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CYTOF Panel

PDF file, 73K, Extended Toxicity Information.

SPADE tree generated by CYTOF analysis

Supplementary Figure 4

Concurrent HER2 therapies for each patient for whom CYTOF analysis was done

Patient cohort schematic

Supplementary Figure 3

Purpose: We conducted a phase I study of dasatinib, an oral SRC-family tyrosine kinase inhibitor, in combination with paclitaxel and carboplatin in the treatment of advanced and recurrent epithelial ovarian cancer.Experimental Design: The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included defining toxicity, response rate (RR), pharmacokinetics, and pharmacodynamics. Using a “3+3” design, cohorts of three to six patients received paclitaxel (175 mg/m2) and carboplatin (AUC 6) every 3 weeks with escalating doses of dasatinib (100, 120, and 150 mg daily), followed by an eight-patient expansion cohort.Results: Twenty patients were enrolled between June 2007 and December 2009. The median age was 61 years (range: 42–82) with a median of 2 prior regimens (range: 0–6), and 71% had platinum-sensitive disease. There were three to six patients in each cohort, and eight in the expansion cohort. Pharmacokinetics were observed over the first two cycles of therapy. One DLT was observed in the 100 mg dasatinib cohort (grade 3 myalgia). Other toxicities in all cycles included neutropenia (95% grade 3–4; 91% in the 150 mg dosing cohort), thrombocytopenia (35% grade 3–4), and fatigue (10% grade 3). The RR was 40% [three complete responses, (15%); five partial responses, (25%)],10 patients (50%) had stable disease, and two were not evaluable. The PFS6-month actuarial estimate was 86%. The median PFS and OS were 7.8 and 16.2 months, respectively.Conclusions: Due to the high incidence of myelosuppression with subsequent cycles, the recommended phase II dose of dasatinib is 150 mg daily in combination with paclitaxel and carboplatin. The combination was safe with evidence of clinical activity. Clin Cancer Res; 18(19); 5489–98. ©2012 AACR.

Mutation of HER2 to obscure binding of Trastuzumab and Pertuzumab

Supplementary Table 1

PDF file, 92K, Volcano Plot.

PDF file, 130K, Heatmaps and Safe Plots.

Supplementary Figure 6

Purpose:Despite promising advances in breast cancer immunotherapy, augmenting T-cell infiltration has remained a significant challenge. Although neither individual vaccines nor immune checkpoint blockade (ICB) have had broad success as monotherapies, we hypothesized that targeted vaccination against an oncogenic driver in combination with ICB could direct and enable antitumor immunity in advanced cancers.Experimental Design:Our models of HER2+ breast cancer exhibit molecular signatures that are reflective of advanced human HER2+ breast cancer, with a small numbers of neoepitopes and elevated immunosuppressive markers. Using these, we vaccinated against the oncogenic HER2Δ16 isoform, a nondriver tumor-associated gene (GFP), and specific neoepitopes. We further tested the effect of vaccination or anti–PD-1, alone and in combination.Results:We found that only vaccination targeting HER2Δ16, a driver of oncogenicity and HER2-therapeutic resistance, could elicit significant antitumor responses, while vaccines targeting a nondriver tumor-specific antigen or tumor neoepitopes did not. Vaccine-induced HER2-specific CD8+ T cells were essential for responses, which were more effective early in tumor development. Long-term tumor control of advanced cancers occurred only when HER2Δ16 vaccination was combined with αPD-1. Single-cell RNA sequencing of tumor-infiltrating T cells revealed that while vaccination expanded CD8 T cells, only the combination of vaccine with αPD-1 induced functional gene expression signatures in those CD8 T cells. Furthermore, we show that expanded clones are HER2-reactive, conclusively demonstrating the efficacy of this vaccination strategy in targeting HER2.Conclusions:Combining oncogenic driver targeted vaccines with selective ICB offers a rational paradigm for precision immunotherapy, which we are clinically evaluating in a phase II trial (NCT03632941).

Supplementary Figure 2

Supplementary Figure 1

Individual histograms of perforin expression by memory CD8 T cell cluster pre- and post-vaccination

Supplementary methods

Supplementary Figure 5

Purpose:Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2).Patients and Methods:In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy.Results:Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS.Conclusions:VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.

PDF file, 48K.

Background: Fam-trastuzumab deruxtecan (DS-8201) was recently approved in patients with advanced/metastatic HER2 positive breast cancer who have received two or more prior HER2-based regimens. In DESTINY-Breast01, an unprecedented response rate was observed in 61% of heavily pretreated patients; however, all-grade interstitial lung disease (ILD) developed in almost 14% of patients, which led to death in 2% of patients. Additionally, patients with a medical history of clinically significant lung disease were excluded which is not consistent with the standard community patient population that may receive fam-trastuzumab deruxtecan. Currently, there are no recommended protocols in place or guidance for monitoring patients for interstitial lung disease/pneumonitis while on fam-trastuzumab deruxtecan. This is a retrospective chart review to assess the feasibility of implementing an ILD monitoring protocol in a cohort of patients receiving treatment with fam-trastuzumab deruxtecan at the Duke Cancer Institute. Methods: Patients with HER2-positive or HER2-low metastatic breast cancer who received ≥five cycles of fam-trastuzumab deruxtecan between Jan 1, 2020-June 30, 2020 were included. Chest imaging and pulmonary function testing with diffusing capacity for carbon monoxide (DLCO) were performed at baseline prior to initiation and every six weeks prior to cycle 3 and cycle 5 of fam-trastuzumab deruxtecan to monitor for ILD. DLCO was corrected for hemoglobin. Patients that experienced more than a 10% decrease in corrected DLCO (DLCOc) were recommended to have a pulmonology consult in which DLCOc was reviewed in combination with chest imaging and clinical history to evaluate for ILD and recommend continuance or cessation of the drug. Clinical pulmonary symptoms to include cough, shortness of breath, dyspnea, and new or worsening respiratory symptoms were monitored per chart review. Results: Seven patients with HER2-positive (N=6) and HER2-low (N=1) metastatic breast cancer were monitored per predefined ILD monitoring protocol with 100% completion of chest imaging and pulmonary function testing at baseline and prior to cycle 3 and cycle 5 indicating feasibility. There were no confirmed cases of ILD/pneumonitis within the patient cohort. Two patients (28.6%) experienced DLCOc decreases >10% warranting a pulmonology consult. Upon further assessment, ILD/pneumonitis was ruled out based on chest imaging and/or asymptomatic presentation and both patients continued therapy without treatment delay or development of pneumonitis to date. Both patients with DLCOc decrease >10% had a history of lung disease or significant metastatic disease involvement including asthma and lymphangitic carcinomatosis, respectively. In these patients, DLCOc, imaging and clinical history were able to safely rule out ILD. . Conclusion: Implementation of a comprehensive protocol to monitor and assess for ILD associated with fam-trastuzumab deruxtecan is feasible for patients receiving this therapy and may prevent treatment delay in patients with suspected ILD. In this small cohort, stable DCLOc suggests the absence of ILD onset in patients treated with fam-trastuzumab deruxtecan. DLCOc monitoring may be a reasonable measure to assess for ILD-related changes in patients. Further data collection is underway to understand the value of a comprehensive ILD monitoring protocol. Citation Format: Heather Moore, Scott Shofer, Amy Guisinger, Gloria Broadwater, Jeremy Force, Sarah Sammons, Carey Anders, Kelly Westbrook. Feasibility of a comprehensive monitoring protocol for the prevention and treatment of interstitial lung disease in patients undergoing treatment with fam-trastuzumab deruxtecan [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 PS13-33.

Background: CDK 4/6 inhibitors in patients (pts) with HR+/HER2- ABC has led to significant improvements in clinical outcomes, however our understanding of the impact of these treatments on CV health is unknown. Gains in overall survival should not be offset by increased CV morbidity and mortality; a particular concern given the shared risk factors for both breast cancer and CV disease. Objective: The aim of this study was to describe patient characteristics, treatment patterns and cardiovascular risk factors and disease in pts with ABC treated with endocrine therapy (ET) or ET + CDK 4/6 inhibitor. Methods: We retrospectively studied pts with HR+/HER2- ABC who were receiving first line endocrine therapy. Post-menopausal (PM) women, pre-menopausal women on ovarian suppression (OS), and men were included. Two cohorts were included: Group A - treated with ET alone (2012-2014; prior to US approval of CDK 4/6 inhibitors) and Group B - treated with ET+ CDK 4/6 inhibitor (2015-2017). The following data was extracted from Duke University Health System’s electronic medical record (EPIC) and entered into a REDCap database: demographics, baseline cardiovascular risk factors, and co-morbidities. Pt characteristics are summarized using medians and interquartile ranges for continuous variables and categorical descriptions are summarized using frequencies and percentages. Results: In total 103 patients were included with 57 in Group A (ET alone) and 46 in Group B (ET + CDK 4/6 inhibitor). Median age was 62.0 and 63.5 years in Group A and B, respectively. Fifty-three (93%) of pts in Group A were PM women compared to 37 (80%) PM women and 1 (3%) male in Group B. The groups seemed to be similar in terms of race (white 70% vs 72%), baseline body mass index (28.2 vs 27.6), baseline systolic blood pressure (132.0 vs 135.5) and diastolic blood pressure (79.0 vs 77.5). Similarly, the groups seemed to be similar in baseline hypertension (68% vs 62%); diabetes (23% vs 24%); Hemoglobin A1c (7.2% vs 6.4%) or family history of CV disease (56% vs 55%), Group A versus Group B, respectively. There were slightly more current/past smokers in Group B than Group A (48% vs 35%) and more pts in Group A with a history of hyperlipidemia relative to Group B (52% vs 31%). Conclusions: In this retrospective descriptive cohort study there seemed to be no differences in demographics or baseline CV risk factors between the ET and ET + CDK 4/6 inhibitor cohorts with the exception of more baseline hyperlipidemia in the ET cohort. This might suggest that baseline CV risk factors did not dissuade practioners from prescribing ET + CDK 4/6 inhibitor therapy. We plan to expand our cohort to collect information on type and duration of ET and CDK 4/6 inhibitors, reason for treatment discontinuation, and CV events (eg heart failure, arrhythmias, stroke, myocardial infarction), to better understand the impact that cardiovascular risk factors have on outcomes in breast cancer patients taking ET+ CDk 4/6 inhibitor. Table 1: Demographics and CV risk factors in ABC patients treated with ET or ET + CDK4/6 inhibitor Median (IQR) unless otherwise indicatedGroup A ET (n=57)Group B ET+ CDK 4/6 inhibitor (n=46)Age median (range)62.0 (27-84)63.5 (30-82)Menopausal status, n (%) Post menopausal Premenopausal + OS Male53 (93) 4 (7) 037 (80) 8 (17) 1 (3)Race, n (%) White Other40 (70) 17 (30)33 (72) 13 (28)Type of Insurance, n (%) Private Medicare Medicare and Private Medicaid Medicaid and Medicare Vererans Sponsored Self-Pay Unknown19 (33) 14 (25) 16 (28) 1 (2) 4 (7) 0 3 (5) 019 (41) 4 (9) 15 (33) 1 (2) 4 (9) 1 (2) 0 2 (4)BMI (kg/m2)28.2 (24.9, 30.6)27.6 (24.4, 34.5)Baseline BP (mmHg) Systolic Diastolic132.0 (118.0, 145.0) 79.0 (73.0, 84.0)135.5 (124.0, 149.0) 77.5 (72.0, 84.0)HgbA1c (%)7.2 (6.4, 7.4)6.4 (5.4, 6.4)CVRF, n (%) Hypertension Diabetes FH CVD Current/past smokers Hyperlipidemia36 (68) 13 (23) 28 (56) 20 (35) 29 (52)28 (62) 11 (24) 22 (55) 22 (48) 14 (31)OS = ovarian suppression; BMI = body mass index; BP = blood pressure; HgbA1c = hemoglobin A1c; CVRF = cardiovascular risk factors; FH = family history; CVD = cardiovascular disease; IQR = Interquartile range Citation Format: Susan Dent, Gloria Broadwater, Terry Hyslop, Kevin Oeffinger, Michel Khouri, Sanjeev Balu, Gretchen Kimmick. Cardiovascular (CV) risk profile in patients with estrogen receptor (ER) positive HER2 negative advanced breast cancer (ABC): A retrospective cohort study (CAREB) [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 PS10-48.

Purpose: Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2). Patients and Methods: In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy. Results: Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS. Conclusions: VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.

Background: Immune-based therapy for metastatic breast cancer has had limited success. Strategies to augment adaptive immunity include vaccines targeting genomic amplifications like Human Epidermal Growth Factor Type 2 (HER2), an established driver of malignancy. Using a novel alphaviral vector, we constructed a vaccine encoding a portion of HER2 (VRP-HER2). Methods: In preclinical studies, mice were immunized before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and anti-tumor function were assessed. We then translated this vaccine into a phase I clinical trial in which subjects with advanced HER2-overexpressing breast cancers received VRP-HER2 every 2 weeks for a total of three doses (cohort 1). In cohort 2, subjects received the same dose of VRP-HER2 along with a standard HER2 targeted therapy. Results: VRP-HER2 induced HER2-specific T cell and antibody responses while controlling tumor growth in murine models. Vaccination with VRP-HER2 was well tolerated in both patient cohorts. PFS was modest, while median OS was 50.2 months in cohort 1 and 32.7 months in cohort 2. In cohort 2, there is one partial response and two patients with continued stable disease. Vaccine induced anti-HER2 antibodies and T cells were identified. Increased perforin expression by memory CD8 T cells post vaccination significantly correlated with improved PFS. Conclusions: VRP-HER2 led to an increase in perforin expressing HER2-specific memory CD8 T cells in preclinical and clinical studies, and had profound antitumor effects in murine models. The generation of HER2-specific memory CD8 T cells was significantly correlated with increased PFS in patients. Subsequent studies will seek to enhance T cell activity by combination with anti-PD-1/PD-L1 antibodies. Citation Format: Crosby EJ, Gwin WR, Chang S, Maecker HT, Lubkov V, Snyder JC, Broadwater G, Hyslop T, Osada T, Hobeika AC, Hartman ZC, Morse MA, Lyerly HK. CD8 T cells induced by novel alphaviral vector predict improved progression free survival in advanced HER2+ breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-16.

BACKGROUND: Brain metastasis is a complication in advanced breast cancer (ABC) and is associated with poor prognosis. Incidence of breast cancer brain metastasis (BCBM) is increasing with advances in therapy, allowing patients to survive long enough to develop CNS metastasis. Improved outcomes have been documented in ABC over the past decades, largely related to the use of trastuzumab in HER2+ ABC. However, it remains unclear whether survival has improved in HER2- ABC in patients with BCBM. This study asks: has the improvement in systemic and radiotherapies for HER2- breast cancer impacted survival in patients with breast cancer brain metastasis. OBJECTIVES: 1) To estimate whether date of BCBM diagnosis is associated with overall survival (OS) in patients diagnosed between 1996-2015. 2) To estimate whether OS of this patient population depends upon other demographic and clinical factors. METHODS: This is a retrospective chart review of patients with diagnosis of BCBM between 1996-2015. Data collection includes: age at BCBM diagnosis, ethnicity, ER/PR/HER2 status, date of BCBM diagnosis, date of primary breast cancer diagnosis, date of death/last clinical follow-up, and treatment. Kaplan-Meier analysis and the log-rank test compared OS (time from diagnosis of BCBM until death or last clinical FU) between groups diagnosed in 5-year cohorts (1996-2000, 2001-2005, 2006-2010, 2011-2015). A univariate proportional hazards model was used to regress OS on date of diagnosis. A multivariate proportional hazards model was used which included the subset of patients diagnosed with BCBM in 2001 and later. This model adjusted for additional factors: race, time to development of BCBM diagnosis, age at the time of BCBM diagnosis, year of diagnosis as a continuous factor, ER, PR, while testing the significance of HER2 status. A p-value < 0.05 was significant. RESULTS: A total of 165 patients with BCBM were included in this analysis, with a median age of 53.8 (SD 13.0) at time of BCBM diagnosis. Most patients were Caucasian (66%; 109/165) or African-American (29%; 48/165). Although statistical significance was not attained, greater median overall survival was seen for patients diagnosed with BCBM in more recent 5-year cohorts (2011-2016, 9.5 months; 2006-2010, 8 months) than patients in older cohorts (2001-2006, 3.6 months; 1996-2000, 5.3 months), p=0.3. Date of diagnosis of BCBM as a continuous variable is predictive of overall survival (HR 0.83 [95% CI: 0.71-0.97] comparing 5-year intervals, p=0.016). After adjusting for the covariates listed above, HER2 positive status is predictive of overall survival (HR 0.34 [95% CI: 0.34-0.56]; p CONCLUSIONS: While survival has improved by 5.9 months over the past two decades, it remains highly dependent on HER2 status. Novel therapies for BCBM are greatly needed for ER+ and triple negative subtypes. Final results will include an expanded analysis to incorporate additional cases and three other categorical covariates measured during follow-up: whether the patient received radiotherapy, surgery, and/or medical therapy after diagnosis of brain metastases. GRANT FUNDING: TL-1 CTSA Pre-Doctoral Training Grant (5TL1TR001116-03). Citation Format: Narloch JL, Harnden K, Broadwater G, Peterson B, Hyslop T, Kirkpatrick J, Fecci P, Kim G, Blackwell KL. HER2 status remains the primary predictor of improved survival in patients with BCBM over the past 2 decades (1996-2015) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-08-12.

Background: Increased stromal tumor infiltrating lymphocytes (TILs) are predictive and prognostic for improved outcomes from neoadjuvant or adjuvant chemotherapy in triple negative breast cancer. Increased tumor mutational burden may promote neoantigens causing immune system upregulation. Microsatellite instability in gastrointestinal cancer predicts for response to checkpoint inhibition and is associated with inherited cancer predisposition. The immune system response in BRCA mutated breast cancer has not been described. The purpose of this study is to assess tumor infiltrating immune cells in early stage breast cancer patients with and without BRCA gene mutations. Methods: We retrospectively investigated 124 early stage breast cancer patients with BRCA mutations (n=62, BRCA+) and without BRCA mutations (n=62, BRCA WT). The %TILs was measured manually by H&E. Our control group consisted of age, stage, and receptor status matched early stage untreated breast cancer patients who were deemed BRCA WT by extended gene panel testing or were negative for BRCA 1/2 and had a posttest probability of harboring an autosomal dominant mutated gene of ≤ 1% using the Bayes-Mendel algorithm. We used a two-sample binomial arcsin approximation to detect a 20% difference in TILs between cohorts to attain 80% power with a one-side alpha of 0.05. Wilcoxon Rank-Sums test was used to compare differences in the central tendencies for continuous variables. We used the Nanostring PanCancer immune profiling panel to immunophenotype a portion of the BRCA+ and BRCA WT cohorts and used nSolver for quality control, normalization, and bioinformatics analyses. Results: Here we report TILs from the first 21 patients of our study. Thirteen patients harbored BRCA mutations and eight patients did not. All patients were HER2 negative. Eight (61%) and four (50%) patients were hormone receptor positive (HR+) in the BRCA+ and BRCA WT cohorts, respectively. Median %TILs were not significantly different between the BRCA+ (15, range 0-70) and BRCA WT (17.5, range 5-60; p=0.7) groups. Median %TILs in the HR+/BRCA+ (12.5, range 0-50) and HR-/BRCA+ (15, range 5-70) cohorts were not statistically different when compared to HR+/BRCA WT (10, range 5-15; p=0.4) and HR-/BRCA WT (30, range 20-60; p=0.2) cohorts, respectively. There were 2 patients with lymphocyte predominant breast cancer (n=1, HR-/BRCA+; n=1, HR-/BRCA WT). Conclusions: This is the first study to characterize TILs and a tumor immune microenvironment phenotype in early stage breast cancer patients with BRCA mutations. These results suggest harboring a BRCA mutation is not associated with increased TILs in early stage untreated breast cancer patients. This conclusion stayed true regardless of hormone receptor status. However, a trend of decreased TILs was seen in HR-/BRCA+ patients when compared to those with HR-/BRCA WT disease. Moreover, the median and range of TILs were higher in the HR+/BRCA+ group compared to the HR+/BRCA WT group. This suggests increased TILs may exist in some HR+ patients with a BRCA mutation. Further investigation of TILs and immune profiling of early stage untreated breast cancer patients with and without BRCA mutations is warranted. Citation Format: Force J, Abbott S, Broadwater G, Kimmick G, Westbrook K, Hwang S, Kauff N, Stashko I, Weinhold K, Nair S, Hyslop T, Blackwell K, Castellar E, Marcom PK. Elucidating the tumor immune microenvironment phenotype in early stage untreated BRCA mutated breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-19.

Despite promising advances, overcoming immune suppression and driving productive immune responses in the tumor microenvironment remains a significant challenge. Using a spontaneous breast cancer model, we found that vaccination targeting HER2d16, a highly expressed driver of oncogenicity and HER2-therapeutic resistance, elicited significant anti-tumor responses. In contrast, vaccines targeting a non-driver tumor-specific antigen (GFP) or unique non-driver tumor neoepitopes had no impact on tumor occurrence or progression. While vaccine-induced HER2-specific CD8+ T cells were essential for responses, tumors treated therapeutically with a vaccine alone ultimately progressed. However, long-term tumor control and complete tumor regression was only achieved when vaccine was combined with immune-checkpoint blockade (anti-PD1). Single cell RNAsequencing of tumor-infiltrating T cells (TILs) revealed that while vaccination expanded CD8 T cells within the tumor, only the combination of vaccine with anti-PD1 therapy induced a tumor rejection activation signature that was identified in the expanded T cell clones. We go on to use the single cell data to clone and reexpress the TCRs from expanded TILs from vaccinated mice and show that they are HER2-reactive. This data conclusively demonstrates the efficacy of this vaccination strategy in expanding tumor rejection T cells and supports its further evaluation in an ongoing Phase II trial (NCT03632941). The workflow used to identify and clone expanded, tumor specific T cells has broad potential applications across tumor types and treatment platforms. Citation Format: Erika J. Crosby, Chaitanya Acharya, Christopher Rabiola, William J. Muller, Lewis A. Chodosh, Gloria Broadwater, Jonathan Shepherd, Daniel Hollern, Xiaping He, Charles M. Perou, Benjamin K. Ashby, Benjamin G. Vincent, Michael A. Morse, Herbert K. Lyerly, Zachary C. Hartman. Stimulation and expansion of oncogene-reactive tumor infiltrating T cells through combined Ad-HER2Δ16 vaccination and anti-PD1 enable anti-tumor responses against established HER2 BC [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 904.

Purpose/Objectives: RT is associated with acute treatment-related complications that can lead to poor quality of life (QOL) and fatigue. Exercise has been shown in other cancer treatment settings to improve negative outcomes. We conducted a prospective pilot study to explore the association between exercise, patient-reported outcomes (PROs), and radiation therapy (RT) toxicities. Materials/Methods: Patients with surgically excised ductal carcinoma in situ or invasive breast carcinoma receiving curative radiation were enrolled. Each patient completed an exercise behavior/QOL survey at two time points: before the patient’s 5th fraction of radiation and during the last week of treatment. Limb girth measurements to evaluate lymphedema and assessment of shoulder range of motion (ROM) were completed on all patients at the same two time points. Skin toxicity was assessed weekly throughout radiotherapy. Exercise behavior was quantified with the Godin Leisure Time Exercise Questionnaire (metabolic equivalent [MET] hours per week). Patients with >7 METs/week were designated the exercise cohort (n=10) and those with Results: Median patient age was 56 (range 28-70) years. Median MET in the exercise cohort was 12.9/week (range 7.5-35.0, n=10); 0.0/week in the non-exercise cohort (range 0-5.8, n=10). Women in the exercise cohort experienced significant improvement in depression scores over the course of treatment as compared to those who did not exercise (p=0.013). Those in the exercise cohort also reported less fatigue on the FACT-Fatigue subscale at treatment completion (exercise: 133.0; non-exercise: 121.0) (p=0.6). In addition, only 30% of exercisers suffered from grade 2 dermatitis compared to 70% of non-exercisers (p=0.2), despite a similar body mass index (26.4 exercise cohort versus 28.1 non-exercise). Exercisers also had greater ROM in the affected (91.7 vs. 85.2%, p=0.1) and contralateral shoulder (95 vs. 90%, p=0.048) at treatment completion. No differences in pain or sleep scores were noted and lymphedema was mild ( Conclusion: The vast majority of current exercise oncology literature indicates that physical activity is an independent predictor of quality of life metrics in cancer patients. Our study notes a trend towards improved outcomes with increased exercise during radiation therapy, suggesting that accrual of additional patients to our pilot study is worthwhile. Ultimately, a concurrent exercise intervention may improve quality of life and reduce acute toxicity in patients undergoing breast radiation treatment. Citation Format: Ritu Arya, Lee W Jones, Rachel C Blitzblau, Manisha Palta, Lisa Massa, Gloria Broadwater, Janet K Horton. The association between exercise behavior and patient-reported outcomes in women with early breast cancer receiving locoregional radiation therapy [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 P2-12-07.

Purpose/Objectives(s): Women with biologically favorable early stage breast cancer are increasingly treated with accelerated partial breast techniques. However, many alternative techniques require costly specialized equipment not routinely available in most radiation oncology facilities. In addition, suboptimal cosmetic outcomes have been reported with the external beam technique, possibly related to large post-operative treatment volumes. To address these issues, we designed a phase I dose-escalation protocol to determine the maximally tolerated dose (MTD) of a single radiosurgery treatment delivered preoperatively to the intact tumor plus a small margin. Materials/Methods: Women aged 55 or older with clinically node negative, ER and/or PR+, HER2-, T1 invasive carcinomas were enrolled (n = 26). Patients with low/intermediate grade in situ disease Tumor tissue was obtained from diagnostic and lumpectomy specimens. Immunohistochemistry (IHC) for Fas was performed on paraffin-embedded samples before and after radiation. A histoscore was created using the average membrane and cytoplasmic staining intensity multiplied by the percentage of positive cells. Results: Thirty-two women were treated, 8 each at the 15, 18, and 21Gy dose levels with an additional expansion cohort at the final 21Gy dose level. The maximally tolerated dose was not reached. Three patients required post-operative conventional radiation due to high-risk tumor features (ex. larger primary, nodal involvement). At a median follow-up of 6.8 months, primarily mild toxicities (grade 1-2 dermatitis, fibrosis, and pain) were noted. At 6 months (n = 20), all reported cosmetic outcomes are excellent or good. At 12 months (n = 10), 80% are excellent or good. Both patients with a fair/poor cosmetic outcome received radiosurgery plus post-operative conventional treatment; one experienced grade 3 breast atrophy. There have been no local or distant recurrences to date. Post-treatment MRIs were obtained in 20/32 patients, with early indicators of decreased cell density and increased vascular permeability. Sixteen patients had evaluable paired IHC and six demonstrated significant Fas up-regulation after radiation. The mean combined post-treatment histoscore was about twice as high as the mean pre-treatment score. Conclusion: Preoperative stereotactic radiotherapy to the intact breast tumor can be delivered with widely available clinical tools in a convenient single fraction, and provides a unique opportunity to study breast cancer radiation response. 21Gy did not yield dose-limiting toxicity and will be utilized for future studies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-04.

Aerobic exercise training (AET) is an effective adjunct therapy to attenuate the adverse side-effects of adjuvant chemotherapy in women with early breast cancer. Whether AET interacts with the antitumor efficacy of chemotherapy has received scant attention. We carried out a pilot study to explore the effects of AET in combination with neoadjuvant doxorubicin–cyclophosphamide (AC+AET), relative to AC alone, on: (i) host physiology [exercise capacity (VO2 peak), brachial artery flow-mediated dilation (BA-FMD)], (ii) host-related circulating factors [circulating endothelial progenitor cells (CEP) cytokines and angiogenic factors (CAF)], and (iii) tumor phenotype [tumor blood flow (15O–water PET), tissue markers (hypoxia and proliferation), and gene expression] in 20 women with operable breast cancer. AET consisted of three supervised cycle ergometry sessions/week at 60% to 100% of VO2 peak, 30 to 45 min/session, for 12 weeks. There was significant time × group interactions for VO2 peak and BA-FMD, favoring the AC+AET group (P < 0.001 and P = 0.07, respectively). These changes were accompanied by significant time × group interactions in CEPs and select CAFs [placenta growth factor, interleukin (IL)-1β, and IL-2], also favoring the AC+AET group (P < 0.05). 15O–water positron emission tomography (PET) imaging revealed a 38% decrease in tumor blood flow in the AC+AET group. There were no differences in any tumor tissue markers (P > 0.05). Whole-genome microarray tumor analysis revealed significant differential modulation of 57 pathways (P < 0.01), including many that converge on NF-κB. Data from this exploratory study provide initial evidence that AET can modulate several host- and tumor-related pathways during standard chemotherapy. The biologic and clinical implications remain to be determined. Cancer Prev Res; 6(9); 925–37. ©2013 AACR.

Purpose: We conducted a phase I study of dasatinib, an oral SRC-family tyrosine kinase inhibitor, in combination with paclitaxel and carboplatin in the treatment of advanced and recurrent epithelial ovarian cancer. Experimental Design: The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included defining toxicity, response rate (RR), pharmacokinetics, and pharmacodynamics. Using a “3+3” design, cohorts of three to six patients received paclitaxel (175 mg/m2) and carboplatin (AUC 6) every 3 weeks with escalating doses of dasatinib (100, 120, and 150 mg daily), followed by an eight-patient expansion cohort. Results: Twenty patients were enrolled between June 2007 and December 2009. The median age was 61 years (range: 42–82) with a median of 2 prior regimens (range: 0–6), and 71% had platinum-sensitive disease. There were three to six patients in each cohort, and eight in the expansion cohort. Pharmacokinetics were observed over the first two cycles of therapy. One DLT was observed in the 100 mg dasatinib cohort (grade 3 myalgia). Other toxicities in all cycles included neutropenia (95% grade 3–4; 91% in the 150 mg dosing cohort), thrombocytopenia (35% grade 3–4), and fatigue (10% grade 3). The RR was 40% [three complete responses, (15%); five partial responses, (25%)],10 patients (50%) had stable disease, and two were not evaluable. The PFS6-month actuarial estimate was 86%. The median PFS and OS were 7.8 and 16.2 months, respectively. Conclusions: Due to the high incidence of myelosuppression with subsequent cycles, the recommended phase II dose of dasatinib is 150 mg daily in combination with paclitaxel and carboplatin. The combination was safe with evidence of clinical activity. Clin Cancer Res; 18(19); 5489–98. ©2012 AACR.

Purpose: p53 as a prognostic and predictive factor in early-stage breast cancer has had mixed results. We studied p53 protein expression, by immunohistochemistry, in a randomized clinical trial of stage II patients treated with adjuvant doxorubicin and cyclophosphamide with or without paclitaxel [Cancer and Leukemia Group B (CALGB) 9344, INT0148]. Patients and Methods: Epithelial p53 expression was evaluated using two immunohistochemical antibodies (DO7 and 1801) in formalin-fixed, paraffin-embedded tissue from patients with node-positive breast cancer who were randomized to four cycles of cyclophosphamide and one of three doses of doxorubicin (60, 75, or 90 mg/m2; AC) and to receive four subsequent cycles of paclitaxel (T) or not. Prognostic and predictive value of p53 protein expression was assessed, independent of treatment assignment, for escalating doses of doxorubicin or addition of T with endpoints of relapse-free (RFS) and overall survival (OS). Results: Of 3,121 patients, 1,887 patient specimens treated on C9344 were obtained, passed quality control, and evaluated for p53 expression. Expression was 23% and 27% for mAbs 1801 and D07, respectively, with 92% concordance. In univariate analysis, p53 positivity was associated with worse OS with either antibody, but only p53 staining with monoclonal antibody 1801 had significantly worse RFS. In multivariate analysis, p53 was not predictive of RFS or OS from either doxorubicin dose escalation or addition of paclitaxel regardless of the antibody. Conclusion: Nuclear staining of p53 by immunohistochemistry is associated with worse prognosis in node-positive patients treated with adjuvant doxorubicin-based chemotherapy but is not a useful predictor of benefit from doxorubicin dose escalation or the addition of paclitaxel. Clin Cancer Res; 17(15); 5170–8. ©2011 AACR.

Background: Random periareolar fine needle aspiration (RPFNA) is a research technique developed to assess short-term breast cancer risk in women at increased risk of breast cancer. Although there is increasing acceptance of RPFNA, neither the reproducibility nor the inter–institutional compatibility of RPFNA has been established. To address these key limitations, the Cancer and Leukemia Group B (CALGB) Prevention Group tested the reproducibility of RPFNA in a multi-institutional cross-sectional study. Methods: Sixty-three high-risk women from five CALGB institutions (Duke, Ohio State, Roswell Park, Dana Farber, and Vermont) underwent RPFNA from July 1, 2007 to June 30, 2008. Duplicate bilateral RPFNA was performed on each woman by a single investigator on a single day. Masood Cytology Index score was assessed by a single blinded cytopathologist. Results: There was a high degree of statistical agreement in the Masood Cytology Index scores of duplicate RPFNA samples from the same breast, with a Spearman correlation coefficient of 0.8312 (P < 0.0001). Importantly, although there was agreement in duplicate samples from the same breast, there was lack of agreement between duplicate samples from the opposite breast. Conclusions: This multi-institutional study shows that RPFNA is a highly reproducible measure of breast cytology in a cooperative group cross-sectional trial. RPFNA did not show a high degree of agreement between breasts, suggesting that breast cancer risk and progression may occur at different rates in individual breasts from a single woman. These studies provide proof-of-principle for future RPFNA-based cooperative group prevention studies. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1379–85)

Purpose: Currently, we lack biomarkers to predict whether high-risk women with mammary atypia will respond to tamoxifen chemoprevention. Experimental Design: Thirty-four women with cytologic mammary atypia from the Duke University High-Risk clinic were offered tamoxifen chemoprevention. We tested whether ESR1 promoter hypermethylation and/or estrogen receptor (ER) protein expression by immunohistochemistry predicted persistent atypia in 18 women who were treated with tamoxifen for 12 months and in 16 untreated controls. Results: We observed a statistically significant decrease in the Masood score of women on tamoxifen chemoprevention for 12 months compared with control women. This was a significant interaction effect of time (0, 6, and 12 months) and treatment group (tamoxifen versus control) P = 0.0007. However, neither ESR1 promoter hypermethylation nor low ER expression predicted persistent atypia in Random Periareolar Fine Needle Aspiration after 12 months tamoxifen prevention. Conclusions: Results from this single institution pilot study provide evidence that, unlike for invasive breast cancer, ESR1 promoter hypermethylation and/or low ER expression is not a reliable marker of tamoxifen-resistant atypia. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1884–90)

Purpose: This study investigated the connection among HER-2 gene amplification, HER-2 protein expression, and markers of tumor angiogenesis and oxygenation in patients with operable, invasive breast tumors. Experimental Design: From 1988 to 1995, 425 patients with metastatic breast cancer were enrolled in a study of high-dose chemotherapy with autologous transplant. Primary tumor blocks were obtained and evaluated using immunohistochemistry (IHC) staining of vessels with von Willebrand factor antibody. Mean microvessel densities (MVD) were determined by counting von Willebrand factor stained cells in three separate “vascular hot spots” using image analysis. Tumor samples were also stained for HER-2 by IHC, HER-2 gene amplification by fluorescence in situ hybridization, carbonic anhydrase 9 by IHC, and vascular endothelial growth factor (VEGF) by IHC. Plasma from 36 patients with primary tumor samples had VEGF (R&D Systems, MN) and d-dimer (American Diagnostica, Greenwich, CT) levels determined. Results: There was a significant positive correlation between HER-2 gene amplification and both maximum and average MVD (Spearman coefficient = 0.51 and 0.50; P = 0.03 and 0.05, respectively). There was an inverse correlation with HER-2 gene amplification and expression of the tumor hypoxia marker CA-9 (χ2 P = 0.02). The level of HER-2 gene amplification correlated with plasma d-dimer levels (Spearman coefficient = 0.43; P = 0.021). Interestingly, tumors with HER-2 by IHC had decreased amounts of VEGF staining (χ2 = 5.81; P = 0.01). There was no correlation between HER-2 by IHC and MVD or d-dimer. Of all of the variables examined, only average (P = 0.0016) and maximum MVD (P = 0.0128) predicted disease-free survival (Cox univariate model). Conclusions: HER-2-amplified breast cancers have increased amounts of angiogenesis, decreased amounts of hypoxia, and increased markers of fibrin degradation. These findings have prognostic, predictive, and therapeutic implications in breast cancer treatment.

Background: Women with deleterious mutations in BRCA1/2 have a 9- to 36-fold increased risk of breast cancer compared with general population rates. However, there are a significant number of women whose families have multi-generation premenopausal breast cancers and lack an identified BRCA-mutation. Currently we lack strategies to predict the development of breast cancer in women from BRCA-negative families. Random Periareolar Fine Needle Aspiration (RPFNA) is a research technique developed to test for “field effects” in high-risk women. Carol Fabian previously demonstrated that atypia in RPFNA predicted breast cancer risk, however, many women in this cohort were known BRCA mutation carriers. We investigated whether cytological atypia in RPFNA predicts the subsequent development of breast cancer in women from BRCA-negative breast cancer families. Results: From 01/01/03 to 07/01/12, we performed RPFNA in 254 high-risk BRCA-negative women. Women underwent yearly mammogram/Magnetic Resonance Imaging (MRI) and bi-annual clinical breast exam. During 6.5 women-years of follow-up, 24 new breast cancers were diagnosed. The median age of the 254 women in this study was 48, and the median BMI was 24. Eighty-six percent (218) of the women were Caucasian, 13% (32) were African-American, and 2 women were Native Americans. Sixty-eight women presented with cytologic-atypia in at least one breast; 16 of these women had cytologic-atypia in both breasts. Fourteen (21%) of the 68 women with baseline cytologic-atypia later developed cancer in a breast that had had baseline cytologic-atypia; none of the 68 women developed cancer in a breast without baseline cytologic-atypia. One hundred eighty-six women did not have cytologic-atypia at baseline; 10 (5%) of these women developed cancer. Ten women had a prophylactic mastectomy and were censored at the date of mastectomy. The median length of follow-up among the uncensored patients was 74 months. In the baseline atypic and non-atypic groups, the 48-month cumulative incidence of cancer development was 0.22 (95% confidence interval [CI], 0.13-0.34) and 0.04 (95% CI, 0.02-0.08), respectively. Atypia was significantly associated with cancer development both with and without controlling for covariates (p < 0.001). The covariate-adjusted hazard ratio (CV-AHR) for cytologic-atypia was 6.3 (95% CI, 1.7-24.2). The hazard ratios for the covariates were as follows: age (1.01; 95% CI, 0.89-1.16), BMI (1.02; 95% CI, 0.91-1.15), menopausal status (0.16; 95% CI, 0.01-2.60), Gail Risk (1.18; 95% CI, 1.03-1.35) and mammographic density for scattered (1.7; 95% CI, 0.27-10.9) and heterogenetic (1.10; 95% CI, 0.20-6.01) compared to the reference type of extreme. Conclusions: This is the first demonstration that in women from high-risk BRCA mutation-negative families, cytologic-atypia in RPFNA is associated with the subsequent development of breast cancer. Citation Format: Victoria L. Seewaldt, Bercedis Peterson, Gloria Broadwater. Cytological atypia predicts short-term breast cancer risk in women from high-risk families that lack a BRCA mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-191. doi:10.1158/1538-7445.AM2013-LB-191

Background: Currently we lack adequate prevention strategies for triple-negative breast cancer; progress in designing targeted strategies is limited by a lack of knowledge of the biology of cancer initiation. The origins of triple-negative breast cancer in premenopausal women are poorly understood. Importantly, we do not understand whether the molecular pathways that underlie the aggressive behavior of triple-negative breast cancer can be detected in premalignant breast lesions. Growing evidence indicates that early mammary carcinogenesis is regulated by integrated signaling networks and not by isolated genes. Interleukin-6 (IL6) plays a role in stem cell regeneration, promotes epithelial to mesenchymal transition (EMT), and high IL6 serum levels predict a poor outcome in premenopausal women with triple-negative breast cancer. Methods and Results: We used Reverse-Phase Protein Microarray (RPPM) profiling to test for activation of phosphoprotein signaling in premalignant Random Periareolar Fine Needle Aspiration (RPFNA) mammary epithelial cytology from 65 high-risk African American women. Unsupervised hierarchical clustering of RPPM proteomic analysis RPFNA aspirates identified three activated ER− signaling pathways including co-activation of Akt/mTOR/PI3K and IL6/pStat3/vimentin. Analysis of interstitial breast tissue demonstrated that high interstitial IL6/VEGF cytokine production predicted high epithelial expression of pStat3/vimentin. Based on these observations we are currently launching a prevention trial to target Akt/mTor using metformin 500 mg bid. Conclusion: This is the first demonstration that Akt/mTor and IL6/Stat3/vimentin signaling is activated in pre-cancerous mammary epithelial cells from high-risk premenopausal women. Currently we are testing whether trials are on-going to test whether metformin can inhibit activation of mammary epithelial Akt/mTor and perhaps IL6/Stat3/vimentin signaling. Citation Information: Cancer Prev Res 2011;4(10 Suppl):CN07-03.

Background: Currently we lack the ability to rapidly test new agents to prevent breast cancer. Targeted agents are in clinical testing for treatment of breast cancer but are not being adequately explored for prevention. This is because, without biomarkers to select women that are most likely to respond and rapidly evaluate response, Phase I/II testing of targeted agents for prevention will be too risky and expensive. Single biomarkers have been traditionally used to evaluate response to chemotherapeutic agents. However, single biomarkers may not be adequate to evaluate the complex phosphorylation network signaling events that occur during breast cancer initiation. Furthermore, emerging evidence suggests that resistance to targeted agents may occur through paradoxical activation of quiescent signaling pathway. As a result, it has become increasingly important to monitoring network signaling in women receiving targeted agents. Targeted agents that have low-toxicity profiles and have inhibitory activity against breast cancer cell proliferation and/or invasion may show promise as a prevention agent. However, before any given targeted agent could be tested for pilot prevention trials, we first need to identify 1) signaling pathway(s) that are affected by the drug in preclinical models of breast cancer, 2) short-term and long-term consequences of treatment, 3) potential biomarker(s) to closely track response. Methods and Results: We tested for protein network signaling in cytological specimens in women at high-risk for breast cancer. Our group used reverse-phase protein microarray (RPPM) platform to test for proteomic signatures of short-term risk to breast cancer. RPPM was developed to provide reproducible assessment of 50 phospho-proteins in 5,000 epithelial cells. Initial signature testing was performed in mammary cytology obtained from two groups of 50 high-risk women undergoing Random Periareolar Fine Needle Aspiration (RPFNA). Unsupervised heirachal clustering of RPPM proteomic analysis RPFNA aspirates from high-risk women identified three activated signaling pathways 1) Akt/mTOR/PI3K/cSrc, 2) EGRF/MEK/ERK, and 3) HER2/bcl-2. Conclusion: Here we demonstrate our ability to identify activated phospho-protein signaling pathways in limited RPFNA cytology. The power of this approach is that we can use a combination of RPPM and RPFNA to track response to any new targeted prevention agents. Importantly, we are able to validate our proteomic signatures in parallel nanobiosensor testing of live atypical RPFNA cytology from women in our high-risk cohort. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A108.

Over the last 50 years, the number of cancer related deaths has decreased by only 2%. One of the most promising approaches to reduce breast cancer mortality is to develop tools for early detection and early intervention of breast cancers. Normal mammary gland homeostasis requires the coordinated regulation of signaling networks; whereas, dysregulation of signaling networks occurs during breast cancer initiation. Reverse-phase protein microarray (RPPM) is a high-throughput proteomic tool, developed to test for dysregulation of protein signaling networks in human biopsy specimens. For example, ErbB2/HER2/neu, Erb3, EGFR, Akt, Erk1/2 receptor tyrosine kinases all play a role in cancer cell growth and survival, and developing targeted therapies against these pathways is a promising tailored approach to breast cancer treatment. To test which tyrosine kinases are activated in early mammary carcinogenesis, reverse phase protein microarray analysis (RPPM) was performed on 31 random periareolar fine needle aspirates (RPFNA) obtained from a cohort of asymptomatic high-risk women, testing 59 antibody endpoints related to cell growth and survival pathways, including ER, EGFR, Her2, Erb3, Akt and Erk1/2, to test for patterns of differential protein expression. RPFNA allows for serial sampling of breast cytology and subsequent histology from asymptomatic women at high risk for developing breast cancer and also provides the ability to monitor response to preventative treatments. Epithelial cell clusters from RPFNA samples were isolated using AutoPix automated laser capture microdissection. RPPM uses denatured lysate so antigen retrieval, which is a limitation for tissue arrays, is not a problem. In addition, each sample is printed in serial dilution, providing an internal standard, and RPPM does not require direct labeling of the sample, thus improving reproducibility, sensitivity, and robustness. An interrogation of phosphorylated versus non-phosphorylated state of proteins was performed using a Wilcoxon rank sum test. Spearman rank correlation coefficients were used to estimate the association between RPPM total- and phospho-protein expression as a function of cytology; a 2-sided p value < 0.05 was used to determine if a significant positive or negative association exists. Four distinct clusters of phospho-proteins were identified amongst the 31 RPFNA samples. Varying cell lines, including 15hTert, MDA231, T47D, were then treated with inhibitors based on the protein pathways identified in these clusters. Fluoxetine, a common antidepressant, was shown to affect cancer cell viability as well as the phosphorylation of Erk1/2 pathway in a concentration and time dependent manner. Diagnosing cancers based on proteomic signatures in addition to histopathology will allow for individualized selection of therapeutic combinations that can best target the entire disease-specific errant protein network of a patient. This proteomic signature has great potential for daily clinical practice considering the RPPM technology is commercially available through Theranostics Health and could be used every time a woman undergoes a routine biopsy. In addition, understanding a key protein pathway and how it is wired could have a profound effect on both functional biology and on the understanding of cancer mechanisms as a whole. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A15.

Background:Emerging data suggest that RNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue can produce reliable gene expression profiles. Archived material from two taxane monotherapy studies, CALGB 9342 (comparison of 3 doses of P) and 9840 (weekly vs. q3 week P)were profiled for gene expression and DNA copy number.Methods: A total of 238 patients had primary tumor blocks available from a combined sample size of 680. DNA and RNA was extracted from 1.5mm punch cores and the Ambion Recover-All kit™. quality was measured by spectrophotometric analysis, Bioanalyzer RNA Integrity Number (RIN), and housekeeping genes (RPL13A and Actin). A custom DASL™ array containing 779 genes in two-fold redundancy was designed with genes selected to represent the PAM50 intrinsic subtypes, the Oncotype Dx Score, the Netherlands prognostic signature and the genes most frequently found on recurrent breast cancer amplicons. Several methods for identifying outliers were evaluated, including principal components analysis, pairwise correlations as well as the reproducibility of the platform based on replicate samples.Results: Adequate RNA was obtained from 237/238 of these cases which ranged in age from 12-18 years. Of these, 215/237DASL arrays passed further quality control measures. Adequate DNA for CGH was obtained from 227/238 samples. Analysis of PAM50 intrinsic subtypes showed an excess of basal-like tumors (30%) in the primary tumors of this metastatic cohort compared with expected frequency in an early stage population. Luminal A tumors were less frequent than expected (20%). Patients with basal-like tumors did far worse than other tumor types for both PFS on P (p=0.015) and OS (p=2.7X10-6), which persisted in multivariable analysis (p=0.0047), however the interaction term was not significant (Wald p=0.26). While basal-like tumors had similar PFS and OS on both weekly and q3 week P, luminal A tumors appear to achieve more benefit from weekly P (p= 0.0041).The HER2-enriched expression subtype had a similar prognosis to Luminal A and B tumors. This appeared to be due to the presence of T, as the addition of this agent improved PFS (p=0.026) and OS (p=2.0X10-4). Of note, some centrally confirmed HER2 FISH amplified tumors were classified into luminal A, B, and basal-like subtypes. These tumors have similar prognoses to the overall group, for example the basal-like and HER2 tumors had a poor prognosis despite T (p=0.00086). This suggests that HER2 FISH positive tumors may behave based on the underlying tumor subtype. Sawtooth genomes (45% vs 15%) were more frequent than predicted by an early stage tumor dataset as were simplex genomes (3% vs 24%). HER2 by FISH and CGH were highly concordant suggesting data on gene amplification from this platform is robust.Conclusions: Gene expression and copy number profiling of FFPE material from archived tumor blocks (>10 years) produces quality data for biomarker discovery in CALGB clinical trial datasets. These tools allow discovery of novel patterns of gene expression and genomic aberrations that are associated with differential response to P and T. Further studies using these platforms should be performed. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4032.

Abstract #6046 Purpose: The recent ACO/CAP guidelines revised the cut-point for HER2 expression by immunohistochemistry (IHC) from 10% up to 30%. Recently we showed that heterogeneity of expression is a function of the assessed biomarker and, for HER2, >80% area of expression was most prognostic and predictive. Here we test this hypothesis in CALGB 9840, a prospective cooperative group clinical trial of Paclitaxel (P) and Trastuzumab (T). Experimental Design: Tissue from 274 cases selected from cases with available slides from the TH arm were first scored by pathologists as 0-3+, but then again scored to assess percentage of tissue staining with prospectively chosen cut points of >10%, >30% or >80%. Response was assessed by modified RECIST criteria, including complete and partial response. Stable disease and progressive disease were considered non-response. Results: Amongst the 176 cases treated with P+T there were 90 (52%) responses (PR+CR), 3 cases were unevaluable for response. In the subset of cases treated with T, there was inconsistent differences in the >10% vs the >30% categories but the >80% category was uniformly statistically significantly predictive of response. Receiver Operator Characteristic (ROC) curves constructed to assess prediction of response showed areas under the curves (AUC) of 0.59 for IHC and 0.60 for FISH suggesting both are failed tests for defining an optimal cut-point. However, selecting only the cases scored as 3+ shows an AUC of 0.72 and the optimal cut-point is for the test is at 95% positive 3+ HER2 staining. This modification of the cut-point would have denied T to 6 of the 49 patients who responded to therapy in the 3+ group. However, there were also 41 (of 86) patients in the 0,1+ and 2+ groups that responded to therapy, raising the possibility that the response in those 6 patients may have been unrelated to T therapy. Conclusions: When using clinical outcome to assess the value of the current tests for prediction of response to T, ROC curves suggest that both the old and new HER2 tests fail at defining an optimal cut-point. However, using only strongly positive cases (3+), an acceptable test can be achieved which is maximized at 95% area of expression. This cut-point, while statistically rigorous, is limited by the fact that the patients were treated with both P and T. This result suggests a similar analysis is warranted on a larger cohort of T treated patients, perhaps in the adjuvant or neoadjuvant setting. The availability of other options for HER2 amplified cases (ie lapatinib) and the increased use of T in the adjuvant setting warrant optimization of tests for the best possible patient selection. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6046.