Ingle, James N.

Abstract Background: The use of anti-estrogen (AE) therapies (selective estrogen receptor modulators [SERMs], aromatase inhibitors [AIs]) in breast cancer (BC) patients was recently associated with risk of developing rheumatoid arthritis (RA) (J Rheum 2015;42:55-9). We attempted to replicate and extend these results using electronic health record (EHR) enhanced cancer registry data that assessed specific AE therapies and also accounted for potential confounding by age, smoking status, and chemotherapy use. Methods: Using cancer registry and EHR data, we identified a cohort of BC patients who were newly diagnosed and treated at Mayo Clinic Rochester from 1998-2011 and had no previous diagnosis of RA. Smoking status at diagnosis, BC treatments and RA at diagnosis and during follow-up were identified using validated EHR-based algorithms. Hazard ratios (HRs) and 95% confidence intervals (CI) from a multivariate Cox model were used to estimate the association of AE use with risk of RA, controlling for age, smoking status at BC diagnosis, and BC chemotherapy use. Results: The analytic cohort of 9,244 newly diagnosed BC patients treated and followed at Mayo Clinic had a median age at diagnosis of 59 years (range 18-97); 32% were smokers; and 29% were treated with chemotherapy. During a median follow-up of 49 months (range 1-191), 19 patients developed RA. Compared to BC patients not receiving any AE therapy, those receiving AI monotherapy, but not SERM monotherapy or both SERMs and AIs, were at significantly increased risk of developing RA (Table). AE Therapy# of RA Cases / # patientsHR* (95% CI)P-valueNo AE therapy6/4,0711.00 (reference)SERMs only5/2,5371.20 (0.36 - 3.95)0.77AIs only5/7126.37 (1.77 - 22.92)0.005SERMs then AIs3/1,8750.90AI then SERMs0/490.91 (0.21 - 3.86)*Adjusted for age, smoking status and use of chemotherapy Conclusions: Our findings indicate that the use of AIs for BC therapy, but not SERMs or SERMs followed by AIs, is associated with increased risk for development of RA, when controlling for smoking status and use of chemotherapy. While needing replication, these findings suggest a specific role for AIs in RA development and suggest pathways that could be targeted to prevent this potential treatment complication. Citation Format: Matthew K. Breitenstein, Ming-Fen Ho, Richard M. Weinshilboum, James R. Cerhan, Jyotishman Pathak, Tim Bongartz, Liewei Wang, James N. Ingle. Association of anti-estrogen therapy in breast cancer patients and subsequent risk of rheumatoid arthritis: An electronic health record study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-191. doi:10.1158/1538-7445.AM2015-LB-191

Abstract Introduction: Triple negative breast cancers (TNBC) are characterized as lacking estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression and TNBC patients have higher rates of recurrence and death compared with other breast cancer subtypes. For TNBC patients who fail standard chemotherapy, there are a lack of novel drug therapies, given the absence of well-defined molecular targets. Recently, a microarray meta-analysis identified 7 triple negative subtypes, including the validation of the luminal androgen receptor (LAR) positive subtype [Lehmann, 2011]. However, microarray technology is dependent on probe-target specificity and the 7 subtypes have yet to be validated using RNA sequencing data, and the presences of recurrent genomic alterations in the 7 subtypes are unknown. METHODS: We obtained 1106 breast cancer RNA-Seq bam files from The Cancer Genome Atlas (TCGA) and aligned with Tophat v1.3. The PAM50 intrinsic gene signature was used to extract a cohort of 128 TNBC samples. Consensus clustering of genes, greater than 75th percentile of variance, was performed using Kmeans clustering in Spearman's correlation space. A nearest centroid prediction model was developed from genes differentially expressed among the clusters. Eighty independent TNBC RNA sequencing samples were obtained (British Colombia; BC) [Shah, 2012] which were calibrated to our TNBC conditional quantile normalized cohort and sub-typed by our model. RESULTS: Using RNA-Seq gene expression count data, we identified 5 clusters, all of which were stable, including the LAR cluster. Signaling pathway impact analysis (SPIA) implicated cytokine-cytokine receptor interaction, leukocyte transendothelial migration, and regulation of actin cytoskeleton pathways commonly altered in the non-LAR TNBC subtypes. In contrast, cell cycle, ECM-receptor interaction, endocrine regulated calcium reabsorption, and insulin signaling pathways were altered in the LAR versus non-LAR subtypes. Neuroactive ligand-receptor interactions were observed to be altered commonly between all sub-types. We then applied our model to the Shah, et al cohort. In this cohort, the LAR subtype was consistent with Shah's classification of ‘other’ TNBC and contained no basal samples by PAM50 intrinsic modeling. Analysis of sub-type specific mutation data from the BC cohort demonstrates an increased mutational load in ECM-related proteins, particularly the myosins, along with increased TP53 clonality in the non-LAR subtypes. CONCLUSIONS: Using TCGA RNASeq data, we have confirmed the presence of 5 major TNBC subtypes, including the LAR; which was negligible in basal composition by PAM50 intrinsic modeling. SPIA pathway analysis indicates a core set of pathways demonstrating altered expression across the TNBC sub-types and the identification of molecular targets within each subtype is ongoing. Citation Format: Kevin J. Thompson, Xiaojia Tang, Zhifu Sun, Jason P. Sinnwell, Hugues Sicotte, Douglas W. Mahoney, Steven Hart, Peter T. Vedell, Poulami Barman, Jeanette E. Eckel Passow, Eric D. Wieben, James N. Ingle, Judy C. Boughey, Liewei Wang, Richard Weinshilboum, Krishna R. Kalari, Matthew P. Goetz. Molecular classification of triple negative breast cancer via RNA-sequencing data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5592. doi:10.1158/1538-7445.AM2014-5592

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2014-4185

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-3.

Abstract Background: The ECOG E5194 study was a prospective trial designed to evaluate surgical excision (lumpectomy) without radiation for selected women with ductal carcinoma in situ (DCIS) of the breast with low risk clinical and pathologic features. Methods: Eligible patients were enrolled on two study cohorts (not randomized): (1) low or intermediate grade DCIS, tumor size < 2.5 cm; or (2) high grade DCIS, tumor size < 1.0 cm. Cohort assignment was based on pathology assessment from the treating institution. Protocol specifications included surgical excision of the DCIS tumor with a minimum negative margin width of at least 3 mm or no tumor on re-excision. Radiation treatment was not allowed. From April 1997 to October 2002, 665 evaluable patients were enrolled through ECOG or NCCTG (561 in Cohort 1; 104 in Cohort 2). Tamoxifen was optional (not randomized) beginning in May 2000, and was given to 30% of the patients. The primary study endpoint was the rate of developing an ipsilateral breast event (IBE), defined as local recurrence of DCIS or invasive carcinoma in the treated breast. The median follow-up was 12.3 years. We have previously reported 7-year results (L. Hughes, J Clin Oncol 27:5319, 2009; median follow-up 6.3 years; 66 IBE’s), and we herein provide 12-year results. Results: Median patient age was 60 years and 58 years for Cohort 1 and Cohort 2, respectively. Tumor size was < 10 mm for 79% and 80% of patients, respectively. The minimum negative margin width was > 5 mm for 64% and 69% of patients, respectively. There were 99 IBE’s, of which 51 (52%) were an invasive IBE. The IBE and invasive IBE rates increased over time in both cohorts (see Table). The 12-year rates of an IBE were 14.4% for Cohort 1 and 24.6% for Cohort 2 (p = 0.003), and for an invasive IBE, 7.5% and 13.4%, respectively (p = 0.08). No difference was seen for the 12-year rates of overall survival (84.0% vs 82.8%; p = .96) or contralateral breast events (6.7% vs 12.0%; p = 0.16). On multivariate analysis, study cohort (hazard ratio = 1.81; p = 0.01) and tumor size (p = 0.01) were statistically significant for an IBE, and study cohort was borderline statistically significant for an invasive IBE (p = 0.08). On central pathology review (75% of cases), neither grade nor comedo necrosis was associated with the risk of an IBE or invasive IBE (all p > 0.15). Salvage treatment at the time of an IBE included mastectomy for 42% (31/74) and 64% (16/25) of the patients, respectively. Conclusions: For these selected patients with favorable DCIS based on clinical and pathologic characteristics treated with surgical excision without radiation, the rates of an IBE and an invasive IBE continued to increase through at least 12 years of follow-up. IBE Rates According To Study Cohort. Cohort 1 (Low or Intermediate Grade)Cohort 2 (High Grade)TimeIBEInvasive IBEIBEInvasive IBEAt 5 years6.0% (4.0%, 8.1%)2.7% (1.3%, 4.1%)15.0% (7.7%, 21.7%)5.3% (0.8%, 9.7%)At 7 years9.5% (7.0%, 12.0%)4.8% (2.9%, 6.6%)18.2% (10.6%, 25.8%)7.6% (2.2%, 13.0%)At 10 years12.5% (9.5%, 15.4%)6.4% (4.2%, 8.6%)24.6% (15.7%, 33.4%)13.4% (5.9%, 20.9%)At 12 years14.4% (11.2%, 17.6%)7.5% (5.1%, 10.0%)24.6% (15.7%, 33.4%)13.4% (5.9%, 20.9%) Citation Format: Lawrence J Solin, Robert Gray, Lorie L Hughes, William C Wood, Mary Ann Lowen, Sunil Badve, Frederick L Baehner, James N Ingle, Edith A Perez, Abram Recht, Joseph Sparano, Kathy Miller, Nancy E Davidson. Local excision without radiation for ductal carcinoma in situ: 12-year results from the ECOG E5194 study [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 P6-13-01.

Abstract Purpose: Tamoxifen and raloxifene, are primary prevention strategies for women at high risk of breast cancer (Visvanathan, 2013). Recent advances in genetic studies of breast cancer risk have identified common susceptibility loci that explain 14% of familial risk for breast cancer in the general population (Michailidou, 2013). However, it is not known if these loci are risk factors for breast cancer among high-risk women treated with SERMs for breast cancer prevention. We hypothesized that the large risk reduction associated with SERMs, coupled with the fact that several breast cancer loci correlate with family history, may limit the contribution of these common genetic loci to breast cancer in this high risk population. We present the first report to evaluate 75 established breast cancer susceptibility loci, in the context of a polygenic risk score (PRS), as a risk factor for breast cancer among high risk women taking raloxifene and tamoxifen for breast cancer prevention. Methods: We conducted a matched case-control study of 594 cases (i.e., participants who developed breast cancer while on SERM therapy) and 1,171 matched controls selected from the 33,000 participants enrolled in the NSABP P-1 and P-2 breast cancer prevention trials. Genotypes of 75 single nucleotide polymorphisms (SNPs) were available from a genome-wide association study conducted at the RIKEN Center for Genomic Medicine. We formed a quantitative PRS from reported per-SNP odds ratios (OR) for the 75 susceptibility loci. Conditional logistic regression was used to examine the PRS as a risk factor for breast cancer and to assess whether the PRS and breast cancer association differed by treatment type, family history, or other clinical characteristics. Analyses also examined associations of PRS with invasive vs. in situ cancer and ER-positive vs. ER-negative cancer. Results: The PRS ranged from 3.98 to 7.74, and a one unit change in PRS was associated with a 42% increase in breast cancer (OR=1.42; 95% CI: 1.18-1.70; P = 0.0002). There was evidence of a stronger association of PRS with breast cancer among women with no first-degree family history (OR=1.62, 95% CI: 1.18-2.21) compared to those with a positive family history (OR=1.32, 95% CI: 1.06-1.66) (Pintx<0.05). The PRS also appeared a stronger risk factor for ER-positive (OR=1.59, 95% CI: 1.25-2.02, P < 0.0002) vs. ER-negative (OR=1.05, 95% CI: 0.68-1.62, P=0.84) breast cancer, although differences did not reach statistical significance (Pintx=0.10). PRS and breast cancer associations were similar across tamoxifen and raloxifene treatments, age at trial entry, 5-year predicted Gail model risk, hysterectomy status, BMI, presence of atypical hyperplasia and invasive vs. in situ cancer. Conclusion: A polygenic risk score composed of 75 loci was a risk factor for ER-positive breast cancer, especially in the absence of a first-degree family history of breast cancer. Further, the PRS associations with breast cancer were similar for women taking tamoxifen or raloxifene for prevention. These data suggest that common genetic variation adds information on risk of ER-positive breast cancer in a high-risk population receiving SERMs. Citation Format: Celine M Vachon, Daniel J Schaid, James N Ingle, Matthew P Goetz, Donald L Wickerham, Michiaki Kubo, Erin E Carlson, Soonmyung Paik, Norman Wolmark, Yusuke Nakamura, Liewei Wang, Richard M Weinshilboum, Fergus J Couch. The contribution of common genetic variation to breast cancer risk among women receiving tamoxifen or raloxifene within the National Surgical Adjuvant Breast and Bowel Project (NSABP) P-1 and P-2 trials [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 P6-10-03.

Abstract Background: While the biological functions and clinical importance of ERα are well understood in breast cancer, much less is known about its most closely related family member, ERβ, particularly in the setting of triple negative disease. Additionally, the ability to therapeutically target ERβ in triple negative breast cancer (TNBC) has not been fully explored. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in a cohort of 71 TNBCs. To further define the biological functions of ERβ in TNBC, novel ERβ expressing triple negative cell lines (MDA-MB-231 and Hs578T) were developed and comprehensively characterized at the level of global gene expression profiling, modulation of important biological pathways, cellular proliferation and response to targeted therapies. Results: In TNBCs from 71 patients, nuclear and cytoplasmic ERβ was detected at moderate to high levels in 24% and 32% of cases respectively. This moderate to high expression of both nuclear and cytoplasmic ERβ was associated with higher levels of Ki67. Of the 17 tumors expressing ERβ, 13 (76%) were negative for androgen receptor expression. In the triple negative MDA-MB-231-ERβ and Hs578T-ERβ cell lines, expression of ERβ led to inhibition of proliferation in response to both estrogen and multiple ERβ specific agonists. Microarray analysis and RT-PCR profiling of these cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors, while suppressing the expression of many interleukins. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells inhibited the proliferation rates and blocked TGFβ signaling in non-ERβ expressing TNBC cells, effects that were completely reversed following depletion of cystatins from the conditioned media. Conclusions: ERβ is expressed in a substantial proportion of TNBCs, and most do not express the androgen receptor. In TNBC, where targeted therapies are lacking, our data suggest that estrogen or ERβ specific agonists would be expected to elicit anti-tumor effects when ERβ is expressed. These tumor suppressive effects of ERβ appear to be mediated in part through the actions of cystatins, and their inhibition of TGFβ signaling, suggesting that this family of secreted proteins may represent novel biomarkers for monitoring ERβ specific drug responsiveness and/or patient outcomes. Citation Format: Jordan M Reese, Malayannan Subramaniam, Vera J Suman, Xianglin Wu, Vivian Negron, Anne Gingery, Kevin S Pitel, Sejal S Shah, Heather E Cunliffe, Ann E McCullough, Barbara A Pockaj, Thomas C Spelsberg, Matthew P Goetz, James N Ingle, John R Hawse. Therapeutic targeting of ERβ in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-06.

Abstract Background: Triple negative breast cancer (TNBC) represents 15% of all breast cancers, and is characterized by an aggressive clinical course. While efforts are ongoing to characterize the molecular basis for variation in TNBC, there are conflicting data regarding the impact of prognostic factors such as age and Ki-67 typically used for treatment decisions in other breast cancer subtypes. We identified a retrospective cohort of women with ER- breast cancer with long term follow-up, and performed central confirmation of ER, PR, HER2, Ki-67 and histological classification to assess the association of Ki-67 and histologic subtype on clinical outcome in TNBC pateints (pts). Methods: 9,836 women who underwent breast cancer surgery at Mayo Clinic Rochester from 1985-2005 were identified. Pts were excluded due to: ER+ disease (7363); prior cancer (553); non-invasive disease (465); bilateral breast cancer (167); metastatic disease [at diagnosis or within 60 days of surgery (110)]; ER- disease treated with neoadjuvant chemotherapy (121) or adjuvant hormonal therapy (112); or tumor block exhausted (94). For all others, centralized ER/PR/HER2 identified the following for exclusion, >1% ER staining (n=76), > 1% PR (n=14) and HER2+ (n=144) by IHC (3+) or FISH amplification. 225 cases are still undergoing review. For the centrally confirmed pts with TNBC (n=392), Ki-67 and histological characterization (WHO subtypes) were assessed. Results: Patient characteristics are listed in the table. The median age for all TNBC’s was 45 (range 29-88) and nearly all tumors were grade 3 (91%) with high proliferation (63% with Ki-67 > 30%). The median follow-up was >10 years, where 139 pts had a disease event: progression (86) or second primary (53) and 53 died without a disease event. The 10 yr DFS rate was 55.8% (95%CI: 48.8-63.9%) among the 238 pts who did not receive adjuvant chemotherapy (AdjCT) and 58.9% (95% CI: 48.9-70.9%) among the 102 pts administered AdjCT. In pts without adjCT, DFS was found to differ with respect to number of positive LNs (0 vs. 1-4 vs. 4+; log rank p=0.003) and medullary histology (log rank p=0.015) but not with age (< 50 vs. ≥ 50), tumor size (< 2.0 vs. 2-5 vs. ≥ 5.0 cm) or Ki-67 (0-15% vs. 15.1-30% vs. >30 %). Conclusions: Our findings confirm the poor prognosis of TNBC. The medullary histological subtype is associated with significantly better prognosis than other TNBC subtypes (10 year DFS rate of 78.5%; 95%CI: 59.1-99.9% in pts not treated with adjCT). After central confirmation, neither age nor Ki-67 provide additional prognostic information. Patient characteristicsMedian Age (range)45(29-88) n%Histology Apocrine287.1Carcinoma, anaplastic or undiffrentiated9323.7Central scar/necrotizing348.7Clear Cell297.4Infiltrating ductal ca7118.1Large Cell neuroendocrine143.6Medullary and atypical medullary4311.0Metaplastic ca246.1Trabecular ca338.4other histologies235.9Grade 130.82317.9335891.3Tumor Size 0.1-2 cm19650> 2.0 cm19549.6not stated10.3Nodal status 0251641-38220.94-9338.410+225.6not evaluated41.0Ki-67 0-157719.615.1-306516.6>30%24763.3not done20.5Adjuvant Chemotherapy Yes10226.0No23860.7unknown5213.3 Citation Format: Ahmed Elkhanany, Vera J Suman, Victoria Cafourek, Judith A Gilbert, James N Ingle, Fergus Couch, Daniel W Vissscher, Matthew P Goetz. Triple negative breast cancer: The role of classic histological and prognostic factors on disease free survival [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 P5-02-11.

Abstract [Background] Patient advocates bring a unique, valuable perspective and presence to SPOREs. However, although patient advocates are recommended members of SPORE grants, guidelines are not given on the role of advocates or on measurement of the performance of advocates. [Methods] The Mayo Clinic Breast Cancer SPORE Patient Advocate Advisory Committee has developed a participation measurement tool. This tool will underpin and augment evaluations of the individual advocate contributions by the program PI and the SPORE project leaders. The Advocate Advisory Committee Chair will review the evaluations with the individual advocates. Annually, the advocate committee will review the performance of the group by the standard of the metrics. [Results] Advocates kept a log of their SPORE activities for four months then worked with a statistician to discern patterns of measurable, evaluable behaviors. The determined evaluable areas are training, scientific activity, meeting attendance, communication. Currently, those metrics are being tracked. Advocates developed the metrics together and report activities at the monthly advocate meetings. Currently, activities are simply tracked and categorized. No minimum standards are set. This will be the third step in the development of the metrics. [Conclusions] This approach is the first step in the development of meaningful evaluation of advocate SPORE activities. It quantifies the participation of advocates in the SPORE and provides the basis for developing a qualitative performance evaluation. The SPORE PI and the Committee Chair will use the evaluations to interpret the involvement of advocates in SPORE research and activities and to plan interventions to strengthen the advocate contributions to the SPORE. Citation Format: Cynthia Chauhan, Wayland Eppard, Lori Denison, Debra Gearhart, Lawanna Holmes, Ruth Kraft, Linda Miller, Mary A Sitta, Mary L Smith, James Ingle, Matthew Goetz. Measurements of advocate participation in and contributions to the Mayo Clinic breast cancer SPORE [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 P2-09-04.

Abstract Endocrine therapy, with SERMs and AIs, is the most important treatment modality for the 70% of patients with ER+ early breast cancer. Clinically, there is marked variability between patients in response to treatment and adverse events (AEs) such as musculoskeletal (MS) AEs with AIs. In 649 women with early-stage breast cancer we identified large inter-individual variations in pretreatment and on-anastrozole (A) plasma estrogens as well as plasma A concentrations, suggesting that the approved A dose of 1 mg per day may not be optimal for all patients. We performed a series of genome-wide association studies (GWAS) and identified a genome-wide significant variant SNP associated with concentrations of E2 that created an estrogen response element (ERE), and a significant SNP associated with E1-conjugates, providing potential insights into the variability. Pharmacogenomics (PGx) is the study of the role of germline and somatic genetic variation in individual variability in drug response phenotypes, i.e., disease response and AEs. The scientific goal is to understand mechanisms for the phenotype and the clinical goal is the determination of the right drug at the right dose for the right patient. The Mayo PGx Research Network Center and Breast SPORE and the RIKEN Center for Integrative Medical Science have worked collaboratively with cooperative groups to perform GWAS in women treated with both SERMs and AIs. The PGx paradigm consists of the GWAS as the initial step in a process that goes beyond the identification of associations to study the relationship of the single nucleotide polymorphisms (SNPs) to genes and the relationships of these SNPs and genes to the drug effect and the phenotype under study. Using the phenotype of AI-related MS-AEs, the major reason women stop therapy, a case-control GWAS was performed with patients from MA.27. The GWAS identified a SNP on chromosome (Chr) 14 that was associated with MS-AEs, near a gene (TCL1A), and created an ERE. Importantly, TCL1A expression was related to expression of multiple cytokine receptors, NFκB, and chemokines (related to rheumatoid arthritis) in an E2- and SNP-dependent fashion. These results provide a pharmacogenomic basis for MS-AEs with AIs. Using the phenotype of development of breast cancer in high-risk women receiving SERMs, a case-control GWAS was performed with patients entered on the NSABP P-1 and P-2 trials. The GWAS revealed variant SNPs on Chr 4 near CTSO (deleterious) and Chr 16 in ZNF423 (protective). The OR for differences in risk of breast cancer for participant homozygous for both protective or both deleterious alleles was 5.71. Both CTSO and ZNF423 were shown to participate in E2-dependent induction of BRCA1 expression in a SNP-dependent fashion. Most remarkable was that the presence of a SERM reversed the E2-inducible expression of ZNF423 and BRCA1 in a SNP-dependent fashion. In addition, ZNF423 was shown to be an E2-inducible BRCA1 transcription factor. The deleterious variant SNP near CTSO was found to disrupt an ERE. The GWAS described led to functional genomic studies that produced new knowledge relating to endocrine biology that provide a focus for further research to move towards precision endocrine therapy for women with breast cancer. Citation Format: James N. Ingle. Pharmacogenomics in the Quest for Precision Endocrine Therapy of Breast Cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr ML-1.