Citations

Abstract Support: Alliance U10CA180821; Alliance Statistical Center grant U10CA180882; ACOSOG grant U10CA76001 HER2 gene amplification and its corresponding overexpression are present in approximately 12% of invasive breast cancers. While HER2-targeted agents (e.g. trastuzumab, pertuzumab, and lapatinib) are effective treatments, resistance remains a major cause of death from HER2-positive breast cancer. Mechanisms of resistance are poorly understood. Without a molecular understanding of these mechanisms, therapeutic advances will be delayed. We have generated molecular profiles of primary HER2-positive breast cancers treated on a neoadjuvant clinical trial, and compared features associated with response to treatment. The American College of Surgeons Oncology Group (ACOSOG) Z1041 trial in HER2-positive breast cancer was designed to compare the pathologic complete response (pCR) rate of a regimen of paclitaxel and trastuzumab, followed by trastuzumab administered with fluorouracil, epirubicin, and cyclophosphamide (FEC-75) to a regimen of FEC-75 alone followed by paclitaxel and trastuzumab. The trial identified no difference in pCR rates between the regimens (Buzdar et al., The Lancet Oncology 2013). In supplement to the tissues obtained from 37 of the patients enrolled in the Z1041 trial, an additional 11 cases were obtained from a single institution study (201101961) of patients treated with neoadjuvant trastuzumab that had pre-treatment core biopsies suitable for genomic studies. We have extracted genomic DNA from both pretreatment tumor biopsies and blood samples of these 48 patients and performed whole genome (WGS) and exome sequencing. Coincident with these efforts, we have extracted high quality RNA from 42 of the 48 biopsies, and have processed RNA-seq profiles of the tumors. Among patients in this cohort, 24 (50%) achieved a pCR. Because no difference was observed between arms of the Z1041 trial, patients with or without a pCR were directly compared without adjusting for treatment regimen. On average, each tumor and normal sample pair were sequenced to a depth of 49.4x and 32.5x by WGS respectively. In total, 15,027 candidate somatic variants were identified in known genes, including 11,606 missense, 860 nonsense, and 418 frameshift insertions or deletions. Preliminary results identified mutations in HER2 that were associated with the failure to achieve pCR in several cases. Furthermore, tumors assigned to the HER2-enriched subtype by RNA-seq analysis were more likely to achieve a pCR (19 compared to 8) than tumors with genomic features indicative of either the luminal or basal-like subtypes (3 compared to 12); a significant difference in the proportion of cases that achieve pCR (Fisher's exact test p-value = 0.0032). The identification of these features suggests that it may be possible to predict, at the time of diagnosis, those patients who will not respond to the current standard of care for HER2-positive breast cancer. Citation Format: Lesurf R, Griffith O, Griffith M, Watson MA, Hoog J, Ellis MJ, Ota D, Suman VJ, Meric-Bernstam F, Leitch AM, Boughey JC, Unzeitig G, Buzdar AU, Hunt KK, Mardis ER. The genomics of response to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast cancer – Results from the ACOSOG Z1041 (Alliance) trial. [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 PD6-02.

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 Activating mutations in PIK3CA occur in approximately 40% ER+BC. MK-2206 (M), a pan-AKT inhibitor, induced apoptosis of ER+ BC under estrogen deprivation in preclinical studies. We conducted this neoadjuvant trial to determine the pathologic complete response (pCR) rate of M plus anastrozole (A) for PIK3CA mutant (Mut) ER+ BC. Methods This single arm open label study of M+A used a 2-stage Simon phase II design (stage 1, n=16; stage 2, n=13, alpha=0.10, power=0.90) to test whether pCR rate <1% (based on historical data with A alone), against the alternative that pCR rate ≥15% in PIK3CA Mut ER+ BC. At least 1 pCR in stage 1 was required to proceed to stage 2. Eligible patients (pts) with clinical stage II or III ER+HER2- BC were pre-registered and proceeded to a research tumor biopsy for PIK3CA sequencing, followed by treatment with daily A monotherapy for 28 days (cycle 0). Pts with PIK3CA Mut BC were subsequently registered, underwent a second biopsy, and started M (150mg PO weekly) with daily A on cycle 1 day 1 (C1D1) for a maximum of four 28-day cycles followed by surgery. Goserelin was added for premenopausal pts. A tumor biopsy on C1D17, 17 days post the start of M, was performed. Those with C1D17 Ki67 >10% discontinued study treatment. pCR was defined as no invasive cancer in the breast and the lymph nodes. Tumor specimens collected at all timepoints are being analyzed for markers of proliferation, apoptosis, and PI3K pathway activity, gene expression microarray, intrinsic subtypes, and next generation sequencing of 83 genes. Results Of the 51 pts pre-registered, 35 pts did not register due to no PIK3CA mutation (n=22), inadequate specimen for testing (n=6), physician/pt decision (n=7). The remaining 16 pts (median age: 58, range: 40-77 years) received combination therapy. Three pts did not complete 4 cycles due to C1D17 Ki67 >10% (n=2) and intolerability (grade (Gr) 4 transaminase elevation in C1, n=1). Other severe toxicities possibly related to M included Gr 3 rash (25%) and pruritus (12.5%). Of the 13 pts completed study therapy and underwent surgery, all had residual disease in the breast and 7 also had positive nodes. Table 1 summarized changes in Ki67 during treatment. ComparisonsnAbsolute changes in Ki67 median (range)Wilcoxon signed rank p-valueC1D1 relative to pre-registration11-17.0% (-49.8 to 4.1%)0.0020C1D17 relative to pre-registration14-16.4% (-51.4 to 4.1%)0.0004C1D17 relative to C1D112-1.5% (-18.6 to 15.8%)0.9697C1D1, biopsy post 28 days of A alone; C1D17 biopsy post 17 days on combination therapy Although Ki67 levels post A monotherapy (C1D1) or M+A (C1D17) were significantly lower than that of pre-registration samples, Ki67 did not differ between C1D17 and C1D1 samples. Other correlative studies are ongoing and results will be presented. Conclusion Despite the small sample size, biomarker analysis on serial biopsy specimens demonstrated that M+A is unlikely to be more effective than A alone in PIK3CA Mut ER+ BC. This trial demonstrated the feasibility of genomic sequencing for pt selection and the value of a small, well-designed proof-of-principle neoadjuvant trial for the evaluation of targeted agents. Citation Format: Ma CX, Suman VJ, Goetz M, Northfelt D, Burkard M, Ademuyiwa F, Naughton M, Margenthaler J, Aft R, Gray R, Tavaarwerk A, Wilke L, Haddad T, Moynihan T, Loprinzi C, Hieken T, Hoog J, Guo Z, Han J, Vij K, Mardis E, Sanati S, Al-Kateb H, Doyle L, Erlichman C, Ellis MJ. A phase II neoadjuvant trial of MK-2206, an AKT inhibitor, in combination with anastrozole for clinical stage 2 or 3 PIK3CA mutant estrogen receptor positive HER2 negative (ER+HER2-) breast cancer (BC). [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 P5-13-04.

Abstract Background: Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses. Clinical management of TNBC is limited to surgery, chemotherapy and radiation due to lack of estrogen receptor alpha and HER2 expression. Recently, we have shown that approximately 40% of TNBCs express estrogen receptor beta (ERβ) and have begun to explore the possibility that this receptor could be utilized as a novel therapeutic target for this disease. Methods: To examine the biological functions of ERβ in TNBC, novel ERβ expressing TN cell lines (MDA-MB-231 and Hs578T) were developed. In vitro experiments were employed to determine alterations in the global gene expression profiles, biological pathways, proliferation rates, and cell cycle progression following estrogen or ERβ-specific agonist treatment. Cell line xenografts were also established in athymic ovariectomized nude mice to examine tumoral responses to ERβ targeting agents and to investigate gene and protein expression patterns as well as potential serum biomarkers indicative of therapeutic response. Additionally, using the resources of the Mayo Clinic Breast Cancer Genome Guided Therapy Study (BEAUTY), we have identified, and begun to analyze, ERβ+ and ERβ- patient derived xenografts (PDX) established from women with TNBC. Results: Our studies have revealed that both estrogen and multiple ERβ-specific agonists elicit significant anti-proliferative effects in ERβ+ TNBC cells primarily through a G1/S phase cell cycle arrest. These anti-proliferative effects appear to be mediated by cystatins, a family of small secreted cysteine protease inhibitors which are highly induced following estrogen and ERβ-specific agonist treatment. Conditioned media isolated from estrogen or ERβ-specific agonist treated cells decreased the proliferation rates of multiple non-ERβ expressing cell lines; effects that were completely reversed when cystatins were depleted from the media. In addition, we have shown that activation of ERβ, and the subsequent induction of cystatin gene expression, leads to suppression of canonical TGFβ signaling through multiple mechanisms including suppression of TGFβR2 expression, induction of Smad7 expression and blockade of TGFβ ligand-mediated activation of this pathway both in vitro and in vivo. Finally, ERβ+ TNBC PDXs exhibit significantly decreased tumor growth rates in estrogen-treated mice compared to ERβ- TN breast tumors. Conclusions: Our in vitro and in vivo data show that estrogen and ERβ-specific agonists elicit anti-cancer effects in ERβ+ TNBC. These effects appear to be mediated, in part, by cystatins through their inhibitory effects on canonical TGFB signaling, a pathway known to drive TNBC progression. Importantly, these data lay the foundation for studies aimed at examining the ability to therapeutically target ERβ in TNBC patients. Citation Format: Reese J, Bruinsma E, Subramaniam M, Suman V, Pitel K, Kalari K, Yu J, Wang L, Goetz M, Ingle J, Hawse J. ERβ elicits tumor suppressive effects in triple negative breast cancer through the induction of cystatins and suppression of TGFβ signaling. [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 P5-04-01.

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: Recent studies have indicated that vaccination can protect against cancer development. One key aspect of developing vaccines is circumventing peripheral tolerance by identifying subdominant epitopes that are unique to the deregulated tumor microenvironment. While existing subdominant epitope vaccines are showing efficacy in preventing cancer, these vaccines are applicable only for subsets of patients with specific HLA subtypes. Therefore, we recently developed a degenerate HER2 subdominant epitope-based vaccine that should be useful in approximately 85% of all patients. The vaccine consists of a pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) that are naturally processed and are designed to elicit helper T cell immunity, the cornerstone of immune surveillance. Here we present Phase I trial results of this multi-peptide HER2 vaccine. Methods: Eligible women had HER2+ breast cancer (Stages II-III) and had completed standard treatment (i.e. surgery, chemotherapy, and trastuzumab) at least 90 days prior to enrollment and were rendered disease free. Vaccine included the above epitope pool along with adjuvant GM-CSF. Patients were vaccinated six times over six months and were monitored for toxicity at each visit. Peripheral blood samples were collected for immune responses evaluating for T cell and antibody immunity. Endpoints were safety and immunogenicity leading to the development CD4 helper T cells that recognized naturally-processed HER2. Results: Twenty-two subjects (age 33 to 69 years) were enrolled. At the present analysis, 21 have completed all 6 vaccination cycles; one patient withdrew after developing a grade 1 injection site reaction during the first vaccination cycle. Twenty patients have had LVEF measured after vaccination; only 2 patients had an LVEF drop of 10% or more but remained in the normal LEVF range. One severe toxicity was reported: a grade 3 INR increase considered unrelated to treatment. Mild to moderate (grade 1-2) toxicities included injection site reactions, fatigue, and white blood cell count decreases. All patients were alive at analysis and only one experienced a recurrence (median follow-up 507 days, range 22 – 844). Twenty patients have had immune response assessments. Vaccine induced T cell immunity was observed in 94% of patients to p59, in 94% of patients to p88, in 82% of patients to p422, and in 74% of patients to p885. Importantly, T cell immunity to naturally processed HER2 proteins occurred in 94% of patients. The mean number of T cells specific for each peptide, post vaccination, ranged from 349–528 T cells per million peripheral blood mononuclear cells (PBMCs). The mean number of T cells specific for whole HER2 protein was 783 T cells per million PBMCs compared to a mean of 898 T cells/million PBMCs specific for the foreign tetanus toxin. In contrast to T cell responses, modestly increased antibody immunity to HER2 occurred in 35% of patients, consistent with the T cell-inducing design of the vaccine. Conclusion: Our results show that it is possible to develop vaccines with broad HLA coverage that circumvent natural tolerance and induce tumor antigen-specific immunity in the vast majority of patients. Citation Format: Knutson KL, Kalli KR, Block MS, Hobday TJ, Padley DJ, Erskine CL, Dockter T, Suman VJ, Wilson G, Degnim AC. Robust generation of T cell immunity to HER2 in HER2+ breast cancer patients with a degenerate subdominant HLA-DR epitope vaccine. [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 P2-11-02.

Abstract Background ACOSOG Z1041 (Alliance) found pathological complete response rates in women with operable HER2-positive breast cancer were similar with FEC → P+T (Arm 1) and P+T → FEC+T (Arm 2) where treatment was administered as 5-FU 500 mg/m2, epirubicin 75 mg/m2 and cyclophosphamide 500 mg/m2 day 1 of a 21-day cycle x 4; paclitaxel 80 mg/m2 weekly x 12 and trastuzumab 4 mg/kg once then 2 mg/kg weekly x 11. We now report DFS and OS results with a median follow up of 4.4 years (range: 26 days to 6.2 years). Methods All patients who began study treatment were included in the analyses. Stratified log rank tests and stratified proportional hazard models were used to assess differences in DFS and OS from randomization between treatment arms. Results From September 15, 2007 to December 15, 2011, 282 women with HER2-positive breast cancer were enrolled. Two patients randomized to arm 1 withdrew consent prior to treatment and are excluded from these analyses. Patient and disease characteristics of the remaining 280 women (arm 1, n=138; arm 2, n=142) were similar between treatment arms. Recurrences and deaths are shown in the table. DFS were not found to differ with respect to treatment arm (stratified logrank p=0.6870; HR stratified (Arm2/Arm1)=0.88; 95%CI: 0.48-1.61). Also, OS was not found to differ with respect to treatment arm (stratified logrank p=0.8790; HR stratified (Arm2/Arm1)=1.07; 95%CI: 0.43-2.69). Conclusions Concurrent administration of trastuzumab with anthracyclines offers no additional benefit in terms of achieving pathologic complete response or improving survival and is not needed. FEC → P+TP+T → FEC+T Arm 1Arm 2n138142Recurrences2022Deaths811 Grant Support: U10 CA76001 The study is registered with ClinicalTrials.gov, number NCT00513292. Citation Format: Buzdar AU, Suman VJ, Meric-Bernstam F, Leitch AM, Ellis MJ, Boughey JC, Unzeitig GW, Royce ME, Ewer MS, Hunt KK. Disease-free (DFS) and overall survival (OS) data from ACOSOG Z1041 (Alliance) a randomized neoadjuvant trial comparing FEC followed by paclitaxel plus trastuzumab (FEC → P+T) with paclitaxel plus trastuzumab followed by FEC plus trastuzumab (P+T → FEC+T) in HER2-positive operable 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 P1-14-02.