Goetz, Matthew P.

5 Background: Triple-negative breast cancer (TNBC) is an immunogenic breast cancer subtype, with a greater percentage of tumors containing tumor-infiltrating lymphocytes (TILs) compared to non-TNBC. TILs are prognostic for recurrence and predictive for chemotherapy benefit in TNBC, but not in estrogen receptor (ER)-positive tumors. A subset of TNBC expresses the androgen receptor (AR), and resembles ER-positive breast cancer in terms of demographics (older age) and clinical course (later recurrences that tend to involve bone). However, little is known regarding the association between TILs and AR expression in TNBC. Methods: From a cohort of 9982 women with surgically-treated non-metastatic breast cancer, patients who met criteria for TNBC (ER/PR < 1% and HER2-negative) by centralized-pathology review were included as previously published [Leon-Ferre et al, Breast Cancer Res Treat 2017]. Stromal TILs in these tumors were quantified on full-face hematoxylin and eosin sections from the surgical specimen, following the 2014 TIL Working Group recommendations [Salgado et al, Ann Oncol 2014]. The expression of AR [EPR1535(2), Abcam] was scored as a continuous variable (1% increments), and categorized as absent (0%), low (1-25% ), low-moderate (26-50%) moderate (51-75%) and high (> 75%) and correlated with TIL density. Results: 605 patients met criteria for TNBC. The median age was 56 years. Most tumors were T1-2 (95%), N0-1 (86%), and high grade (88%). The median stromal TIL content was 20% (0-90%). Tumor tissue was available for AR staining in 509/605 patients. 165 (32%) tumors expressed AR as follows: low: 12%, low-moderate: 7%, moderate: 5%, and high: 8%. Apocrine tumors (n = 30) expressed AR most frequently (86%) and at higher levels (AR high: 50%). For all 509 tumors, the median stromal TIL content according to AR expression was AR absent: 20%, AR low: 20%, AR low-moderate: 15%, AR high: 10%. AR and stromal TILs (both as continuous variables) demonstrated a weak inverse correlation (correlation coefficient -0.1064, p = 0.017). Conclusions: AR expression in TNBC is associated with lower levels of stromal TILs. Additional data correlating the AR, stromal TILs and outcomes in this cohort will be presented at the meeting.

Tamoxifen is biotransformed by CYP2D6 to 4‐hydroxytamoxifen and 4‐hydroxy N‐desmethyl tamoxifen (endoxifen), both with greater antiestrogenic potency than the parent drug. Patients with certain CYP2D6 genetic polymorphisms and patients who receive strong CYP2D6 inhibitors exhibit lower endoxifen concentrations and a higher risk of disease recurrence in some studies of tamoxifen adjuvant therapy of early breast cancer. We summarize evidence from the literature and provide therapeutic recommendations for tamoxifen based on CYP2D6 genotype.

Purpose Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, demonstrated efficacy as monotherapy and in combination with fulvestrant in women with hormone receptor (HR)–positive, human epidermal growth factor receptor 2 (HER2)–negative advanced breast cancer previously treated with endocrine therapy. Methods MONARCH 3 is a double-blind, randomized phase III study of abemaciclib or placebo plus a nonsteroidal aromatase inhibitor in 493 postmenopausal women with HR-positive, HER2-negative advanced breast cancer who had no prior systemic therapy in the advanced setting. Patients received abemaciclib or placebo (150 mg twice daily continuous schedule) plus either 1 mg anastrozole or 2.5 mg letrozole, daily. The primary objective was investigator-assessed progression-free survival. Secondary objectives included response evaluation and safety. A planned interim analysis occurred after 189 events. Results Median progression-free survival was significantly prolonged in the abemaciclib arm (hazard ratio, 0.54; 95% CI, 0.41 to 0.72; P = .000021; median: not reached in the abemaciclib arm, 14.7 months in the placebo arm). In patients with measurable disease, the objective response rate was 59% in the abemaciclib arm and 44% in the placebo arm ( P = .004). In the abemaciclib arm, diarrhea was the most frequent adverse effect (81.3%) but was mainly grade 1 (44.6%). Comparing abemaciclib and placebo, the most frequent grade 3 or 4 adverse events were neutropenia (21.1% v 1.2%), diarrhea (9.5% v 1.2%), and leukopenia (7.6% v 0.6%). Conclusion Abemaciclib plus a nonsteroidal aromatase inhibitor was effective as initial therapy, significantly improving progression-free survival and objective response rate and demonstrating a tolerable safety profile in women with HR-positive, HER2-negative advanced breast cancer.

Abstract Purpose: Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor–positive (ER+) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in PIK3CA-mutant ER+ breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in PIK3CA mutant ER+ breast cancer. Experimental Design: Potential eligible patients with clinical stage II/III ER+/HER2− breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor PIK3CA sequencing. Patients positive for PIK3CA mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17. Results: Fifty-one patients preregistered and 16 of 22 with PIK3CA-mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 &gt;10% (n = 2) and toxicity (n = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an ESR1 mutation at surgery. Conclusions: MK-2206 is unlikely to add to the efficacy of anastrozole alone in PIK3CA-mutant ER+ breast cancer and should not be studied further in the target patient population. Clin Cancer Res; 23(22); 6823–32. ©2017 AACR.

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor–positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Abstract Introduction: MutHER is a phase II trial that demonstrated the anti-tumor activity of the pan-HER inhibitor neratinib in HER2mut, non-amplified MBC. The major challenges to accrue to this trial were the large number of pts to screen for the 2-3% HER2mut population and the high rate (24%) of poor quality tumor DNA for sequencing. The goals of this ctDNA study were: 1) the concordance of HER2mut detected by ctDNA versus tumor testing; 2) the incidence of ctDNA HER2mut in HER2 non-amplified MBC; 3) changes in HER2mut variant allele frequency (VAF) on neratinib therapy. Methods: A sample size of 30 negative (neg) controls was needed to ensure 90% confidence if ctDNA testing has &gt;90% specificity in detecting HER2mut. Thus, plasma from MBC pts obtained at screening for MutHER trial (Neg control: 40 pts without HER2mut on tumor testing; Positive (pos) control: 14 pts with known HER2mut who received neratinib) were subjected to Guardant360 ctDNA 70-gene panel sequencing (all exons of HER2 included). ctDNA from the 14 neratinib treated pts were also analyzed at week (wk) 4 and upon progression. ctDNA data from MBC pts clinically tested at Guardant Health were interrogated for HER2mut incidence. Results: Among the 14 pts with tumor pos for HER2mut, ctDNA sequencing identified the same HER2mut in 11, discrepant HER2mut in 1, and neg in 2. The 2 pts with ctDNA neg for HER2mut had progressive disease (PD) and stable disease (SD &gt; 6 months) on neratinib, respectively. Among the 40 neg controls, 8 were not evaluable (no detectable ctDNA or assay unsuccessful) and all 32 successfully sequenced cases were neg for HER2mut. The sensitivity and specificity of ctDNA for HER2mut detection was 11/14 (79%, 90% CI: 53-94%) and 32/32 (100%, 90% CI: 91-100%), respectively. Among the 11 paired baseline and wk 4 samples, 9 (82%) had lower HER2mut VAFs at wk 4 than at baseline, with 1 complete response (CR), 1 partial response (PR), 5 SD, and 4 PD at wk 8 as best tumor response. Two pts had higher wk 4 ctDNA HER2mut VAFs and both had radiographic PD at wk 8. The absolute HER2mut VAF levels at wk 4 were significantly associated with TTP (Spearman rho=-0.69, p=0.02) and tumor size change (rho=0.67, p=0.05). The HER2mut VAFs were significantly higher at progression than wk 4 in all pts (p&lt;0.01). One pt acquired a new HER2mut T798I, which is analogous to the gate-keeper mutation EGFR T790M. The incidence of HER2mut without amplification in unselected consecutive MBC clinically tested by Guardant360 was 3% (48/1,584), with mutation pattern similar to published tumor testing data. Conclusions: ctDNA sequencing is sensitive and highly specific in detecting HER2mut, offering a non-invasive method to identify pts for trials of HER2mut-targeted therapy. Decreased HER2mut VAFs at wk 4 was observed in 82% of cases, consistent with the on-target effect of neratinib. Increased HER2mut VAFs at wk 4 is a potential early marker of progression. Citation Format: Cynthia Ma, Ron Bose, Feng Gao, Rachel Freedman, Melinda Telli, Gretchen Kimmick, Eric Winer, Michael Naughton, Matthew Goetz, Christy Russell, Debu Tripathy, Melody Cobleigh, Andres Forero, Timothy Pluard, Carey Anders, Shana Thomas, Jill Anderson, Caroline Bumb, Kimberly Banks, Richard Lanman, Richard Bryce, Alshad Lalani, John Pfeifer, Daniel Hays, Mark Pegram, Kimberly Blackwell, Philippe Bedard, Hussam Al-Kateb, Matthew Ellis. Circulating tumor DNA (ctDNA) sequencing for HER2 mutation (HER2mut) screening and response monitoring to neratinib in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT011. doi:10.1158/1538-7445.AM2017-CT011

Abstract Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ- secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain (NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1 transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming. Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray. Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling in MDA-MB 231 cells. Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1 xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities. MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72 hours viable cells were injected into the tail vein of immune-compromised mice to develop lung metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases, those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions. Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”, these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor stemness and early onset of distant metastases. Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2017-4851

Abstract Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection. Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut). Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression. Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687–95. ©2017 AACR.