjuvant anti-HER2 therapies. Our data suggest that upregulation of ECM/integrin/Src signaling contributes to resistance to combinations of HER2 and PI3K inhibitors, and strongly support the growing body of literature indicating that components of the tumor microenvironment promote resistance to targeted therapies. Citation Format: Ariella B. Hanker, Monica Valeria Estrada, Junfei Zhao, Feixiong Cheng, Preston D. Moore, Darren Tyson, Violeta Sanchez, Brent N. Rexer, Melinda Sanders, Zhongming Zhao, Thomas P. Stricker, Carlos L. Arteaga. ECM/Integrin signaling promotes resistance to the combination of HER2 and PI3K inhibitors in HER2+, PIK3CA-mutant breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 302.

Approximately 25% of human breast cancers overexpress the HER2 (ErbB2) proto-oncogene, which confers a more aggressive tumor phenotype and associates with a poor prognosis in patients with this disease. Two approved therapies targeting HER2, the monoclonal antibody trastuzumab and the tyrosine kinase inhibitor lapatinib, are clinically active against this type of breast cancer. However, a significant fraction of patients with HER2+ breast cancer treated with these agents eventually relapse or develop progressive disease. This suggests that tumors acquire or possess intrinsic mechanisms of resistance that allow escape from HER2 inhibition. This review focuses on mechanisms of intrinsic and/or acquired resistance to HER2-targeted therapies that have been identified in preclinical and clinical studies. These mechanisms involve alterations to HER2 itself, coexpression or acquisition of bypass signaling through other receptor or intracellular signaling pathways, defects in mechanisms of cell cycle regulation or apoptosis, and host factors that may modulate drug response. Emerging clinical evidence already suggests that combinations of therapies targeting HER2 as well as these resistance pathways will be effective in overcoming or preventing resistance.

Tyrosine kinase inhibitors (TKIs) are effective anti-cancer therapies but resistance to these agents eventually develops. Several models of resistance to TKIs have been studied including resistance to EGFR inhibitors. Recent studies in EGFR-dependent A431 cells found upregulation of the IGF1R pathway as a mechanism to overcome blockade of EGFR. This was associated with amplification of IGF1R signaling and recovery of downstream PI3K-AKT activation. This work adds to a growing body of cell lines in culture that have provided insights into mechanisms of resistance that can be interrogated in primary tumors in patients. In this review, these model systems and their applicability to human cancers, as well as strategies to identify and overcome resistance to TKIs, are discussed.

522 Background: Abemaciclib, an inhibitor of cyclin dependent kinases CDK4 and CDK6, demonstrated safety and clinical activity as a single agent for hormone receptor positive (HR+) metastatic breas...

Background: We have previously shown in animal models that combination anti-HER2 therapy leads to complete tumor regression of HER2+ breast cancer (BC). We translated these findings in a neoadjuvant trial of 12 weeks (wks) of L+T (TBCRC006) that demonstrated a meaningful pathologic complete response (pCR) and near pCR (in ER+ tumors) in patients with locally advanced HER2+ BC. In the present trial, we sought to determine whether longer treatment would lead to a higher rate of pCR without the use of chemotherapy by converting near pCR to pCR especially in the ER+ subset. Methods: TBCRC023 (NCT00999804) is a randomized phase II trial combining a Simon Phase 2 design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Women with HER2+ BC measuring 2 cm or larger were eligible and were randomized in a 1:2 ratio to 12 vs. 24 wks of L+T. Letrozole (along with ovarian suppression if premenopausal) was also administered in patients whose tumors were also ER+. Serial tumor biopsies were obtained at baseline, wk 1, wk 12, and at the time of surgery. All evaluable patients were assessed for pCR, defined as no residual invasive carcinoma in the breast. Patients did not undergo surgery, withdrew consent, or received additional neoadjuvant therapy were counted as non-responders. Results: Ninety-seven patients were enrolled (33 in 12-wk arm and 64 in 24-wk arm), of whom 95 were evaluable. Seventy seven percent of patients were white and 18% were black. Twenty percent were of Hispanic ethnicity. Median age was 51 and 55% were postmenopausal. Median tumor size was 5 cm and 65% were ER+. Study treatment was well tolerated with grade 1-2 diarrhea (24% in 12-wk arm, 31% in 24-wk arm) and grade 1-2 acneform rash (12% in 12-wk arm, 19% in 24-wk arm) being the most common toxicities. Grade 3 toxicities were uncommon and were mostly in the 24-wk arm (elevated liver function test: 9%, diarrhea 2%, mucositis 2%), while the 12-wk arm had one grade 3 anemia and the study’s only serious adverse event (acute kidney injury). There were no grade 4 toxicities. The experimental arm completed stage 2 accrual and pCR rate was numerically superior to control, entirely due to better results in ER+ (Table 1), but lower than the expected pCR rate of 36% needed in the planned analysis to conclude in favor of enhanced efficacy with extended therapy. pCR rates were also lower in the control arm than in the previous study. Table 1. pCR rates 12-week arm % (n)24-week arm % (n)ER-positive8.7% (2/23)33.2% (13/39)ER-negative20% (2/10)8.7% (2/23)Overall pCR12.2% (4/33)24.2% (15/62) Conclusions: Treatment with L+T (with endocrine therapy in ER+ tumors) for 24 weeks leads to doubling of the pCR rate in women with HER2+ breast cancer without using cytotoxic chemotherapy. This approach is effective and well tolerated and warrants study as part of a de-escalation strategy that may spare some patients the cost and toxicity of chemotherapy. Tissue obtained on this trial will provide a valuable resource to validate correlative findings from our prior studies and discover new biomarkers to help guide proper patient selection for treatment. Citation Format: Mothaffar F Rimawi, Polly A Niravath, Tao Wang, Brent Rexer, Andres Forero, Antonio C Wolff, Rita Nanda, Anna M Storniolo, Ian Krop, Matthew P Goetz, Julie R Nangia, Sao Jiralerspong, Anne C Pavlick, Carolina Gutierrez, Rachel Schiff, Susan G Hilsenbeck, C Kent Osborne. TBCRC023: A randomized multicenter phase II neoadjuvant trial of lapatinib plus trastuzumab, with endcorine therapy and without chemotherapy, for 12 vs. 24 weeks in patients with HER2 overexpressing breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S6-02.

A recent study by Niepel and colleagues describes a novel approach to predicting response to targeted anti-cancer therapies. The authors used biochemical profiling of signaling activity in basal and ligand-stimulated states for a panel of receptor and intracellular kinases to develop predictive models of drug sensitivity. In some cases, the response to ligand stimulation predicted drug response better than did target abundance or genomic alterations in the targeted pathway. Furthermore, combining biochemical profiles with genomic information was better at predicting drug response. This work suggests that incorporating biochemical signaling profiles with genomic alterations should provide powerful predictors of response to molecularly targeted therapies.

Mutations in genes that constitute the phosphatidylinositol 3-kinase (PI3K) pathway occur in >70% of breast cancers. Clinical and experimental evidence suggest that PI3K pathway activation promotes resistance to some of the current breast cancer therapies. PI3K is a major signaling hub downstream of human epidermal growth factor receptor (HER)2 and other receptor tyrosine kinases. PI3K activates AKT, serum/glucocorticoid regulated kinase (SGK), phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and several other molecules involved in cell cycle progression and survival. In estrogen receptor (ER)+ breast cancer cells, PI3K activation promotes estrogen-dependent and -independent ER transcriptional activity, which, in turn, may contribute to anti-estrogen resistance. Activation of this pathway also confers resistance to HER2-targeted therapies. In experimental models of resistance to anti-estrogens and HER2 inhibitors, pharmacological inhibition of PI3K/AKT/mTOR has been shown to overcome drug resistance. Early clinical data suggest that combined inhibition of either HER2 or ER plus inhibition of the PI3K pathway might be an effective strategy for treatment of respective HER2+ and ER+ breast cancers resistant to standard therapies. Here, we review alterations in the PI3K pathway in breast cancer, their association with therapeutic resistance, and the state of clinical development of PI3K pathway inhibitors.

We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, SUM190, and UACC893) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots remained inhibited in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein substrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in four of the LR lines: BT-474, MDA-MB-361, HCC1954, and UACC893. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases, whereas Yes was the predominant SFK in MDA-MB-361 and UACC893 cells. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 or MDA-MB-361 sensitive cells with Src inhibitors and Lap for 21 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-6.

We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, and SUM190) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots was undetectable in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein subtrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in BT-474 and HCC1954 LR cells compared to drug-sensitive cells. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 sensitive cells with AZD0530 and Lap for 14 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 707.

Abstract #4054 The anti-tumor effect of HER2 antagonists in HER2-dependent breast cancer cells has been proposed to rely on inhibition of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Mutations in the p110α catalytic subunit of PI3K occur in up to 40% of breast cancers and activation of this pathway has been implicated in resistance to the HER2 antibody trastuzumab (T). PIK3CA mutations cluster in two regions in the helical (E542K, E545K; exon 9) and catalytic (H1047R; exon 20) domains. We studied the role of these mutants in resistance to HER2 inhibitors in breast cancer cells with HER2 amplification. Two lines with endogenous H1047R p110 (PI3K), SUM190 and HCC1954, and SKBR3, SUM225, and BT474 cells stably transduced with retroviral vectors encoding HA-tagged wild-type, E545K, or H1047R p110, were studied for their response to the HER2 tyrosine kinase inhibitor lapatinib ditosylate (L; GW-572016) and T. In monolayer and 3D cell proliferation assays, partial resistance to L and T was conferred by either mutation compared to WT p110. After prolonged L treatment, Ser473 phosphorylation of Akt recovered in all cells with endogenous or ectopic p110 mutants in spite of continued inhibition of Y1248 P-HER2 and Y1289 P-HER3 by L. Further, BT474 cells with either p110 mutant could be passaged in the continued presence of L. Immunoprecipitation of p85, the regulatory subunit of PI3K, showed that PI3K association with HER3 was abrogated by T and L. RNAi of HER3 in MCF10A/HER2/PI3KE545K and MCF10A/HER2/PI3KH1047R cells markedly but not completely inhibited growth suggesting that the PI3K mutants may still depend on HER3 for full activation. We hypothesized that in cells with mutant PI3K, the mutants coexist in a pool with WT enzyme, and that mutant and WT p110α are bound to p85 in variable proportions. Experiments to measure whether the ratio of mutant to WT p110 bound to p85, assayed by mass spectrometry, will increase in the PI3K-mutant cells with acquired resistance to L are in progress. To test the role of these mutants on resistance to anti-HER2 therapies in vivo, athymic mice bearing BT474 xenografts with ectopic WT, E545K, or H1047R p110 are undergoing treatment with L and T. Finally, we analyzed mutations of PIK3CA in a cohort of 40 patients with locally advanced HER2+ breast cancer treated with weekly single-agent T for 3 weeks, followed by T with docetaxel for a total of 12 weeks before surgery (Mohsin et al. JCO 23:2460, 2005). Sequential core biopsies at weeks 1 and 3 after initiation of T were taken. Eight of 40 tumors (20%) expressed mutant PI3K, 2 in exon 9 and 6 in exon 20. PIK3CA mutations did not correlate with change in Ki67 (p=0.97) or cleaved caspase 3 (p=0.51) in weeks 1 or 3 of treatment nor with pathologic complete response (p=0.21). These data suggest that, if mutant PI3K confers relative resistance to T, a shorter time to recurrence may be a more robust endpoint as the initial cellular or clinical response may not be a good indicator of this resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4054.