Ingle, JN

Abstract BACKGROUND: In SWOG-8814A, pts with ER+ node+ breast cancer and low 21 gene recurrence scores (RS) had good prognosis and no CAF benefit, but high RS predicted longer survival from CAF followed by T (CAF-T) vs T (Albain, Lancet Oncol 2010). The aims of SWOG-8814B were to identify novel genes and networks for 1) prognosis of early and late relapse and 2) prediction of CAF benefit, using whole transcriptome expression analysis with next generation RNA sequencing (NGS). METHODS: Stored RNA previously extracted for SWOG-8814A (T, CAF-T arms; T, 5 yrs) was analyzed for RNA/library yield (see companion abstract Cherbavaz et al. for methods). Genes were sequenced and expression of mRNA species was related to disease-free survival (DFS) using Cox proportional hazards. Discovery analyses controlled false discovery rate (FDR) at 10%. Genes were identified for prognosis on T and prediction on CAF-T vs T. Networks of genes/pathways were explored. Early (0-5 yrs) and late (5-13+ yrs) time periods were studied. Gene Ontology, Cytoscape, pathway and hierarchical clustering were used for functional gene and metagene analyses. RESULTS: Of 367 samples, 354 (96%; 142 T, 212 CAF-T; 141 DFS events) had sufficient RNA/library yield, with 20,101 genes sequenced. For prognosis on T, there were 2327 and 568 genes discovered in early and all-yrs follow-up, with only 9 genes prognostic after 5 yrs. Prognosis analyses for residual risk after CAF-T were uninformative. Functional mapping found that genes prognostic for worse DFS were enriched for proliferation (G2M, M-phase), cellular metabolism, DNA repair, stress response and EMT; whereas, those with better DFS involved transcription regulation/repression via zinc finger proteins. Hierarchical clustering (T arm) found significant DFS prognostic metagene signatures for ER-related genes, immune response, ECM/stroma, chromatin remodeling-transcription factor activity and TGFb pathway. All varied for early vs late DFS events. For example, low ER/high stroma expression signatures correlated with high proliferation gene expression and were strongly associated with early events (standardized [st] HR 2.94, p<0.001). Late recurrence was associated with high proliferation, both individually (stHR 1.51, p=.035) and in combination with higher ER expression (stHR 1.51, p=0.09). Fifteen genes predicted CAF benefit (9 better DFS, 6 worse), or 129 genes if FDR relaxed to 20%. Cluster analysis for CAF prediction is ongoing. CONCLUSIONS: Unique genes, clusters and pathways were identified by NGS of archival material in ER+ N+ breast cancer, including previously unreported signatures. While ER, stroma and proliferation-related signatures were associated with early prognosis, proliferation best predicted worse DFS after 5 yrs. NGS of the primary tumor is most informative for early events in pts with only 5 years of T, with few genes selecting only for late relapse. If validated, these signatures may identify pts with excellent DFS despite positive nodes for endocrine therapy alone as well as others for whom chemotherapy and/or biologics are also required . SUPPORT: NCI CA 180888, 180819, 180821, 180820, 180863; in part, Genomic Health, Inc. Citation Format: Albain KS, Crager MR, Barlow WE, Baehner FL, Bergamaschi A, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Cherbavaz DB, Sing AP, Shak S, Hortobagyi GN, Hayes DF. Molecular predictors of outcome on adjuvant CAF plus tamoxifen (T) vs T in postmenopausal patients (pts) with ER+, node+ breast cancer – Transcriptome expression analysis of the phase III trial SWOG-8814. [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 S3-02.

Abstract Background: AI's are more effective than TAM in ER+ breast cancer. In AI refractory MBC, the response rate to TAM is 0% (Osborne 2011). Z-Endx is an active metabolite of TAM and among TAM treated women in the adjuvant and metastatic settings, reduced CYP2D6 metabolism and low Endx concentrations (Css <20 nM) have been associated with increased likelihood of disease recurrence. Preclinical studies have demonstrated greater Z-Endx exposure and anti-tumor activity with oral Z-Endx compared to equivalent doses of oral TAM (Reid 2014) Methods: We conducted a phase I trial to determine the maximum-tolerated dose (MTD) and evaluate the toxicities, clinical activity, and pharmacokinetics (PK) of Z-Endx in patients (pts) with ER+, AI refractory MBC. Unlimited prior endocrine regimens were allowed. An accelerated titration schedule was applied (2 pts/dose level) until moderate toxicity or DLT, followed by a 3+3 design and then to expansion cohorts (40, 80, and 100 mg/day). Z-Endx was administered orally once daily (28 day cycle). Eye exams were performed at baseline, and end of cycles 2 and 6. PK was performed during cycle 1 and prior to subsequent cycles. For pts in the expansion cohorts, tumor biopsies were obtained at baseline for DNA sequencing (Foundation Medicine). Plasma cholesterol levels were obtained at baseline and after 1 cycle. Results: From March 2011 to Dec 2014, 41 pts (38 evaluable), median age 60, received Z-Endx once daily encompassing 7 dose levels (20-160 mg/daily). The median number of prior hormonal regimens was 2 and 3 for the dose escalation and expansion cohorts, respectively. Dose escalation was stopped at 160 mg/day given MTD not reached and attainment of mean Endx Css of 3.6 uM. Cycle 1 DLT (PE) was observed in one patient (60 mg). No eye toxicity was observed. PK demonstrated mean Endx Css of > 1 uM at all dose levels ≥ 40 mg/day. Antitumor activity was observed at multiple dose levels including 3 confirmed partial responses and an additional 7 with stable disease for ≥6 cycles. Of these 10 pts, 9 had prior progression on both AI and fulvestrant and 3 additionally on TAM. After 1 cycle, total and LDL cholesterol decreased > 20 points in 54% and 40% of pts, respectively. Tumor sequencing in the expansion cohorts (n=14) did not identify ESR1 mutations; however, ESR1 amplification was identified in 1 pt with prolonged stable disease (>200 days). Of 6 pts with rapid progression (≤2 cycles), 4/6 had either CCND1 amplification (n=1) or at least one of the following activating mutations: ERBB2 L755S (n=1), AKT1 E17K (n=1), MTOR E1799K (n=1). Conclusions: The direct administration of Z-END provides substantial drug exposure, acceptable toxicity, and "proof of principle" antitumor activity in endocrine resistant MBC. While the MTD was not determined, the goal of achieving Endx Css concentrations of > 1 uM was achieved. Tumor sequencing identified pts with predicted and confirmed endocrine resistance. A randomized phase II comparing endoxifen (80 mg/day) with TAM in AI refractory MBC was recently activated (NCT02311933). Supported in part by CA 133049, CA186686, CA15083, CA116201, and CA15083. Citation Format: Goetz MP, Suman VJ, Reid JM, Northfelt DW, Mahr MA, Dockter T, Kuffel M, Buhrow SA, Safgren SL, McGovern RM, Collins JM, Streicher H, Hawse JR, Haddad TC, Erlichman C, Ames MM, Ingle JN. Final results of a first-in-human phase I study of the tamoxifen (TAM) metabolite, Z-Endoxifen hydrochloride (Z-Endx) in women with aromatase inhibitor (AI) refractory metastatic breast cancer (MBC) (NCT01327781). [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 PD2-03.

Abstract Background: During tumor progression, activation of Aurora A kinase (AURKA) is associated with epithelial to mesenchymal transition (EMT) reprogramming and expansion of a subpopulation of tumor initiating cells harboring a CD44+/CD24low/- phenotype [D'Assoro, Oncogene 2014]. These cells are characterized by their capacity to self-renew, resist drug therapies, and promote distant metastases. In ER+ breast cancer (BC) models, activation of AURKA is associated with down-regulation of ERα expression and resistance to endocrine therapy. Alisertib, a selective inhibitor of AURKA, can reverse EMT and restore tumor ERα expression and sensitivity to endocrine therapy [Opyrchal, PLoS One 2014]. As a single agent in HR+ advanced BC, alisertib was associated with a 6-month clinical benefit rate of 54% and median PFS of 7.9 months [Melichar, Lancet Oncol 2015]. The objectives of this phase I trial were to determine the maximum-tolerated dose (MTD) and evaluate the toxicities and clinical activity of alisertib with fulvestrant in patients (pts) with HR+ advanced BC. Methods: In this standard 3+3 dose-escalation phase I study, pts were assigned to two different oral doses of alisertib (40-50 mg BID on days 1-3, 8-10, 15-17 q 28-day cycle) in combination with standard dose fulvestrant (500 mg IM on day 1 and 15 of cycle 1 and then day 1 q 28-day cycle thereafter). Eligibility included HR+ advanced BC, postmenopausal status, measurable disease or nonmeasurable bone disease by RECIST v1.1, ECOG performance status ≤ 1, unlimited prior endocrine therapies, and ≤ 2 chemotherapy regimens in the metastatic setting. Results: Ten pts enrolled September 2014 - April 2015, and 9 were evaluable for the primary endpoint (one excluded due to ineligibility). The median pt age was 59 (range 48, 73). Prior endocrine therapies included AI (9, 100%), fulvestrant (6, 67%), and everolimus/exemestane (5, 56%). Eight pts (89%) had prior chemotherapy. A median of 4 cycles of therapy have been administered (range 1+, 9+). There were no severe (grade 3+) toxicities reported during cycle 1 at either dose level, thus the MTD was not reached. The cycle 1 grade 1/2 adverse events regardless of attribution were fatigue (6, 67%), neutropenia (5, 56%), anemia (5, 56%), leukopenia (4, 44%), diarrhea (3, 33%), nausea (3, 33%), and mucositis (1, 11%). As of June 3, 2015, 2 pts have discontinued treatment due to disease progression, and 7 remain on treatment with stable disease (Table). One pt with bone only disease had a near CR on PET scan. Dose LevelAlisertib Dose (BID)Treatment Cycles≥ Grade 3 Toxicity, All CyclesProgression-Free Survival (days)1 (n=3)40 mg4, 7+, 9+ 117, 170+, 223+2 (n=6)50 mg1+, 2, 3+, 3+, 4+, 5+grade 4 neutropenia (1 pt)28+, 56, 56+, 57+, 112+, 116++ indicates patients still receiving treatment Conclusion: Alisertib in combination with fulvestrant was well-tolerated. The recommended phase II dose is 50 mg twice daily on days 1-3, 8-10, and 15-17 q 28-day cycle with standard dose fulvestrant. Promising antitumor activity was observed. Correlative tissue evaluation of AURKA expression and other EMT biomarkers is underway. Funding: This work was funded by Takeda Oncology and supported by NIH Grant K12 CA90628 [TCH]. Citation Format: Haddad TC, D'Assoro AB, Suman VJ, Opyrchal M, Goetz MP, Ingle JN. A phase I trial to evaluate the safety of the addition of alisertib to fulvestrant in hormone receptor positive (HR+), advanced breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-04.

Abstract BACKGROUND: We previously reported that low 21 gene Recurrence Scores (RS) identify patients with ER-positive, lymph node-positive breast cancer who may not benefit from anthracycline-based adjuvant chemotherapy added to tamoxifen (SWOG-8814A NCI correlative science study; Albain et al. Lancet Oncol 2010). New exploratory and comprehensive quantitative analyses now permit whole transcriptome NGS on residual RNA extracted from FFPE blocks 12-18 years post-fixation. Herein, we report methodology details and feasibility results (see companion abstract, Albain et al., for clinical outcomes correlations). METHODS and RESULTS: Sequencing was carried out in Illumina HiSeq 2000 instruments, yielding 4.2 trillion data points. Messenger RNA expression was quantified using 3rd quartile normalization. Both Library (RNA) and Sequencing Standards showed high quality coverage as measured by median uniquely mapped reads over a 13 month window (168M and 182M, respectively, including duplicate reads). The median absolute deviation (MAD) of the relative log expression (RLE) of mapped reads for the Library and Sequencing Standards was 0.22 and 0.05, respectively. The Library Standard variation was greater than the Sequencing Standard, as library preparation was manual. Of 360 patient samples with sufficient RNA (≥ 100 ng total RNA), 354 (98.3%) were successfully sequenced and included in the final analysis data set. Average library yield was 39 ng/μL. Only 5 libraries failed yield requirements and one library failed expression quality metrics. The median insert length was 120 bp with the first and third quartiles 93 and 152 bp, respectively. After removal of duplicate reads, 82% of reads were uniquely mapped, and the median library size was 8.95M (number of unique mapped reads). Sequences with counts <10 for all 354 patients were excluded. The medians of the 1st, 2nd and 3rd quartiles for exons mapped to the RefSeq database were 20, 40 and 78 counts, and for introns, 72, 134 and 244 counts, respectively. The majority of exonic (86.7%) and intronic (95%) sequences were mapped. There were 20,101 RefSeq mapped exons with counts ≥10. Of these exons, 988 passed additional filtering criteria and were subjected to hierarchical clustering, with Gene Ontology and pathway analysis performed on selected gene expression patterns (for results, see companion abstract of Albain et al.). CONCLUSIONS: High quality whole transcriptome NGS is feasible from decades-old clinical trial FFPE specimens that have not been stored in any special fashion. Controlled laboratory, bioinformatics and biostatistics methods, with inclusion of appropriate process controls, ensure robustness and reliability of the NGS process. This in turn results in the discovery and validation of biologically and clinically relevant variations from prior landmark clinical trials. SUPPORT: NCI CA 180888, CA180819, CA180821, CA180820, CA180863; in part, Genomic Health, Inc. Citation Format: Cherbavaz DB, Hayes DF, Qu K, Crager MR, Barlow WR, Goddard AD, Beasley EM, Jeong J, Collin F, Liu M-L, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Sing AP, Baehner FL, Hortobagyi GN, Shak S, Albain KS. Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies. [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 P5-07-01.

Abstract Background: Tumor-infiltrating lymphocytes (TIL) have prognostic and potentially predictive significance in the (neo)adjuvant treatment of high-risk breast cancer. However, quantitative TIL measurement is not routinely performed. It is unclear why some tumors attract large quantities of TIL while others do not. We sought to confirm the association between TIL and pathologic complete response rate (pCR) and to further use next generation sequencing (NGS) to identify genes and gene pathways associated with the presence/absence of TIL. Methods: We studied 140 women with high risk stage I-III breast cancer, enrolled in the Breast Cancer Genome Guided Therapy Study (BEAUTY), obtaining serial biopsies for DNA/RNA sequencing and MRI imaging to assess response to neoadjuvant chemotherapy (NAC) with taxane (+/- trastuzumab+/-pertuzumab for HER2+ disease) followed by AC or (F)EC. Diagnostic pre-NAC core needle biopsies and surgical resection specimens post-NAC were available from 110 patients. Stromal TIL were semi-quantitated on a scale of 1-4 (with 1: ≤10/hpf, 2: subtle infiltrate >10/hpf, 3: moderate infiltrate readily visible at low power magnification, 4: dense infiltrate with innumerable lymphocytes). For this analysis, low TIL was defined as scores of 1-2 vs. high defined as 3-4. Using pre-NAC biopsies, RNAseq was performed using the Illumina HiSeq2000 and the Mayo Analysis Pipeline for RNAseq (MAP-Rseq) for quality control, sequence alignment, and gene counts. The quantity of TIL was associated with transcripts across the transcriptome after conditional quantile normalization. Differentially expressed genes were obtained using EdgeR analysis, using a false discovery rate of 0.05, and pathways were evaluated using GAGE methods. Results: The pCR and residual cancer burden (RCB)-0/I rates by stromal TIL status within each molecular subtype are presented in the table. A diverse spectrum of 1344 genes with differential expression between tumors with high vs. low stromal TIL was identified. The genes with >2.0-fold change (FC) and p<1e-09 included S100A7 (4.49 FC), LCN2 (2.48 FC), and ART3 (2.82 FC) (genes known to be involved in immune regulation), as well as TDRD1 (2.71 FC) (a gene related to ERG [ETS-related gene] expression). In addition, the "regulation of actin cytoskeleton" pathway was upregulated in tumors with high TIL, while the "Hedgehog signaling" and "Wnt signaling" pathways were downregulated. Molecular SubtypeStromal TILspCR rate n (%)RCB-0/I rateLuminal AHigh------Luminal ALow0/9 (0%)0/9 (0%)Luminal BHigh1/9 (11.1%)1/8 (12.5%)Luminal BLow3/24 (12.5%)6/23 (26.1%)ER+/HER2+High3/9 (33.3%)4/9 (44.4%)ER+/HER2+Low1/6 (16.7%)1/6 (16.7%)ER-/HER2+High8/9 (88.9%)7/7 (100%)ER-/HER2+Low4/8 (50.0%)6/8 (75.0%)Triple NegativeHigh10/19 (52.6%)13/19 (68.4%)Triple NegativeLow7/14 (50.0%)9/13 (69.2%) Conclusions: We identified genes and gene pathways associated with high TIL expression in breast tumors prior to NAC that provide insight into the interactions between TIL and tumors. TIL can be easily semi-quantitated on H&E and along with these novel biomarkers, may contribute to the personalization of breast cancer therapy. Citation Format: Moyer AM, Boughey JC, Kalari KR, Suman VJ, McLaughlin SA, Moreno-Aspitia A, Northfelt DW, Gray RJ, Sinnwell JP, Carlson EE, Dockter TJ, Jones KN, Felten SJ, Conners AL, Wieben ED, Ingle JN, Wang L, Weinshilboum RM, Visscher DW, Goetz MP. Differential mRNA expression patterns in breast tumors with high vs. low quantity of stromal tumor–Infiltrating lymphocytes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-05.

Abstract Background: Tumorigenesis involves both genetic and epigenetic changes. Epigenetic alterations are reversible and are promising cancer therapeutic targets. Decitabine (5-aza-2'-deoxycytidine), a DNA methyltransferase inhibitor, is FDA approved for hematological malignancies. However, the effect of decitabine in breast cancer is not completely understood. Previous reports indicated that one decitabine mechanism involves regulation of protein levels for DNMT1, the major DNA methyltransferase that methylates hemimethylated CpG di-nucleotides in DNA. However, the E3 ligase involved in this process has not been identified. Whether decitabine also regulates DNMT3A and 3B in a similar fashion remains unclear. Therefore, our goals were to 1) understand mechanisms underlying decitabine action, 2) test the antitumor activity of decitabine in breast cancer models and 3) identify biomarkers associated with response to decitabine. Methods and Results: Western blots of breast cancer cell lines showed that DNMT1, DNMT3A, and DNMT3B protein levels decreased following decitabine treatment without a reduction in mRNA levels. Bioinformatic analysis of DNA methyltransferase sequences revealed a potential TRAF6 binding motif, and the interaction with TRAF6 (TNF receptor-associated factor 6) was confirmed by IP. TRAF6 functions as an E3 ligase. To determine whether TRAF6 might be the E3 ligase responsible for the degradation of DNMTs after decitabine treatment, we knocked down TRAF6 by RNA interference or knocked out the TRAF6 gene by CRISPR/Cas9. Down regulation of TRAF6 attenuated DNMT ubiquitination and increased DNMT protein levels, suggesting that TRAF6 might mediate proteasome-dependent degradation of all three DNMTs. This was further confirmed by reconstituting the knockout cells with WT and a TRAF6-C70A mutant, followed by assessing DNMT protein levels. Global DNA methylation was also increased after TRAF6 depletion and was confirmed in TRAF6 knock out cells in which DNMT levels were unaffected by decitabine. Cell cytotoxicity and colony forming assays showed that TRAF6 knockout cells were resistant to decitabine, suggesting that a major decitabine mechanism of action is through the regulation of TRAF6 which, in turn, degrades DNMTs, leading to decreased global methylation. Finally, decitabine significantly induced TRAF6 at both mRNA and protein levels, a process that might create positive feedback leading to increased degradation of DNMT proteins upon decitabine treatment. Based on these results, we further hypothesized that levels of the three DNMTs might influence decitabine response. Using 18 breast cancer patient derived xenograft (PDX) models, we found a wide range of DNMT protein levels regardless of ER/HER2 status. DNMT levels in the PDX models were directly associated with sensitivity to decitabine treatment, confirming our hypothesis. Conclusion: Our data showed that decitabine might be an effective agent for treating breast cancer and revealed a novel mechanism underlying decitabine treatment. Baseline DNMT protein levels may serve as a biomarker for predicting decitabine drug response. Citation Format: Yu J, Qin B, Boughey JC, Moyer AM, Visscher DW, Sinnwell JP, Yin P, Thompson KJ, Docter TJ, Kalari KR, Suman VJ, Wieben ED, Felten SJ, Conners AL, Jones KN, McLaughlin SA, Copland JA III, Moreno Aspitia A, Northfelt DW, Gray RJ, Ingle JN, Lou Z, Weinshilboum R, Goetz MP, Wang L. Regulation of DNA methyltransferases via TRAF6 determines breast cancer response to decitabine. [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 P3-07-51.

Abstract Background: Both HR deficiency and BRCA mutation status predict response to platinum-based therapy and BRCA mutation status predicts docetaxel resistance. However, the association of either biomarker with response to the individual elements of either AC or taxanes (T) is unknown since T is commonly given concomitantly with or after anthracyclines (A). We evaluated the association of HRD and BRCA mutation status with response to neoadjuvant weekly T followed by AC or (F)EC in high-risk breast cancer. Methods: We studied 140 high risk Stage I-III breast cancer patients (pts), enrolled in the breast cancer genome guided therapy study (BEAUTY), obtaining biopsies for DNA/RNA sequencing and MRI imaging to assess response to neoadjuvant weekly T (+trastuzumab+/-pertuzumab for HER2+ disease) followed by AC or (F)EC. Germline BRCA status and HR status of tumor samples (Myriad laboratories) were obtained. HR deficient tumor was defined as HRD score ≥42 or BRCA mutation. MRI response by changes in tumor size after 12 weeks of T was classified by WHO criteria. pCR was defined as ypT0/Tis ypN0. Both MRI response after T and pCR (after T and AC) were examined in terms of germline BRCA mutation (gBRCAmut vs. gBRCAwt) and tumor HR deficiency. Results: Of 140 pts enrolled, 8 withdrew consent and 2 carboplatin treated pts were excluded. Germline data were available for 124/130 pts. 12 patients had BRCA deleterious germline mutations (4 BRCA1, 8 BRCA2). MRI partial (PR)/complete response (CR) rate to T was 47.3% (95% CI: 37.8-57.0%) in the BRCAwt group and 66.7% (95% CI: 34.9-90.1%) in the BRCAmut group. No MRI CR's were observed in BRCA1 mut pts. In contrast, pCR rate was 50% in the 12 gBRCAmut pts (95% CI: 21.1-78.9%) and 31.3% in the 112 gBRCAwt pts (95% CI: 22.8-40.7%). HR deficiency status has thus far been determined for 74 pts: 26 pts have HD deficient tumors: 18 TNBC, 5 Luminal B, 2 ER-/HER2+; and 1 ER+/HER2+. Determination of HR deficiency is ongoing and will be reported for the full cohort in terms of 12 week MRI response to T and pCR to T+AC. HR deficientMolecular Subtypeyes (%)no (%)TBD (%)Luminal A0/112/11 (18.2)9/11 (81.8)Luminal B5/37 (13.5)13/37 (35.1)19/37 (51.3)Luminal NOS0/21/2 (50)1/2 (50)ER+/Her2+1/17 (5.8)14/17 (82.4)2/17 (11.8)ER-/Her2+2/20 (10)11/20 (55)7/20 (35)Triple Negative18/43 (41.9)6/43 (18.6)17/43 (39.5)germline BRCA statusMRI partial response after T (%)MRI complete response after T (%)pCR after T&AC (%)BRCA11/4 (25)0/42/4 (50)BRCA25/8 (62.5)2/8 (25)4/8 (50)BRCAwt35/112 (31.3)18/112 (16.1)35/112 (31.3) Conclusion: In the setting of neoadjuvant weekly T followed by AC, pCR rates were non-significantly higher in pts with BRCA1 mutations. While we observed no overall association between BRCA mutation status and response rates to taxanes; nearly all MRI responses to taxanes (partial and complete) were observed in the BRCA2 group. Prospective studies are needed to validate these findings and to determine whether BRCA status can be used to select therapy. HR deficiency is uncommon in luminal A and HER2+, frequent in TNBC, and the association of HRD with both MRI response to taxanes and pCR will be reported at the meeting. Citation Format: Boughey JC, Kalari KR, Suman VJ, McLaughlin SA, Moreno Aspitia A, Moyer AM, Northfelt DW, Gray RJ, Vedell PT, Tang X, Dockter TJ, Jones KN, Felten SJ, Conners AL, Hart SN, Visscher DW, Wieben ED, Ingle JN, Hartman A-R, Timms K, Elkin E, Jones J, Wang L, Weinshilboum RW, Goetz MP. Role of germline BRCA status and tumor homologous recombination (HR) deficiency in response to neoadjuvant weekly paclitaxel followed by anthracycline-based chemotherapy. [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 P3-07-29.

Abstract Background: ASP is hypothesized to decrease the risk of breast cancer (BRCA) and BRCA recurrences. Thus we performed an exploratory analysis of ASP and CC use within the NCIC CTG MA.27 adjuvant trial comparing exemestane (E;N=3789) to anastrozole (A; N=3787) with a factorial second randomization to CC or placebo (P). Neither A or E was superior in breast cancer outcomes. Baseline low-dose ASP use is an accepted surrogate of cardiovascular risk factors and was used as a stratification factor across all 4 arms. Randomization to CC-P was discontinued after 18 months (n = 1622) due to concerns of cardiac toxicity. Methods: Patients taking >81mg of ASP daily at baseline were ineligible for randomization and use of >81mg of ASP daily was not allowed. Women enrolled during CC randomization were included in the comparison of E and A, stratified by whether they had been randomized to CC [yes, no;N=1622] and concomitant low-dose ASP [≤81 mg/day (yes, no); N=2209]. Other stratification factors included: lymph-nodes (negative, positive, or unknown); prior adjuvant chemotherapy (yes, no). The primary endpoint, event-free-survival (EFS), was defined as time from randomization to time of locoregional or distant disease recurrence, new primary breast cancer, or death from any cause. Secondary endpoints included overall survival (OS) defined as time from randomization to time of death from any cause and distant disease-free-survival (DDFS), defined as time from randomization to time of distant disease recurrence. Univariate (uni) assessment of CC and ASP use was assessed with stratified log-rank test, adjusting for lymph-node status and adjuvant chemotherapy and applied by intention-to-treat. Exploratory multivariate (multi) analyses (N = 1622) had forced inclusion of treatment and used step-wise forward stratified Cox modeling to examine the effects of CC, ASP use and baseline patient characteristics on outcomes; a factor was added with Wald test statistic p ≤ 0.05. Results: At median follow-up of 4.1 years, 186/1622 (11%) patients had an EFS event; 125 (8%) had died from any cause, and 80 (5%) had distant BRCA relapse. CC did not have significant uni association with outcomes: EFS p-value=0.92; OS p-value p = 0.56; DDFS p-value=0.55. ASP use was associated with worse EFS [p = 0.006, HR 1.48 (95% CI 1.12–1.96)], worse OS [p = 0.0002, HR 1.87 (95% CI (1.35–2.61)], and non-significant difference in DDFS (p = 0.72). CC had no multi association with EFS, OS or DDFS. ASP use had no multi association with EFS and DDFS (p > 0.05). However, ASP use had multi prognostic association with worse OS [p = 0.01; HR 1.67 (95% CI 1.13–2.49)]. Conclusions: Users of either CC or low dose ASP had similar DDFS to non-users in MA.27. As expected, low-dose ASP users (with presumptive cardiovascular risk factors) had worse OS than non-users. Inadequate numbers of patients randomized to CC and lack of randomization to aspirin leaves inadequate evidence from MA.27 to determine whether anti-inflammatories influence breast cancer outcomes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-13-02.

Abstract Background: 1/3 of women with metastatic HER2+ breast cancer will develop central nervous system (CNS) metastases yet evidence-based treatments for women with progressive CNS disease are limited. Neratinib is an irreversible inhibitor of erbB1, HER2, and erbB4 which has promising activity in HER2+ breast cancer. Preclinical evidence suggests it may cross the blood brain barrier. Trial Design: This is a multicenter, phase II, open-label study of neratinib for patients with HER2+ breast cancer and brain metastases. Neratinib is administered at 240 mg orally daily during a 28 day cycle. Two cohorts will be enrolled: Cohort 1 will enroll 40 patients with progressive CNS disease; cohort 2 will enroll ≤5 patients who are candidates for surgical excision of intracranial disease. Surgical candidates receive neratinib 7–21 days preoperatively and resume postoperatively. All patients are re-staged every 2 cycles. Those who develop non-CNS progression have an option to extend therapy with trastuzumab+neratinib. Circulating tumor cells (CTC) are collected at baseline and progression; neurocognitive testing, HADS and EORTC QLQ30/BN20 measures are administered at baseline, cycle 2, cycle 3, and progression (cohort 1). Intracranial tumor, cerebrospinal fluid (CSF), and plasma are collected at surgery (cohort 2). Specific Aims: The primary endpoint is CNS objective response rate (ORR) by composite criteria. Additional endpoints include: non-CNS ORR, progression-free survival, overall survival (OS), site of 1st progression, and toxicity. Correlative and exploratory endpoints include association of CTC count and OS and longitudinal neurocognitive function and quality of life. In an exploratory analysis (cohort 2), we will quantify neratinib concentrations in CSF, intracranial tissue, and plasma and examine associations with response. Eligibility: Patients must have confirmed HER2+ metastatic disease with ≥1 parenchymal brain lesion measuring ≥10 mm that is new or progressed after completing ≥1 line of standard CNS-directed treatment (cohort 1) or CNS disease that is amenable for surgery, including those without prior CNS treatments (cohort 2). Additional eligibility criteria (cohorts 1,2) include: adequate performance status and end organ/marrow function, and ejection fraction ≥50%. Any number of prior lines of therapy is allowed, including prior lapatinib. Statistical Methods: Cohort 1 has a 2-stage design with up to 40 patients. CNS ORR is defined as ≥50% reduction in sum volume of CNS target lesions, without evidence of new lesions, progression of non-target CNS lesions, non-CNS disease progression, worsening neurological symptoms, or increase in corticosteroids. CNS lesion measurements are performed centrally by the Harvard Tumor Imaging Metrics Core. If 1/18 patients have a CNS response in the 1st stage, another 22 patients will enroll. With this design, if ≥5 of 40 patients achieve a CNS response, the drug will be deemed worthy of future study. This 2-stage design has 92% power to distinguish between a true CNS ORR of 20% and a null of 6% (one-sided type I error rate=9%). Accrual: Accrual has begun. Target=45 (cohort 1=40, cohort 2=5) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT1-1-11.

Abstract Background: In AI refractory MBC, TAM anti-tumor activity is limited (median PFS 4.5 months). In patients (pts) receiving TAM in the adjuvant and metastatic setting, breast cancer recurrence has been shown to differ based upon CYP2D6 genotype and Endx exposure. In collaboration with NCI, Z-Endx (NSC #750393) was synthesized and preclinical pharmacology studies demonstrated that oral Z- Endx resulted in > 20 fold Endx exposure compared to an equivalent dose of TAM and significantly greater anti-tumor activity than TAM in AI sensitive and resistant MCF7 xenografts. We conducted a phase I study of Z-Endx in women with ER+, AI refractory MBC to identify dose-limiting toxicities (DLT), a maximum tolerated dose (MTD) and a dose associated with steady state concentrations (Css) of ≥ 2 uM, based on the IC50 of recently identified Endx substrates: PKC and PI3K. Methods: Pts were enrolled to an accelerated titration schedule (2 pts/dose level) until moderate toxicity or DLT, and then to a 3+3 design. Z-Endx was administered orally once daily (28 day cycle). Eye exams were performed at baseline, end of cycle 2, and after 6 cycles. PK was performed on days 1-2, 3, 7, 14, and 28, and prior to subsequent cycles. Results: 23 women with AI refractory MBC (median 56 yrs, range 41-83) received Z-Endx once daily encompassing 7 dose levels (Table 1). The median number of prior chemotherapies and hormonal therapies in the metastatic setting were 2 and 2, respectively. Dose escalation continues at 160 mg/day and the MTD has yet to be determined. Cmax and AUC increased in a dose-dependent manner. A 20 and 100 mg/day dose yields Css of 0.39 and 2.48 uM, respectively. Significant anti-tumor activity was observed including 1 pt (100 mg/day) with a PR lasting 225 days; 1 pt (80 mg/day) with a 30% reduction in tumor size (PFS 169 days) and 2 pts (60 and 80 mg/day) with stable disease for >270 days. No eye toxicity was observed. Conclusions: In women with AI refractory MBC, the MTD of Z-Endx HCl has not been determined but Endx Css concentrations of > 2 uM and substantial anti-tumor activity has been observed. Following completion of the 160 mg/day dose, expansion at 20 and 100 mg/day will commence to perform translational studies and further characterize Endx pk. A randomized phase II examining two different doses of Z-Endx is planned in AI refractory MBC. Supported in part by CA 133049, CA69912, CA15083, CA116201, and CA15083. Table 1Dose Level (mg/day)Number of Treatment CyclesModerate or Severe Toxicities ReportedTumor ResponseProgression-free Survival (days)28 day Z-Endx Css (uM) 20 (n = 2)2; 3Gr 2 hot flashes (1 pt)----60; 850.39 40 (n = 2)2; 6noneStable (1 pt)61; 1670.66 60 (n = 6)1; 2; 2; 5; 8; 14Gr 4 Triglycerides (1 pt);Gr 3 thromboembolic event (1 pt); Gr 2 hot flashes, anemia, and hypoalbuminemia (1 pt)Stable (3 pts)125+; 56; 57; 132; 232; 4331.01 80 (n = 3)4; 6; 10Gr 2 hot flashesStable (3 pts)113; 169; 2961.61 100 (n = 3)1; 2; 8Gr 2 nausea (1 pt); Gr 2 irritability (1 pt)PR (1 pt)30; 56; 2252.48 120 (n = 3)1; 2; 4Gr 2 hypersomnia, paresthesia, and peripheral sensory neuropathy (1 pt)—39; 54; 1082.18 * Bold face toxicities occurred during cycle 1 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD3-4.