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Background The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. Methods We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. Results Intralaboratory (CCC 0.96–0.99) and interlaboratory (CCC 0.98–0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83–0.93) and between laboratories (Kappa 0.87–0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). Conclusion Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Purpose The Herceptin Adjuvant study is an international multicenter randomized trial that compared 1 or 2 years of trastuzumab given every 3 weeks with observation in women with human epidermal growth factor 2–positive (HER2+) breast cancer after chemotherapy. Identification of biomarkers predictive of a benefit from trastuzumab will minimize overtreatment and lower health care costs. Methods To identify possible single-gene biomarkers, an exploratory analysis of 3,669 gene probes not expected to be expressed in normal breast tissue was conducted. Disease-free survival (DFS) was used as the end point in a Cox regression model, with the interaction term between C8A mRNA and treatment as a categorical variable split on the cohort mean. Results A significant interaction between C8A mRNA and treatment was detected ( P < .001), indicating a predictive response to trastuzumab treatment. For the C8A-low subgroup (mRNA expression lower than the cohort mean), no significant treatment benefit was observed ( P = .73). In the C8A-high subgroup, patients receiving trastuzumab experienced a lower hazard of a DFS event by approximately 75% compared with those in the observation arm (hazard ratio [HR], 0.25; P < .001). A significant prognostic effect of C8A mRNA also was seen ( P < .001) in the observation arm, where the C8A-high group hazard of a DFS event was three times the respective hazard of the C8A-low group (HR, 3.27; P < .001). C8A mRNA is highly prognostic in the Hungarian Academy of Science HER2+ gastric cancer cohort (HR, 1.72; P < .001). Conclusion C8A as a single-gene biomarker prognostic of DFS and predictive of a benefit from trastuzumab has the potential to improve the standard of care in HER2+ breast cancer if validated by additional studies. Understanding the advantage of overexpression of C8A related to the innate immune response can give insight into the mechanisms that drive cancer.

Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Background: RNA-Seq from total RNA in FFPE tissue can be more challenging due to limited capture of partially degraded RNA. Exome-capture based RNA-Seq may circumvent such problems and allow reproducible complete molecular characterization of low-quality RNA from small clinical samples. Methods: HER2 negative patients within the GeparQuinto trial were treated with neoadjuvant anthracycline-taxane-based chemotherapy +/- bevacizumab. Patients with bevacizumab therapy had a significantly higher pCR rate, especially within the triple negative subgroup. We performed exome-capture RNA-Seq on 5µm FFPE sections from pre-therapeutic cores of 400 HER2 negative samples from this trial. In a prospectively planned, blinded study we correlated molecular subtypes and metagenes for proliferation, stroma, MHC2, and VEGFA with clinical and histopathological data. Molecular subtypes were defined using the AIMS methods. Metagenes were calculated as mean values corresponding to previously described gene clusters after platform transfer (Rody et al. 2011 PMID 21978456, Hu et al. 2009 PMID 19291283) and then z-transformed. Results: 296 samples with RNA-Seq data were classified as either of high (n=226) or of limited quality (n=70). For 22 samples RNA yield was insufficient and 82 did not pass initial QC. 121 (41%), 63 (21%), 34 (11.5%), 46 (15.5%), and 32 (11%) samples were defined as basal-like, HER2-enriched, luminal A, luminal B, and normal-like, respectively. Subtyping was robust with regard to gene filtering, normalization, and sample quality. ER and PR status from local IHC strongly correlated with gene expression (overall correctness 84% and 80% for ER, and 85% and 74% for PR, in samples with high and limited quality, respectively) and luminal subtypes (95% ER positive). Proliferation metagene correlated with histological grade (median -0.73, -0.39, and 0.53 in G1, G2, and G3, respectively; P Conclusions: Exome-capture RNA-Seq allows robust genomic characterization of clinical samples with limited FFPE material from core biopsies, and molecular subtypes and immune metagenes are predictive for pCR. The VEGFA metagene is a specific predictor for response to neoadjuvant bevacizumab treatment. Citation Format: Karn T, Meissner T, Weber K, Sinn B, Denkert C, Budczies J, Nekljudova V, Fasching PA, Holtrich U, Schem C, Solbach C, Hartmann A, Röcken C, Untch M, Young BM, Willis S, Leyland-Jones B, von Minckwitz G, Loibl S. Blinded molecular subtyping analysis from RNA-Seq of FFPE samples in the GeparQuinto trial reveals predictive value of VEGFA metagene for bevacizumab treatment [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 P2-09-02.

Introduction Next generation sequencing has facilitated the understanding of pathogenesis and molecular heterogeneity of breast cancer (BC) as well as accelerated the path towards precision medicine. DNA sequencing (DNA-Seq) based assays for the detection of mutations and alterations in solid and hematologic cancers are finding their way into clinical practice and are readily available as clinical products. RNA sequencing (RNA-Seq), so far being vastly applied in the research context, promises to expand the diagnostic, prognostic and therapeutic use of this technology in cancer. Beyond mutational status, RNA-Seq enables the detection of fusions, quantification of gene expression level, detection of differentially expressed genes, molecular based subtyping, and risk-stratification. In this study we analyzed RNA-Seq and copy number data from BC patients that had undergone DNA-Seq based diagnostics through commercial providers with the goal to detect additional actionable targets. Materials and Methods We included 18 BC patients (5/18 triple negative) that had previously undergone DNA-based targeted (321 genes) sequencing. RNA-Seq to a minimum of 75M reads (75pb) was performed using 100 ng of total RNA on the Illumina NextSeq 500 platform. STAR was used for alignment (hg19) and gene expression quantification (RefSeq). Fusions were detected using STAR-Fusion. DESeq2 was utilized to identify patient specific differentially expressed genes by analyzing samples individually against a set of 13 controls from healthy breast tissue generated in-house. Copy number variations (CNVs) were detected using the Nanostring CNV Cancer panel (89 genes) on the Nanostring nCounter platform. Differentially upregulated or amplified genes were queried against DGIdb and Gene Drug Knowledge database for suitable drug matches, limiting the queries to clinically actionable antineoplastic drugs. Results Analyzing the cohort of 18 BC patients, we detected on average 26 BC relevant genes (526 total, log2 FC > 2) to be upregulated per patient. Querying the upregulated genes against DGIdb, we found a total of 18 genes that had drug matches and fulfilled the criteria of being actionable antineoplastic drugs, with 17/18 samples having a minimum of two gene targets (avg: 4). Most frequent upregulated genes were TOP2A (83%), AURKA (61%), AURKB (56%), RET (39%)and FGFR3 (28%). In the case of CNVs, 12/18 patients showed at least one gene target with clinically actionable drugs associated. This was observed across 12 gene targets that were amplified (avg: 3) and 4 gene targets that underwent deletions (avg: 1). Most frequent CNVs included MYC (14%) and CCND1 (12%). 4/7 patients having an AURKA overexpression also showed an AURKA amplification on the CNV assay. 10/18 patients had fusions events, with an average of three fusions per patient, including GAB2-WNT11, PAK1-TENM4 and FGFR2-CEP55 fusions. Conclusions We show that RNA-Seq and copy number assays provide additional clinical value by detecting suitable drug targets beyond traditional DNA-based approaches. We are conducting further analysis on how these additionally derived drug targets could improve the current treatment schedule of those patients. Citation Format: Meissner T, Amallraja A, Mark A, Andrews A, Connolly C, Young B, De P, Williams C, Leyland-Jones B. The value of RNA-Seq for the detection of clinically actionable targets in breast cancer - A small cohort analysis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-22.

Purpose: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug. Experimental Design: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays. Results: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21–2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00–4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41–2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28–4.51; P = 0.006) with a significant interaction with bevacizumab treatment (P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected (P = 0.025). Conclusions: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition.

Background: The prognostic significance of increased levels of CD8+ tumor infiltrating lymphocytes(TILs) in ER- breast cancer has been described. We sought to identify possible immune-related biomarkers for predicting benefit from letrozol(LET) or tamoxifen(TAM) for recurrence in ER+ breast cancer. Patient and Methods: We used Illumina DASL Assay to measure gene expression in FFPE primary breast cancers from a subset of postmenopausal patients enrolled in the BIG 1-98 randomized phase 3 trial comparing 5 years LET (n=344) vs TAM (n=381) as adjuvant endocrine therapy. Gene sets (n=1910) that represent cell states and perturbations within the immune system from the Human Immunology Project Consortium were used in an exploratory analysis to identify possible predictive signatures. Results: We identified five distinct gene signatures from previously reported laboratory experiments associated with immune cell differentiation that are highly predictive of benefit (reduced breast cancer recurrence risk) of LET over TAM, each with gene signature p-values Conclusion: The role of selective estrogen receptor modulators on immune response has been well described, where TAM has been shown to prevent differentiation and activation of dendritic cells (Naibandian 2005). Similarly, it has been shown that MET inhibitors negatively regulate neutrophils suggesting that anti-MET drugs in cancer could impact immune response (Finisquerra 2015). These findings suggest that if TAM is a negative regulator of immune response why in the ATAC clinical trial, the combination therapy of anastrozole plus TAM were not significantly different from TAM alone were anastrozole was superior. With the increasing importance of understanding the role of immune response on outcome and the use of combination therapies the assessment of TILs in the neoadjuvant setting will be critical for guiding therapy. Citation Format: Willis S, Gray KP, Regan MM, Rae JM, Kammler R, Young B, Ditzel HJ, Lyng MB, Colleoni M, Viale G, Leyland-Jones B. Immune related gene expression signatures predict benefit of letrozole over tamoxifen in BIG 1-98. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-36.

The precision medicine (PM) revolution is well underway, and next-generation sequencing (NGS) is helping take cancer treatment to new levels. Finding actionable targets in real time is now a reality, and data from several clinical trials based on identified molecular alterations can be assessed and can help address the question of whether personalized treatment based on genomics produces superior survival outcomes compared with unselected treatment. Targeted treatment-based clinical trials have shown benefit in molecular subgroups of patients. Of further interest, it appears that genomics and immunotherapy are coupled to each other, since the immune system recognizes neo-antigens produced by the mutanome. Hence, some of the most important markers predicting response to immunotherapy are genomic markers, PDL1 amplification (for PD-1/PD-L1 checkpoint inhibitors), and tumor mutational burden. However, a number of challenges remain to be addressed if the use of molecular profiling-guided therapy in PM-designed clinical trials is to become the standard on which cancer treatment is based. In this chapter, we explore what it will take for PM trials that use molecular profiling as part of the eligibility criteria to be successful and the remaining hurdles that need to be overcome.

// Michael Forster 1 , Adam Mark 1, 2 , Friederike Egberts 3 , Elisa Rosati 1 , Elke Rodriguez 3 , Martin Stanulla 4 , Dirk Bauerschlag 5 , Christian Schem 6 , Nicolai Maass 5 , Anu Amallraja 7 , Karla K. Murphy 8 , Bruce R. Prouse 8 , Raed A. Sulaiman 8 , Brandon M. Young 7 , Micaela Mathiak 9 , Georg Hemmrich-Stanisak 1 , David Ellinghaus 1 , Stephan Weidinger 3 , Philip Rosenstiel 1 , Norbert Arnold 1, 5 , Brian Leyland-Jones 7 , Casey B. Williams 7 , Andre Franke 1, * and Tobias Meisner 7, * 1 Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany 2 Current address: Center for Computational Biology and Bioinformatics, Department of Medicine, University of California, San Diego, CA, USA 3 Department of Dermatology, Schleswig-Holstein University Hospital, Kiel, Germany 4 Department of Pediatric Haematology and Oncology, Hannover Medical School, Hannover, Germany 5 Department of Gynaecology and Obstetrics, Schleswig-Holstein University Hospital, Kiel, Germany 6 Mammazentrum Hamburg, Hamburg, Germany 7 Department of Molecular and Experimental Medicine, Avera Cancer Institute, Sioux Falls, SD, USA 8 Pathology, Avera McKennan, Sioux Falls, SD, USA 9 Department of Pathology, Schleswig-Holstein University Hospital, Kiel, Germany * These authors share last authorship Correspondence to: Michael Forster, email: m.forster@ikmb.uni-kiel.de Keywords: breast cancer sequencing; normal RNA expression; drug selection; formalin fixed paraffin embedded; fresh frozen Received: March 07, 2018 Accepted: August 04, 2018 Published: August 17, 2018 ABSTRACT Background: While standard RNA expression tests stratify patients into risk groups, RNA-Seq can guide personalized drug selection based on expressed mutations, fusion genes, and differential expression (DE) between tumor and normal tissue. However, patient-matched normal tissue may be unavailable. Additionally, biological variability in normal tissue and technological biases may confound results. Therefore, we present normal expression reference data for two sequencing methods that are suitable for breast biopsies. Results: We identified breast cancer related and drug related genes that are expressed uniformly across our normal samples. Large subsets of these genes are identical for formalin fixed paraffin embedded samples and fresh frozen samples. Adipocyte signatures were detected in frozen compared to formalin samples, prepared by surgeons and pathologists, respectively. Gene expression confounded by adipocytes was identified using fat tissue samples. Finally, immune repertoire statistics were obtained for healthy breast, tumor and fat tissues. Conclusions: Our reference data can be used with patient tumor samples that are asservated and sequenced with a matching aforementioned method. Coefficients of variation are given for normal gene expression. Thus, potential drug selection can be based on confidently overexpressed genes and immune repertoire statistics. Materials and Methods: Normal expression from formalin and frozen healthy breast tissue samples using Roche Kapa RiboErase (total RNA) (19 formalin, 9 frozen) and Illumina TruSeq RNA Access (targeted RNA-Seq, aka TruSeq RNA Exome) (11 formalin, 1 frozen), and fat tissue (6 frozen Access). Tumor DE using 10 formalin total RNA tumor samples and 1 frozen targeted RNA tumor sample.

Background: The optimal treatment strategy for patients with metastatic breast cancer (MBC) is currently unknown. Resistance to standard therapies like anthracyclines and taxanes limit the number of treatment options in many patients to a small number of non-cross-resistant regimens. We hypothesized that genomic and proteomic profiling of clinical MBC samples would identify genomic alterations that are linked to targeted therapies, and that this could facilitate a personalized approach to therapy for our patients. Methods: We retrospectively analyzed 31 consecutive metastatic breast cancer patients that had genomic and/or proteomic studies sent over a 4 month period from February to May 2014. All patients were seen in our Genomic Oncology Clinic and subsequently underwent a rebiopsy of a metastatic site. Formalin-fixed, paraffin-embedded (FFPE) tissue was sent to either Foundation Medicine for genomic profiling, Theranostics for reverse-phase protein microarray, or both. Standard immunohistochemistry for ER, PR, and HER2 was also performed on all patients. Results: Genomic or proteomic alterations were identified in all 31 patients. All patients harbored at least one actionable target and a treatment recommendation for a currently available FDA approved drug or drug combination was able to be suggested in all but one patient. The most commonly observed genomic alterations were within PIK3CA (26%), TP53 (23%), CCND1 (19%), and MYC (16%). Over 30 distinct genomic alterations were identified. Proteomic results were available from 16 patients. Activation of the AKT/mTOR pathway was evident in a majority of patients. A change in HER2 status was also found in 26% of patients. 16% of cases underwent a negative to positive conversion in HER2 status while 10% of cases underwent a positive to negative conversion. It is notable that all 5 patients that underwent a negative to positive conversion in HER2 status had biopsies taken from metastatic disease in the liver. Conclusions: All patients in this retrospective study harbored genomic or proteomic alterations that are associated with targeted therapies. Treatment recommendations were suggested in all but one patient and a majority of patients are receiving the suggested therapy. Our data demonstrate that routine genomic and proteomic analysis in a clinical setting makes a significant positive impact for patients. Citation Format: Casey B Williams, Pradip De, Nandini Dey, Kirstin A Williams, Jessica Klein, Michelle King, Misty Small, Brigitte Cyr, Scooter Willis, Yuliang Sun, Jennifer Carlson, Brandon M Young, Amy Krie, Brian Leyland-Jones. New treatment options for metastatic breast cancer revealed by reverse-phase protein microarray and genomic profiling [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 P2-05-14.

HERA is an international multi-center randomized trial comparing 1 or 2 years trastuzumab, given every 3 weeks, with observation in women with HER2+ breast cancer after standard neoadjuvant or adjuvant chemotherapy. From December 2001 to June 2005, 5102 patients were randomized. Comparing 1 year trastuzumab with observation at 8 years of follow-up, statistically significant differences in disease-free survival (DFS) and overall survival, despite crossover to trastuzumab of observation arm patients, were found. No benefit in DFS of 2 years compared to 1 year trastuzumab was found[Goldhirsch 2013]. To determine possible predictive single-gene biomarkers, an exploratory ITT analysis, with DFS as the primary endpoint, was conducted using mRNA expression data from 610 TransHERA FFPE samplesprofiled on Illumina Whole-Genome DASL cubic spline normalization was applied to the data. Outcome was obtained from the HERA database with 8 years median follow-up and clinical cut-off date April 12, 2012. Characteristics were well balanced between treatment groups. The exploratory analysis of 20,464 genes using DFS as the endpoint identified C8A as a possible biomarker that is prognostic and predictive of response to treatment. Cox regression was used to model DFS, with the interaction term between treatment and C8A as a continuous and a categorical variable split on the cohort mean. The observation arm consists of 199 samples with 66 events and the trastuzumab arm(1&2-year combined) of 411 samples with 108 events. A statistically significant interaction between C8A mRNA and treatment was detected (p C8A is a member of the membrane attack complex and is part of the innate immune system. C8A inserts into the membrane of the target cell and binds with multiple copies of the pore-forming C9 leading to cell lysis. From the GeneAtlas, C8A is highly expressed in liver tissue suggesting an advantage for tumors with high expression of C8A and innate immune response. The Cancer Cell Line Encyclopedia indicates a wide range of C8A mRNA expression. C8A as a single gene biomarker that is prognostic of DFS and predictive of benefit from trastuzumab has the potential to improve the standard of care in HER2+ breast cancer. Understanding the advantage of over expression of C8A related to the innate immune response can give insight into the mechanisms that drive cancer. We note with caution that this finding is the result of an exploratory analysis and is being pursued in additional trastuzumab cohorts for further validation. Citation Format: Scooter Willis, Varvara Polydoropoulou, Zoi Tsourti, Urania Dafni, Brandon Young, Bradley Long, Pradip De, Nandini Dey, Casey Williams, Mitch Dowsett, Brian Leyland-Jones. Exploratory analysis of single gene predictive biomarkers in TransHERA DASL cohort reveals that C8A mRNA expression is prognostic of outcome and predictive of benefit of trastuzumab [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 P3-06-02.

Background In prior published work, the Worldwide Innovative Network (WIN) Consortium described the Simplified Interventional Mapping System (SIMS) algorithm, based on the hypothesis that both genomic and transcriptomic data are important for matching to a tri-therapy regimen. To account for variation in transcription levels between different tissues, gene by gene tumor RNA levels are normalized against RNA levels in analogous biopsied normal tissue. SPRING is the first trial to assess a SIMS-based tri-therapy regimen in advanced NSCLC. Methods Patients with metastatic NSCLC (no EGFR or ALK alterations; no ROS1 alteration if tested; PD-L1 unrestricted; ≤2 prior therapy lines) were treated with avelumab, palbociclib, and axitinib (3 + 3 dose escalation design). After consent, biopsies of tumor and normal endobronchial mucosa were obtained on all patients for analysis on a central genomics/transcriptomics platform and retrospective SIMS algorithm validation. A safety monitoring committee reviews study conduct at least weekly. Results Twelve patients have been treated (3 at dose level 1; 6, dose level 2; 3, dose level 3). Three dose-limiting toxicities (DLTs) at least possibly drug related occurred: 1 DLT at dose level 2 (Grade 3 (G3) infusion reaction); 2 DLTs at dose level 3 (G3 hand/foot syndrome and a G5 respiratory failure). The partial response (PR) rate was 44% (4/9 patients who have reached restaging including 2/3 patients at dose level 1; two PRs are in patients who failed prior pembrolizumab). The maximum tolerated dose was avelumab 10 mg/kg IV q2weeks, axitinib 5 mg po bid, palbociclib 75 mg po daily (7 days off; 21 days on) (dose level 2). Conclusions The tri-therapy combination of avelumab, axitinib, and palbociclib is tolerable with early evidence of activity including in NSCLC patients who failed a prior checkpoint inhibitor. Expansion cohorts are being added to further explore safety of a recommended phase II dose. Transcriptomic and genomic correlates of response are being assessed. Clinical trial identification NCT03386929; First posted: December 29, 2017. Legal entity responsible for the study Worldwide Innovative Network (WIN) Association - WIN Consortium. Funding ARC Foundation for Cancer Research and Pfizer. Disclosure B. Solomon: Travel / Accommodation / Expenses: Elekta. A. Callejo: Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Boerhinger; Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: MSD; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: BMS; Speaker Bureau / Expert testimony: KYOWA KIRIN ; Travel / Accommodation / Expenses: Pfizer; Travel / Accommodation / Expenses: Celgene. J. Bar: Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: Genentech; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: AbbVie; Advisory / Consultancy, Research grant / Funding (institution): MSD; Advisory / Consultancy: Takeda; Advisory / Consultancy: Vascular Biogenics; Research grant / Funding (institution): MedImmune. G. Berchem: Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Celgene. L. Bazhenova: Advisory / Consultancy: Takeda; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: AbbVie; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Loxo Oncology; Shareholder / Stockholder / Stock options: Epic Sciences; Research grant / Funding (institution): Beyondspring Pharma. P. Saintigny: Honoraria (self), Research grant / Funding (self), Travel / Accommodation / Expenses: BMS; Honoraria (self), Research grant / Funding (self), Travel / Accommodation / Expenses: Roche; Honoraria (self), Research grant / Funding (self), Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Research grant / Funding (self): HTG Diagnostics. E. Raymond: Full / Part-time employment: Genoscience; Full / Part-time employment: Scor; Advisory / Consultancy: Pharmaengine; Travel / Accommodation / Expenses: Merck. N. Girard: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Hoffmann-La Roche; Advisory / Consultancy: Lilly; Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy, Travel / Accommodation / Expenses: Bristol-Myers Squibb; Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy: Takeda; Advisory / Consultancy: GSK; Advisory / Consultancy: AbbVie. R. Sulaiman: Full / Part-time employment: Physicians Laboratory . E. Rubin: Travel / Accommodation / Expenses: Roche. V. Lazar: Licensing / Royalties: Worldwide Innovative Network Association. E. Felip: Advisory / Consultancy, Speaker Bureau / Expert testimony: ABBVIE; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy: Blue Print Medicines; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Advisory / Consultancy: Celgene; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy: Guardant Health; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck MGaA; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck Sharp & Domme; Advisory / Consultancy, Speaker Bureau / Expert testimony: NOVARTIS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Takeda; Advisory / Consultancy: JANSSEN; Research grant / Funding (institution): Fundation Merck Salud; Research Grant / Funding (Institution): Grant For Oncology Innovation Emd Serono. A. Onn: Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca. B. Leyland-Jones: Speaker Bureau / Expert testimony: Bayer; Speaker Bureau / Expert testimony: Exelixis; Speaker Bureau / Expert testimony: Genentech; Speaker Bureau / Expert testimony: Puma; Travel / Accommodation / Expenses: NFCR; Travel / Accommodation / Expenses: AkesoGen. R. Kurzrock: Shareholder / Stockholder / Stock options, Co-founder: CureMatch; Shareholder / Stockholder / Stock options: IDbyDNA; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Soluventis; Advisory / Consultancy: Gaido; Advisory / Consultancy: Loxo Oncology; Advisory / Consultancy: Xbiotech; Advisory / Consultancy: Acurate Therapeutics; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy: NeoMed; Research grant / Funding (institution): uardant Health; Research grant / Funding (institution): Grifols; Research grant / Funding (institution): Konica Minolta; Research grant / Funding (institution): OmniSeq; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Merck Serono; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Sequenom; Research grant / Funding (institution): Foundation Medicine. All other authors have declared no conflicts of interest.

Background: NSCLC is the leading cause of cancer death. Recently, much progress has been made to identify and target specific oncogenic drivers such as EGFR mutations and ALK translocations. Unfortunately, the majority of NSCLCs do not have a single driver, but instead harbor complex systems of molecular alterations. The advent of immunooncology has opened newer avenues for treating these patients, but still, many tumors are resistant. Therefore, selection of precise therapies targeting the appropriate pathways in these tumors is an important area of research. In previously published work, the Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) algorithm to match patients to a targeted drug triplet. The SIMS algorithm is based on the hypothesis that the use of both genomic as well as transcriptomic data is important in selecting an ideal tri-therapy regimen. Since the transcriptome varies widely between different types of normal tissue, the relative level of transcription of any given gene in a tumor must be normalized against the expected level of transcription of that gene in the analogous normal tissue. The SPRING trial is the first trial assessing a rational, SIMS-based tri-therapy regimen in advanced NSCLC. The drugs utilized for SPRING–avelumab (anti-PDL1), axitinib (VEGFR inhibitor) and palbociclib (CDK4/6 inhibitor)–were chosen because the SIMS genomic/transcriptomic analysis demonstrated that ~10% of NSCLCs have a combination of these targets. Methods: Patients with locally advanced or metastatic NSCLC without EGFR or ALK alterations are being enrolled and treated with avelumab, palbociclib, and axitinib after informed consent. During phase I, any prior treatment is allowed. SPRING will assess the safety of the tri-therapy combination and determine the recommended phase II dose (RP2D) (3+3 dose escalation). Once the RP2D is found, the study will proceed directly to the phase II portion, which will permit only previously untreated patients in the metastatic setting. A biopsy of both tumor and normal tissue (by endobronchial mucosal biopsy) is obtained on all patients, both of which are analysed in a central genomics and transcriptomics platform. The SIMS algorithm is being validated retrospectively by determining the degree of genomic and transcriptomic matching and correlating with outcome. Results: The phase I study is open in USA, Israel, Spain and Luxembourg and has enrolled 12 patients to date and has progressed to cohort 3. The phase II portion is expected to open later in 2019. This research is supported by the EMD Serono/Pfizer alliance and ARC Foundation for cancer research. Citation Format: Benjamin Solomon, Enriqueta Felip, Jair Bar, Guy Berchem, Lyudmila Bazhenova, Pierre Saintigny, Nicolas Girard, Raed Sulaiman, Catherine Bresson, Fanny Wunder, J Jack Lee, Jacques Raynaud, Eitan Rubin, Brandon Young, Vladimir Lazar, Amir Onn, Brian Leyland Jones, Razelle Kurzrock. Survival Prolongation by Rationale INnovative Genomics (SPRING): An international WIN Consortium Phase I/II proof-of-concept study to explore the safety and efficacy of a tri-therapy approach using avelumab, palbociclib and axitinib in advanced/metastatic non-small cell lung cancer (NSCLC) with integrated genomic and transcriptomic correlates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT223.

A number of studies suggest that response to antihuman epidermal growth factor receptor-2 (currently known as ERBB2, butreferred to asHER2 in this study) agents differs by estrogen receptor (ER) level status. The clinical relevance of this is unknown.To determine the magnitude of trastuzumab benefit according to quantitative levels of ER and HER2 in the HERceptin Adjuvant (HERA) trial.The HERA trial was an international, multicenter, randomized trial that included 5099 patients with early-stage HER2-positive breast cancer, randomized between 2001 and 2005 to receive either no trastuzumab or trastuzumab, after adjuvant chemotherapy. This is a secondary analysis of the HERA study. Local ER immunohistochemical (IHC) analyses, HER2 fluorescence in situ hybridization (FISH) ratio, and copy number results were available for 3037 patients (59.6%) randomized to observation and trastuzumab (1 or 2 years) (cohort 1). Transcript levels of ESR1 and HER2 genes were available for 615 patients (12.1%) (cohort 2).Patients were randomized to receive either no trastuzumab or 1 year vs 2 years of trastuzumab. Endocrine therapy was given to patients with hormone receptor-positive disease as per local guidelines.Disease-free survival (DFS) and overall survival (OS) were the primary and secondary end points in the intent-to-treat population (ITT). Analyses adjusting for crossover (censored and inverse probability weighted [IPW]) were also performed. Interactions among treatment, ER status, and HER2 amplification using predefined cutoffs were assessed in Cox proportional hazards regression models.Median follow-up time was 8 years. Levels of FISH and HER2 copy numbers were significantly higher in ER-negative patients (P .001). In cohort 1, for DFS and OS, a significant treatment effect was found for all ER, IHC, and FISH levels, except for the ER-positive/HER2 low FISH ratio (≥2 to5) group (DFS: 3-way ITT Pvalue for interaction = .07; censored = .02; IPW = .03; OS ITT Pvalue for interaction = .007; censored = .04; IPW = .03). In cohort 2, consistent with cohort 1, a significant predictive effect of the ESR1 gene for both end points was also observed (DFS Pvalue for interaction = .06; OS = .02), indicating that breast cancers with higher ESR1 levels also derive less benefit from trastuzumab.Patients with HER2-positive breast cancers that are ER-positive by IHC analyses with low FISH ratio (≥2 to5), or with higher ESR1 levels derive significantly less benefit from adjuvant trastuzumab after chemotherapy. These data may explain heterogeneity in response to anti-HER2 agents in HER2-positive, ER-positive breast cancers as some may be more luminal-like than HER2 driven.clinicaltrials.gov Identifier: NCT00045032.

Background: Delivery of premature infants outside tertiary care centers is not always preventable. The aim of this study was to compare rates of survival and common morbidities in extremely premature babies transported to a level III facility versus those born at the level III center. Methods: Retrospective chart review was performed on all neonates born at ≤ 28 weeks of gestation with birthweight ≤1500 g who were admitted to the Newborn Intensive Care Unit at Kapi‘olani Medical Center for Women and Children (KMCWC) between 1 January 2000 and 31 December 2005. Infants were divided into two groups, those born at KMCWC (Inborn) and those born at level I institutions and subsequently transported (Transport) to KMCWC. Results: A total of 394 neonates met the study criteria; 349 were inborn while 45 were transported. Survival rates were identical for both groups. However, the Transport group survivors displayed a significantly longer mean length of stay and higher rate of severe retinopathy of prematurity than those in the Inborn group (P≤ 0.01). Conclusion: Identical rates of survival in both groups suggest that community medical professionals are providing satisfactory care to stabilize critical neonates without reducing their chances of survival. However, increased length of stay and higher rate of retinopathy of prematurity in the Transport group suggest that differences in medical management during the first few hours of life may adversely affect outcomes.

Elevation of intraocular pressure (IOP) following injection of intravitreal triamcinolone acetonide (IVTA) is an important clinical problem. The etiology of the steroid response is poorly understood, although a genetic determinant has long been suspected. We performed a pharmacogenomic association study with glucocorticoid receptor polymorphisms.Fifty-two patients (56 eyes) who underwent treatment with IVTA for various retinal diseases were genotyped for six well-studied glucocorticoid receptor polymorphisms (ER22/23EK, N363S, BclI, N766N, and single nucleotide polymorphisms (SNPs) within introns 3 and 4).Three polymorphisms (ER22/23EK, N363S, and the intron 3 SNP) were essentially nonpolymorphic within this population sample and excluded from further analysis. The remaining three polymorphisms (BclI, N766N, and within intron 4) passed the Hardy-Weinberg Equilibrium test, indicating good genotyping quality and normal population distribution of allelic frequency. No statistically significant correlations were found between these three polymorphisms and magnitude of IOP elevation following IVTA, using single point association and haplotype analyses.In this small, pilot study, we found no statistically significant relationship between glucocorticoid receptor polymorphisms and IOP elevation following IVTA. The precise etiology of the steroid response remains obscure. To our knowledge, this is the first published pharmacogenomic study of this common clinical entity.

Estrogen receptor 1 (ESR1) and ESR2 gene polymorphisms have been associated with endocrine-mediated physiological mechanisms, and inconsistently with breast cancer risk and outcomes, bone mineral density changes, and hot flushes/night sweats. DNA was isolated and genotyped for six ESR1 and two ESR2 single-nucleotide polymorphisms (SNPs) from tumor specimens from 3691 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Associations with recurrence and adverse events (AEs) were assessed using Cox proportional hazards models. 3401 samples were successfully genotyped for five SNPs. ESR1 rs9340799(XbaI) (T>C) variants CC or TC were associated with reduced breast cancer risk (HR = 0.82,95% CI = 0.67-1.0), and ESR1 rs2077647 (T>C) variants CC or TC was associated with reduced distant recurrence risk (HR = 0.69, 95% CI = 0.53-0.90), both regardless of the treatments. No differential treatment effects (letrozole vs. tamoxifen) were observed for the association of outcome with any of the SNPs. Letrozole-treated patients with rs2077647 (T>C) variants CC and TC had a reduced risk of bone AE (HR = 0.75, 95% CI = 0.58-0.98, P interaction = 0.08), whereas patients with rs4986938 (G>A) genotype variants AA and AG had an increased risk of bone AE (HR = 1.37, 95% CI = 1.01-1.84, P interaction = 0.07). We observed that (1) rare ESR1 homozygous polymorphisms were associated with lower recurrence, and (2) ESR1 and ESR2 SNPs were associated with bone AEs in letrozole-treated patients. Genes that are involved in estrogen signaling and synthesis have the potential to affect both breast cancer recurrence and side effects, suggesting that individual treatment strategies can incorporate not only oncogenic drivers but also SNPs related to estrogen activity.

To determine whether CYP19A1 polymorphisms are associated with abnormal activity of aromatase and with musculoskeletal and bone side effects of aromatase inhibitors. DNA was isolated from tumor specimens of 4861 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Tumors were genotyped for six CYP19A1 polymorphisms using PCR-based methods. Associations with breast cancer-free interval (BCFI), distant recurrence-free interval (DRFI), musculoskeletal and bone adverse events (AEs) were assessed using Cox proportional hazards models. All statistical tests were two-sided. No association between the CYP19A1 genotypes and BCFI or DRFI was observed overall. A reduced risk of a breast cancer event for tamoxifen-treated patients with rs700518 variants was observed (BCFI CC/TC vs. TT: HR 0.53, 95 % CI 0.34-0.82, interaction P = 0.08), but not observed for letrozole-treated patients. There was an increased risk of musculoskeletal AEs for patients with rs700518 variants CC/TC versus TT (HR 1.22, 95 % CI 1.03-1.45, P = 0.02), regardless of treatment. Tamoxifen-treated patients with rs4646 variants had a reduced risk of bone AEs (AA/CA vs. CC: HR 0.76, 95 % CI 0.59-0.98), whereas an increase of minor allele (C) of rs10046 was associated with an increased risk of bone AEs (HR 1.28, 95 % CI 1.07-1.52). rs936308 variants were associated with a reduced risk of bone AEs in letrozole-treated patients (GG/GC vs. CC: HR 0.73, 95 % CI 0.54-0.99), different from in tamoxifen-treated patients (GG/GC vs. CC: HR 1.32, 95 % CI 0.92-1.90, interaction P = 0.01). CYP19A1 rs700518 variants showed associations with BCFI, DRFI, in tamoxifen treated patients and musculoskeletal AEs regardless of treatment. SNPs rs4646, rs10046, and rs936308 were associated with bone AEs.

Resistance to monoclonal antibody (mAb) therapy limits clinical utility. ADCC is a significant mechanism of action of mAb-elicited tumor response. Postulated mechanisms of mAb resistance include, alteration in cell signaling pathways (PI3K/Akt/Ras-MAPK signaling), over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes. However, most studies on mAb resistance have utilized cancer cells conditioned with mAbs alone, in absence of immune effector cells. We developed an in vitro selection model wherein human HER2 positive breast cancer cell lines (BT474, SKBR3) and EGFR-expressing colorectal cancer cell lines (HT29, DLD1) were subjected to acute ADCC (>90% cell death) with high effector-target (100:1) ratio repeatedly with trastuzumab and cetuximab, respectively, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks for 10 total rounds of ADCC selection. Internal controls included mock-treated parental, isotype-IgG1, effector cells only, and mAb alone. In vitro ADCC assays based on calcein fluorescent labeling of live target cells, demonstrated significant reduction (20%, p Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon M. Young, Mark D. Pegram. Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines. [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 2123.

TPS11617Background: Studies of neoadjuvant chemotherapy in Stage II and III breast cancer patients suggest that some have significant benefit from chemotherapy while others appear to derive much le...

Single-molecule detection (SMD) has demonstrated some attractive benefits for many types of biomolecular analyses including enhanced processing speed by eliminating processing steps, elimination of ensemble averaging and single-molecule sensitivity. However, it's wide spread use has been hampered by the complex instrumentation required for its implementation when using fluorescence as the readout modality. We report herein a simple and compact fluorescence single-molecule instrument that is straightforward to operate and consisted of fiber optics directly coupled to a microfluidic device. The integrated fiber optics served as waveguides to deliver the laser excitation light to the sample and collecting the resulting emission, simplifying the optical requirements associated with traditional SMD instruments by eliminating the need for optical alignment and simplification of the optical train. Additionally, the use of a vertical cavity surface emitting laser and a single photon avalanche diode serving as the excitation source and photon transducer, respectively, as well as a field programmable gate array (FPGA) integrated into the processing electronics assisted in reducing the instrument footprint. This small footprint SMD platform was tested using fluorescent microspheres and single AlexaFluor 660 molecules to determine the optimal operating parameters and system performance. As a demonstration of the utility of this instrument for biomolecular analyses, molecular beacons (MBs) were designed to probe bacterial cells for the gene encoding Gram-positive species. The ability to monitor biomarkers using this simple and portable instrument will have a number of important applications, such as strain-specific detection of pathogenic bacteria or the molecular diagnosis of diseases requiring rapid turn-around-times directly at the point-of-use.

Background The role of shunting and patching during carotid endarterectomy remains controversial. Methods This is a retrospective case series evaluating consecutive patients undergoing carotid endarterectomy with routine shunting and patching. The primary endpoints were perioperative stroke, arterial injury, and lesion recurrence by duplex. Results Of the 220 operations performed, 43% were for symptomatic disease. Successful shunt placement occurred in 98%, with no shunt-related injuries. There was 1 minor perioperative stroke and no major strokes. At a mean follow-up of 24 months (median = 12 months), there was 1 restenosis potentially related to shunt placement. The incidence of asymptomatic >50% stenosis in the patched segment was 8%. Conclusions A combined policy of routine shunting and patching simplifies intraoperative decision making with results that rival or exceed those of trials in which their use was not standardized. Shunts need not be avoided because of concern of arterial injury.

An Ancient Greek aphorism instructs individuals to “know thyself.” But, we aren’t philosophers or poets, we are pharmacists. We must “know pharmacy.” Our curriculum is filled with pharmacology, pharmacotherapy, and pharmacokinetics. These are the concepts that we must master to complete the coursework, graduate, pass the boards, get a job, and serve patients. However, while we are busy learning about pharmacy, we are missing a crucial component of our education. While we are taught about drugs, when do we gain knowledge about ourselves? While we are learning to critically evaluate therapy, when do we critically evaluate ourselves? Is there even time in an already packed curriculum to devote to introspection? In truth there is not only time, but also a great need for self-reflective coursework in the pharmacy curriculum. The time I spent exploring StrengthsFinder ® helped me better understand myself and understand the need for self-reflection during pharmacy education. In addition, the use of the StrengthsFinder ® can help improve team cooperation, promote leadership, and encourage the initiation and continuation of professionalism.

Triple negative (TN) breast cancers that lack expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors, convey a poor prognostic due in part to a lack of targeted therapies. To identify viable targets for the treatment of TN disease, we conducted a Gene Set Enrichment Analysis (GSEA) on four different breast cancer genome expression data sets comparing TN vs ER + PR + HER2-(PPN). The discovery cohorts represent three genome expression platforms and show b-catenin binding proteins and steroid binding proteins as enriched in PPN tumors in all four cohorts. To investigate whether the expression levels of b-catenin or steroid binding proteins are associated with metastasis, an optimal molecular function gene signature was determined in a discovery cohort (GSE25055) and validated in two cohorts (GSE25065, E2100). In the cohort GSE25065 the hazard ratio (HR) of the b-catenin binding gene signature (AR, PXN, SMAD3, TBL1X, CALCOCO1, CHD8, GRIP1, DLG5, GLI3, SLC9A3R1) was 2.36 and in E2100 1.6. The b-catenin binding gene signature was tested in patients who are ER+ and resulted in a HR of 3.51 in GSE25065 and 1.9 in E2100 indicating it is an attribute associated with poor outcome in both TN and ER+ patients. The optimized steroid binding gene signature has a HR = 2.25 in GSE25065 but was not shown to be prognostic in E2100 or the corresponding ER+ patients. Identifying a prognostic gene signature associated with a common molecular function may implicate a mechanism of action in breast cancer. The down regulation of b-catenin binding proteins could increase the b-catenin levels available to drive transcription of b-catenin target genes and mitigate the Wnt-regulatory pathway. To determine a possible transcription factor driver of differential expression of b-catenin binding proteins, promoter analysis was done using the gene set (AR, ESR1, GLI3, LEF1, PTPRT, SLC9A3R1 and SLC9A3R2), all shown to be enriched in PPN in four cohorts. The enriched genes share common promoters associated with ETS1 family of transcription factors that are involved in stem cell development, cell senescence and death, and tumorigenesis. Disclosure All authors have declared no conflicts of interest.

150 Background: The systemic management of metastatic breast cancer (MBC) is mostly based on the ER or HER2 status of the primary tumor. However, the hormonal status or the amplificaction/overexpression HER2 may change in every metastatic site because of the effects of the long-term treatment of metastatic cancer with endocrine therapy, chemotherapy, or targeted agents. The purpose of this study was to investigate the frequency of change in HER2 expression in primary and distant metastatic tumors (especially in liver) in HER2+ breast cancer patients. Methods: We retrospectively analyzed the results of 31 consecutive metastatic breast cancer patients that were seen in our center over 4 months from February 2014 through May 2014. All patients were rebiopsied after consultation and samples were sent for standard immunohistochemistry (IHC) for ER, PR, and HER2 and formalin-fixed, paraffin-embedded (FFPE) samples were sent for genomic (Foundation Medicine) and proteomic analysis (Theranostics). All results from the metastatic samples were compared to the baseline IHC and/or FISH results for HER2. Results: A change in HER2 status was observed in 26% of the cases. 16% of cases underwent a negative to positive conversion in HER2 status while 10% of cases underwent a positive to negative conversion. It is notable that all 5 patients that underwent a negative to positive conversion in HER2 status had biopsies taken from metastatic disease in the liver. Overall, 45% of patients with metastatic disease in the liver had a negative to positive conversion in HER2 status. Conclusions: The results of this study emphasize the significance of confirming HER2 expression in a recurrence lesion. This discordance may be due to the increasing level of genetic instability occurring throughout disease progression that can significantly influence the alterations of the HER2 gene. If feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from non-HER2 amplified breast cancer. Although HER2 status is usually appraised in primary tumor, knowledge of the HER2 status in metastases may be of potential value for therapeutic decision making.

5583 Background: Integrating chromosomal deletions/amplifications with sequencing alterations is increasingly important in the determination of key drivers of outcome in cancer (Leary 2008, Curtis 2012). We introduce a novel computational approach, Gene Set Outcome Analysis, to determine gene signatures constrained to regions with frequent deletion or amplification events in ovarian cancer identified by TCGA. Differential expressions of mRNA from these genes are used as a proxy for loss of function from deletions or amplifications. Methods: Gene signatures from each region were evaluated using log-rank test comparing high and low gene expression groups split by cohort mean. In total, 30,119,708 signatures constrained to 47 deleted and 36 amplified regions were tested for PFS for first line platinum treatment in a random 2/3 split of TCGA ovarian cohort (n=262) resulting in 26,271 gene signatures with p < .001. The remaining 1/3 cohort (n=135) and full cohort (n=397) were used as a replication study where 111 signatures have a p < .01 located in 2 deletion and 1 amplification region. The signature with the lowest p-value from each region that validated in the replication study, were evaluated in GSE9891 (n=179) (Tothill 2008) for PFS when treated with platinum and taxol resulting in gene signatures from 2 deletion regions with p