Northfelt, DW

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 Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma. [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 P6-03-13.

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: Data regarding patients (pts) and physicians' preferences for modern adjuvant chemotherapy (CT) are limited. Prior studies suggested that most pts with early stage breast cancer were willing to receive 6 months of adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) for modest survival benefits (e.g. most women would have accepted 3-6 months extension of life). Methods: E5103 was a phase III trial which randomized node positive or high risk node negative breast cancer pts to receive adjuvant CT (doxorubicin, cyclophosphamide and paclitaxel) with either placebo or bevacizumab. Telephone based surveys were administered to all pts enrolled on E5103 between 01/Jan/10 and 08/Jun/10, as part of a Decision-Making/Quality of Life component. Results presented here are part of the 18 months post-enrollment follow-up. Pts were asked to rate the survival benefit needed to justify 6 months of CT. A complementary survey was sent to all physicians who registered at least one pt on E5103. Results: 465 out of 519 eligible pts (90%) responded to this survey at 18 months. Main reasons for non response were: inability to reach the patient (6%) or patient refusal (2%). Median pts age was 51 (25-76); 42% of pts had at least a college degree. The majority had at least Stage II cancer. 179 (16%) physicians participated, among whom median age was 50 (35-70). The median years in practice was 17 (3-38); 78% of physicians worked on large size practices, 72% saw at least 5 new breast cancer pts/month, and 77% enroll between 1-4 pts on trials/month. We found considerable variation in pts preferences particularly for modest survival benefits: a substantial minority of pts (24%) would consider 6 months of CT definitely worthwhile for 1 month survival benefit, 18% would possibly consider it and 56% would not. The percentage considering CT definitely worthwhile increased with greater benefit, but did not reach 100%, even with 24 months survival benefit. About half of pts considered 6 months of CT definitely worthwhile for 9 months benefit, 70% for 12 months and 84% for 24 months. Physicians were less likely to accept CT for a small chance of benefit (34% of pts vs. 5% of physicians would definitely consider CT worthwhile for 2 months of benefit). For longer benefit, pts and physicians choices were similar (84% of pts vs. 92% of physicians would definitely consider CT worthwhile for 24 months benefit). Table Yes, definitely worthwhileYes, maybeNo, not worthwhileNo answerConsider 6 months of CT to live:PtsPhysiciansPtsPhysiciansPtsPhysiciansPts/Physicians*1 month longer24%3%18%15%56%80%2%2 months longer34%5%23%32%41%60%2%6 months longer44%32%35%54%19%12%2%9 months longer53%51%34%42%11%5%2%12 months longer70%75%23%22%5%1%2%24 months longer84%92%12%5%2%1%2%n Pts= 465; n Physicians= 179; * equal results in both groups Conclusions: This subgroup of pts who had undergone modern adjuvant CT in a large multicenter randomized controlled trial and these physicians who registered pts on the same trial had different cutoffs for acceptable levels of benefits and risks when considering adjuvant chemotherapy. It is important to engage pts in determining whether CT is or is not a "reasonable" option for treatment. Citation Format: Vaz Luis I, O'Neill A, Sepucha K, Miller KD, Baker E, Dang CT, Northfelt DW, Winer EP, Sledge GW, Schneider BP, Partridge A. Survival benefit needed to undergo chemotherapy: Patients and physicians preferences. [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 P5-11-02.

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: The identification of organ-specific targetable signatures may help design more effective treatment for patients with metastatic breast cancer (MBC). We took a multi-OMIC approach to assess whether the AKT-mTOR pathway is globally activated during metastatic progression or whether it represents an organ-specific target. Methods: Snap frozen biopsies from 25 MBC patients enrolled in a prospective phase II trial were used. Sites of metastasis were classified as liver (n=8) and others (n=17), the latter including cutaneous, lung, lymph nodes, and intra-abdominal lesions. Signaling analysis of the 25 cases was performed using Reverse Phase Protein Microarray (RPPA) coupled with Laser Capture Microdissection. Activation of the AKT-mTOR pathway was quantified as phosphorylation of AKT (S473) and the mTOR target p70S6 (T389). Matched exome (WES) and RNASeq data were available for 17 of 25 patients, five with liver metastases. Sequencing data was processed using an in-house developed pipeline to identify somatic events including coding mutations, copy number alterations, gene fusions, and differential expression. Activation of the AKT-mTOR pathway and sequencing data were compared between hepatic and non-hepatic lesions using an integrated RPPA and genomic approach. Results: Among liver metastases, AKT was activated in 4 of the 8 (50.0%) patients, while 6 of the 8 cases (75.0%) showed activation of p70S6. Sequencing data revealed mutation of PIK3CA in 4 of the 5 liver metastases (80.0%). Three of the PIK3CA mutated specimens with catalytic domain mutations (codons 1023 and 147) demonstrated co-activation of AKT and p70S6, while the fourth case, containing a helical domain mutation (E542K), had activation of p70S6 only. The PIK3CA wild-type liver metastasis demonstrated low activation of AKT and p70S6. For non-hepatic metastases AKT was activated in 2 of the 17 cases (11.8%) and p70S6 in 5 of the 17 patients (29.4%). Discussion: Although these results need further validation, activation of the AKT-mTOR pathway appears to be a hepatic specific signature in MBC and could be used for the selection of targeted agents for hepatic lesions. Citation Format: Pierobon M, Wong S, Reeder A, Anthony SP, Robert NJ, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Petricoin EF, Carpten J. The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from 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 P2-05-21.

Abstract Background: Nal-IRI (MM-398, nanoliposomal irinotecan) is designed for extended circulation relative to free irinotecan and to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent conversion to SN-38 in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with response to therapy. In phase I studies of nal-IRI, activity has been shown in metastatic breast cancer (MBC), pancreatic and colorectal cancer. Ferumoxytol (FMX) is an iron-oxide superparamagnetic nanoparticle that has been used off-label for its MRI contrast properties. FMX has long-circulating pharmacokinetics and is taken up by TAMs with similar distribution patterns to nal-IRI in preclinical models. A single site pilot study established the feasibility of performing quantitative FMX MRI. Thirteen patients with advanced cancer (3 with ER/PR+ MBC) were imaged with FMX MRI and treated with nal-IRI. Median tumor lesion FMX uptake in the pilot study was 32.6 and 34.5 μg/mL at 1 h and 24 h, respectively. Lesions with FMX uptake above the median were associated with greater reductions in tumor size following treatment with nal-IRI as determined by CT lesion measurements. The relationship between FMX levels in tumor lesions and nal-IRI activity may serve as a potential biomarker for nal-IRI deposition and response in solid tumors. This study has been expanded to include additional MBC patients to further evaluate the technical feasibility of FMX MRI at multiple study sites, and to evaluate activity of nal-IRI in patients with MBC. Trial Design: Three cohorts of 10 patients with MBC in the following categories will be enrolled: ER and/or PR positive/HER2-negative, triple negative (TNBC) and MBC with brain metastases. An imaging phase will be followed by a treatment phase. The imaging phase consists of a baseline MRI scan, FMX infusion, and follow-up MRI scans at 1-4 and 24 h after infusion. The treatment phase begins 1-6 days after imaging and consists of nal-IRI 80 mg/m2 q2w. A pretreatment biopsy is required for correlative studies. Study Objectives: The primary objective of this multisite expansion is to investigate the feasibility of FMX quantitation in tumor lesions at multiple lesion sites in breast cancer. The secondary objective is to characterize the efficacy of nal-IRI in patients with metastatic breast cancer. Eligibility Criteria: The key inclusion criteria include patients with MBC, ECOG 0 or 1 with adequate bone marrow reserve and no prior topoisomerase 1 inhibitor or anti-VEGF treatment. ER and/or PR positive/HER2-negative and TNBC patients must have had 1-3 prior lines of chemotherapy in the metastatic setting and have at least 2 measurable lesions. Patients with brain metastasis must be neurologically stable and have new or progressive brain metastases after prior radiation therapy with at least one lesion measuring ≥ 1 cm in longest diameter on gadolinium-enhanced MRI. Status: This trial is currently recruiting patients. Citation Format: Sachdev JC, Ramanathan RK, Raghunand N, Anders C, Munster P, Minton S, Northfelt D, Blanchette S, Campbell K, Lee H, Klinz SG, Hendriks BS, Moyo V, Fitzgerald JB, Korn RL. A phase 1 study in patients with metastatic breast cancer to evaluate the feasibility of magnetic resonance imaging with ferrumoxytol as a potential biomarker for response to treatment with nanoliposomal irinotecan (nal-IRI, MM-398). [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 OT3-02-14.

Abstract Background: A previous feasibility trial (E2104 – Ann Oncol 23(2):331–7,2012) suggested incorporation of B into anthracycline-containing adjuvant therapy was feasible but ongoing cardiac monitoring was required to define the true impact of B on cardiac function. Methods: Patients (pts) were assigned 1:2:2 to one of three treatment arms. In addition to doxorubicin and cyclophosphamide followed by weekly paclitaxel, patients received either placebo (Arm A – AC>T) or B during chemotherapy (Arm B - BAC>BT), or B during chemotherapy followed by B monotherapy (15 mg/kg q3wk) for an additional 10 cycles (Arm C – BAC>BT>B). Randomization was stratified and B dose adjusted for choice of AC schedule (classical q3wk − 15 mg/kg; dose dense(dd) q2 wk − 10 mg/kg). When indicated, radiation and hormonal therapy were administered concurrently with B (for Arm C pts). The primary cardiac endpoint was the incidence of clinically apparent cardiac dysfunction (CHF)defined as symptomatic decline in left ventricular ejection fraction (LVEF) to below the lower limit of normal (LLN) or symptomatic diastolic dysfunction as assessed by independent review. Cumulative toxicity data as of Jan 23, 2012 are presented. Results: From 11.07 to 2.11, 4994 pts were enrolled. Median age was 52; 80% received ddAC. Chemotherapy associated toxicities including myelosuppression (Grade 4 neutropenia 16/20/19%) and neuropathy (Grade ≥ 3 8/8/8%) were similar across all arms. Grade ≥ 3 hypertension/thrombosis/proteinuria/hemorrhage was reported by 7/3/<1/<1% of B-treated pts. 99 pts developed CHF, most commonly reported at the post-AC or post-T evaluation. After a median follow-up of 26 months, the cumulative incidence of clinical CHF at 15 months from randomization in Arm A/B/C was 1.0/1.7/2.9% respectively. Median age of CHF pts was 57; median baseline LVEF of CHF pts was 60. Conclusion: Incorporation of B into anthracycline and taxane containing adjuvant therapy results in a significant but small increase in clinical CHF. The rate of clinical CHF is similar to that predicted by E2104 (2.5–2.9%) and reported In the FDA label for anthracycline pre-treated pts(3.8%). No unexpected toxicities were encountered. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-17-01.