Goetz, Matthew P.

Abstract Background: The prognostic value of stromal tumor-infiltrating lymphocytes (TILs) as a biomarker for triple-negative breast cancer (TNBC) has been extensively demonstrated in patients (pts) receiving (neo)adjuvant systemic therapy. In addition, several small studies suggest that a subset of pts with early-stage TNBC and high TILs have excellent long-term outcomes, even in the absence of systemic therapy [1-3]. However, data on the absolute risk of TNBC recurrence according to TIL levels in the absence of systemic therapy are limited and critical to inform the design of future systemic therapy de-escalation clinical trials. Methods: We conducted an individual patient data pooled analysis of 12 international cohorts of pts with TNBC treated with locoregional therapy but no systemic therapy. TNBC was defined as tumors with estrogen and progesterone receptor of < 1% and HER2 negative (IHC 0, 1+ or IHC 2+ and FISH negative) per local evaluation. TILs were locally assessed in hematoxylin & eosin-stained slides according to the International Immuno-Oncology Biomarker Working Group guidelines (www.tilsinbreastcancer.org). We used the Kaplan-Meier method to assess survival outcomes according to prespecified TIL thresholds: 30% and 50%. Confidence intervals (CI) for survival probabilities were calculated using a percentile bootstrap method. The primary endpoint was invasive disease-free survival (iDFS, STEEP 2.0 definition). Key secondary outcomes included recurrence-free survival (RFS), distant disease-free survival (DDFS) and overall survival (OS). Results: 1,835 pts diagnosed with TNBC between 1982 and 2017 who did not receive systemic therapy were included. The median age at diagnosis was 56 (IQR 38-71). Menopausal status was known in 1,184 women, of whom 78% were post-menopausal. The median tumor size was 2.0 cm (IQR 1.2-2.6). Most pts (87%) had no axillary lymph node involvement (N0). Most tumors were invasive ductal carcinoma (74%) and grade 3 (70%). The median level of TILs was 15% (IQR 5-40). The median duration of follow-up was 30.4 years (95% CI 29.9, 31.1). A total of 950 (52%) iDFS, 828 (45%) RFS, 767 (42%) DDFS events, and 604 (33%) deaths were observed. In multivariable analyses, higher TILs were independently associated with improved iDFS, RFS, DDFS, and OS beyond clinicopathological factors (likelihood ratio p< 10e-6). Each 10% increment in stromal TILs was associated with an 8% (95% CI: 6-11), 10% (95% CI: 7-13), and 13% (95% CI: 10-15) reduction in the risk of experiencing an iDFS, RFS or DDFS event, and with a 12% (95% CI: 9-15) reduction in the risk of death. iDFS, RFS, DDFS and OS rates according to different TIL thresholds and nodal status are shown in the Table. Of note, the RFS estimates (which exclude second non-breast primaries and contralateral breast cancers) were consistently higher than the iDFS counterparts (which include both), consistent with a high rate of contralateral breast cancers and second primary tumors in this cohort. Notably, patients with node-negative—and especially stage I—TNBC with high TILs had excellent survival rates at 10-year follow-up. Conclusion: TILs are highly prognostic in pts with systemically untreated early-stage TNBC. Pts with pN0 (and especially stage I) TNBC with high TILs exhibited very favorable long-term outcomes even in the absence of systemic therapy. These data define the natural history of TIL-rich TNBC pts and are crucial to identifying the optimal patient population for future chemotherapy and immunotherapy de-escalation clinical trials. References: [1] Leon-Ferre et al, 2017, PMID: 28913760 [2] Park et al, 2019, PMID: 31566659 [3] de Jong et al, 2022, PMID: 35353548 Table 5 and 10-year survival endpoints according TIL level, nodal status, and stage Citation Format: Roberto A. Leon-Ferre, Sarah Flora Jonas, Roberto Salgado, Sherene Loi, Vincent De Jong, Jodi M. Carter, Torsten Nielson, Samuel Leung, Nazia Riaz, Giuseppe Curigliano, Carmen Criscitiello, Vincent Cockenpot, Matteo Lambertini, Vera Suman, Barbro Linderholm, John WM Martens, Carolien HM van Deurzen, Mieke Timmermans, Tatsunori Shimoi, Shu Yazaki, Masayuki Yoshida, Sung-Bae Kim, Hee Jin Lee, Maria Vittoria Dieci, Guillaume Bataillon, Anne Salomon, Fabrice Andre, Marleen Kok, Sabine Linn, Matthew P. Goetz, Stefan Michiels. Stromal tumor-infiltrating lymphocytes identify early-stage triple-negative breast cancer patients with favorable outcomes at 10-year follow-up in the absence of systemic therapy: a pooled analysis of 1835 patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-05.

Abstract Background: Two poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) are currently FDA-approved for the treatment of HER2-negative metastatic breast cancer (MBC) in carriers of germline pathogenic variants (PVs) in BRCA1 or BRCA2 (BRCA1/2). This study explores the clinical outcomes of MBC patients treated with a PARPi. Methods: In this retrospective study, we included MBC patients treated with a PARPi between January 2017 and February 2022 at Mayo Clinic (Minnesota, Arizona, Florida, and Mayo Clinic Health Systems). We used the Kaplan Meier method to estimate the time-to-treatment-failure (TTF) and the log-rank test to compare different subsets. In addition, predictors of TTF were identified in a multivariate cox-proportional hazard regression model, including age at PARPi initiation, race, ethnicity, histology, estrogen receptor (ER), progesterone receptor (PR), and HER2 expression of the tumor, the number of prior therapies, type of PARPi, and PV carrier status (germline BRCA1/2 or PALB2 vs. somatic BRCA1/2 vs. other). Results: Sixty-five patients treated with PARPi (olaparib: 51; talazoparib: 14) were included in the final analysis. Fifty-five patients were carriers of germline PVs in BRCA1 (n=24, 37%), BRCA2 (n=27, 42%) or PALB2 (n=4, 6%), whereas ten patients (15%) had no germline PVs but the tumor had a somatic mutation in the homologous recombination-related (HRR) genes (7 in BRCA1/2, 2 in ATM, and 1 in CDKN2A and CDH1). At the data cutoff, 48 (74%) patients had discontinued PARPi due to progression or death. Fifteen (23%) patients required a dose reduction due to side effects. Occurrence of grade ≥ 3 side effects: anemia in 8, fatigue in 4, neutropenia in 2, and thrombocytopenia in 2 patients. Eight (15.7%) patients in the olaparib group and seven (50%) patients in the talazoparib group required a dose reduction for side effects. No patient on olaparib required drug discontinuation due to side effects, whereas two patients on talazoparib were switched to olaparib due to cytopenias and could tolerate olaparib. Median TTF in the overall population was 8 months (95% confidence interval [CI]: 6.4 – 9.6), and there was no difference (p=0.64) in TTF between the olaparib and talazoparib groups. Median TTF in the germline BRCA1, BRCA2, and PALB2 PV carriers were 7, 8, and 11 months, respectively (p=0.57). Among patients with somatic BRCA1/2 mutations, the median TTF was 4 months. Numerically, patients with HER2-positive tumors (n=8) had a shorter TTF compared to HER2-negative tumors (Median TTF: 4 vs. 8 months, p=0.098). No significant difference in TTF was observed by ER or PR status of the tumor, age at initiation of PARPi, the number of prior therapies, and prior use of platinum-based chemotherapy or CDK4/6 inhibitors. In multivariate analysis, HER2 positivity (hazard ratio [HR]: 8.0, 95% CI: 2.2 – 29.4, p=0.002), somatic BRCA1/2 mutations (HR: 7.6, 95% CI: 1.2 – 50.0, p=0.03) and somatic mutations in other HRR genes (HR: 19.1, 95% CI: 3.1 – 118.6, p=0.002) were associated with worse TTF. Conclusions: In the real world, PARPi were well-tolerated with promising time-to-treatment-failure (TTF) benefits comparable to data from clinical trials. Notably, relatively shorter TTF was observed in patients with somatic BRCA1/2 and other HRR gene mutations and HER2-positive MBC. These findings improve our understanding of the role of PARPi in MBC and will help to guide treatment decisions with PARPi in the clinical setting. Citation Format: Nusrat Jahan, Jodi Taraba, Karthik V. Giridhar, Roberto A. Leon-Ferre, Amye J. Tevaarwerk, Elizabeth Cathcart-Rake, Ciara C. O’Sullivan, Prema Peethambaram, Timothy J. Hobday, Kathryn Ruddy, Lida A. Mina, Pooja Advani, Felipe Batalini, Matthew P. Goetz, Tufia C. Haddad, Fergus J. Couch, Siddhartha Yadav. Clinical outcomes of metastatic breast cancer patients treated with poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi): the Mayo Clinic experience [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-22.

Abstract Background: In phase I/II clinical trials, the tamoxifen metabolite Z-endoxifen (ENDX) demonstrated substantial oral bioavailability and promising antitumor activity in endocrine-refractory estrogen-receptor positive breast cancer (ER+ BC) and other solid tumors, with ENDX plasma concentrations reaching as high as 5 µM, a concentration that far exceeds the ERα-targeting nanomolar concentrations. We previously identified protein kinase C beta 1 (PKCβ1), an oncogenic signaling kinase which regulates cell proliferation and tumorigenic transformation, as a novel ENDX receptor. ENDX-bound PKCβ1 (KD: 100 nM) and inhibited PKCβ1 kinase activity (IC50: 357 nM) in vitro, at concentrations achieved in phase I/II ENDX studies. Given the observations that ENDX induces antitumor activity in breast cancers resistant to endocrine therapies, including AIs, tamoxifen and fulvestrant, we postulated that ENDX may impact additional signaling pathways. Therefore, in this study we performed unbiased mass spectrometry to analyze the effects of ENDX on the phosphoproteome and total proteome. Methods: In estrogen unstimulated aromatase-expressing ER+/human epidermal growth factor 2 receptor negative (HER2-) MCF7AC1 breast cancer cells, the effects of ENDX on the phosphoproteome and total proteome were analyzed using ENDX concentrations achieved in tamoxifen treated patients (0.01 and 0.1 µM) and concentrations observed in the ENDX phase I/II trials (5 µM). NetWorKIN and RoKAI prediction tools were utilized to identify the upstream kinases of ENDX downregulated phosphoproteins. In estrogen unstimulated MCF7AC1 as well as in the ER+/HER2- T47D breast cancer cells, the ability of ENDX, tamoxifen, fulvestrant and PKC kinase inhibitor enzastaurin to block insulin or PKC agonist phorbol 12-myristate 13-acetate (PMA)-stimulated AKTSer473 phosphorylation was analyzed by immunoblot (IB) assay. Additionally, ENDX effects on AKTSer473 phosphorylation in the MCF7AC1 xenograft model in vivo was analyzed by IB assay. Further, the effects of PKCβ1 silencing in MCF7AC1 cells and the effects of AKT inhibitor MK-2206 treatment on growth and AKTSer473 phosphorylation in the MCF7AC1 as well as long-term estrogen deprived T47D (T47D-LTED) cell lines were analyzed by cell proliferation and IB assays. Results: In MCF7AC1 cells unstimulated with estrogen, ENDX at 5 µM but not at 0.01 and 0.1 µM concentrations inhibited growth, and induced apoptosis, suggesting an ERα-independent effect. Further, ENDX at 5 µM displayed three-fold greater effects in downregulating the phosphoproteome compared to the other concentrations, with minimal impact on the total proteome. Protein kinase C beta (PKCβ) and AKT1 were identified as the prevalent upstream kinases for ENDX downregulated protein phosphorylations. Notably, in estrogen unstimulated MCF7AC1 and T47D cells, ENDX at 5 µM attenuated phosphorylation of AKTSer473 and AKT substrates in vitro in the presence of insulin and in vivo. In MCF7AC1 cells, PMA-induced phosphorylation of PKCβ1 as well as AKTSer473 and AKT substrate phosphorylations were blocked by ENDX at 5 µM, with ENDX inducing PKCβ1 protein degradation both in the presence of insulin and PMA. Further, ENDX effects on growth and AKTSer473 phosphorylation were phenocopied by siRNA-mediated PKCβ1 knockdown as well as treatment with the pan-AKT inhibitor, MK-2206. Notably, the potent PKCβ1 kinase inhibitor, enzastaurin, had no effects either on PKCβ1 protein degradation nor on downstream AKT signaling. Conclusion: Taken together, these findings suggest that ENDX may exert anticancer activity via dual effects on blocking ERα as well as by targeting PKCβ1 for degradation, thus suppressing AKT signaling and induction of apoptosis. These preclinical findings will be studied in a planned neoadjuvant trial comparing ENDX with exemestane and ovarian function suppression (EVANGELINE) that will activate in the Fall of 2022. Citation Format: Swaathi Jayaraman, Xinyan Wu, Krishna R. Kalari, Xiaojia Tang, Mary Kuffel, Elizabeth S. Bruinsma, Santosh Renuse, James N. Ingle, Joel Reid, Matthew Schellenberg, John Hawse, Akhilesh Pandey, Matthew P. Goetz. Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-04.

Abstract Introduction: Acquired ESR1 mutations (mutESR1) after long-term endocrine therapy drive treatment resistance, metastasis, and poor prognosis for patients (pts) with ER+/HER2- metastatic breast cancer (mBC). Lasofoxifene (LAS), a selective estrogen receptor modulator, alone or with a CDK4/6 inhibitor (CDK4/6i) reduced tumor growth better than fulvestrant (Fulv) in mutESR1 BC xenograft models. ELAINE 1 is a randomized trial of LAS vs Fulv in pts with mutESR1 and prior progression on aromatase inhibitor and CDK4/6i. Preliminary results (ESMO 2022) showed that LAS prolonged median progression-free survival (mPFS) compared with Fulv with a favorable safety profile. Here, we report changes in ESR1 ctDNA mutant allele frequency (MAF) from baseline to 8 wks and their associations with clinical benefit (CB) and mPFS. Methods: ELAINE 1 pts were randomized to oral LAS 5 mg daily or IM Fulv 500 mg on days 1, 15, and 29, then every 4 wks, until disease progression or severe toxicity. ctDNA mutESR1 mutations (baseline and 8 wks) were assessed using the Sysmex Inostics OncoBeam or SafeSeq assays—which detect mutESR1 at low allele fractions. MAF changes from baseline to wk 8 were characterized as decreased (decrease in ESR1 MAF or fully cleared), increased (increase in MAF), or equivocal (polyclonal patients [>1 mutESR1] with some increasing and decreasing MAF trends); correlations with PFS and CB were explored. Efficacy measures included objective response rate (ORR), PFS, and CB at 24 wks (CB defined as response or stable disease ≥24 wks). Results: 103 pts received LAS (n=52) or Fulv (n=51). Most common baseline ESR1 variants detected were D538G (56%), Y537S (39%), Y537N (29%), E380Q (22%); 56 (54%) pts were polyclonal. Of the 61 pts with evaluable baseline and wk 8 ctDNA, LAS decreased mutESR1 MAF in 29/35 pts (83% [11 complete clearance]) while Fulv decreased mutESR1 MAF in 16/26 pts (61.5% [6 complete clearance]) (Table). mPFS with LAS was 8 and 4 mos for pts with decreased/cleared MAF and increased MAF, respectively, and with Fulv was 4.5 and 2.8 mos, respectively (Table). LAS decreased the common mutESR1 variants more frequently than Fulv (median relative change -87.1% vs -14.7%). In pts with decreased MAF, CB was observed in 16/29 LAS pts (55%) and 4/16 Fulv pts (25%). The predictiveness of ESR1 MAF clearance for CB was also explored. Of 11 pts with ESR1 MAF clearance taking LAS, 10 achieved CB, yielding a positive predictive value (PPV) of 90.9%. In contrast, 2/6 pts with ESR1 MAF clearance taking Fulv had CB for a PPV of 33.3%. Sensitivity for predicting CB based on direction of ESR1 MAF change was 94% with LAS and 80% with Fulv. In pts with Y537S MAF (n=33), LAS decreased Y537S in 13/15 (87%), with a median relative MAF decrease of 89%. In marked contrast, Fulv increased Y537S MAF in 11/18 pts (61%), corresponding to an MAF relative increase of 82%. LAS and Fulv resulted in complete clearance of Y537S MAF in 33% and 6% of pts, respectively. Conclusion: Our data demonstrate that LAS more effectively decreased or cleared mutESR1 than Fulv. Further, mutESR1 clearance was associated with prolonged PFS and more CB in LAS but not Fulv pts, suggesting that LAS results in robust mutESR1 target engagement. Taken together, our data suggest mutESR1 as a potential liquid biomarker for predicting response to LAS in mutESR1, endocrine-resistant mBC pts. Table. Change from baseline to week 8 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; PFS, progression-free survival. Citation Format: Matthew P. Goetz, Einav Gal-Yam, Daniel Stover, Sarah L. Sammons, Stephanie L. Graff, Grace Wang, Massimo Cristofanilli, Gary Riordan, Hillary S. Sloane, Dominic Carroll, Paul V. Plourde, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene or fulvestrant in the ELAINE 1 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-04.

Abstract Background: SG was approved in 2020 for the treatment of metastatic triple negative breast cancer (TNBC). The most common grade 3/4 adverse event in the ASCENT trial was neutropenia (51.2%) with a 6% incidence of febrile neutropenia. 1 Package insert recommendations do not endorse primary prophylactic growth factor support, rather only initiating if severe neutropenia occurs on treatment.2 Objective: This study retrospectively reviewed the utilization of growth factor support in patients (pts) with metastatic TNBC initiated on SG at each Mayo Clinic Enterprise site. Methods: We performed a multi-center, retrospective review of all pts with TNBC who received SG from January 2021 to December 2021 at Mayo Clinic sites in Minnesota, Florida, Arizona, and its community-based health system network. Data collected included history of neutropenia with previous cycles of SG resulting in a treatment delay, number of cycles, grade of neutropenia and cycle/day of treatment plan when growth factor added. Pts who received only one dose of SG were excluded. The Fisher’s exact test was utilized to compare the difference in the use of primary prophylaxis between sites. Results: 67 pts received at least two doses of SG. Within this cohort, 42 pts (63%) received growth factor support during treatment with SG. Growth factor support was most often added during the first two cycles (59.5%). A total of 12 patients initiated growth factor with no history of delays related to neutropenia and without neutropenia at the time of administration. Eleven of these pts had growth factor support added on Cycle 1 as primary prophylaxis. Primary prophylaxis was most common at Mayo Clinic – Rochester compared to the other sites (Table 1), however there was not a statistically significant difference (p=0.27). There were 26 pts (39%) with a treatment delay due to neutropenia while receiving SG, of which 21 (81%) were managed with the addition of growth factor (13 pegfilgrastim, 8 filgrastim). The median number of cycles for all pts was 5 (range: 1-25). Pts who received growth factor were treated with a median of 5 cycles (range: 1-25) and pts who did not receive growth factor were treated with a median of 4 cycles (range: 1-19) (p=0.10). Conclusions: We observed wide variability in the use of prophylactic growth factor between Mayo Clinic sites with SG. The optimal practice of growth factor use with SG warrants further exploration. References: 1. Bardia A, Hurvitz SA, Tolaney SM, et al. Sacituzumab govitecan in metastatic triple-negative breast cancer. N Engl J Med. 2021;384(16):1529-1541 2. Immunomedics, Inc. Trodelvy (sacituzumab govitecan-hziy) [package insert]. Foster City, CA: Gilead Sciences; 2020. Table 1: Grade of neutropenia for patients receiving SG when growth factor initiated Citation Format: Kaylee Clark, Jamie L. Carroll, Alvaro Moreno-Aspitia, Brenda Ernst, Farah Raheem, Ashley Heil, Beth Boyer, Kristin Mara, Matthew P. Goetz, Roberto A. Leon-Ferre, Karthik V. Giridhar, Jodi Taraba. Mayo Clinic Enterprise patterns of growth-factor utilization for sacituzumab govitecan (SG)-induced neutropenia among patients with metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-56.

Abstract Background: The role of chemotherapy in node positive (N+) luminal breast cancer (BC) is often debated, given low total pathologic complete response (pCR) rates following neoadjuvant chemotherapy (NAC) and discrepancy in adjuvant chemotherapy benefit. A prior single institution study of cN+ luminal BC showed that pts age < 50 and tumor Ki-67 ≥ 20% had high nodal pCR (> 35%). This study’s goals were 1) to validate Ki-67 and age in relation to nodal pCR and 2) evaluate the prognostic impact of nodal pCR on overall survival (OS). Methods: We queried the National Cancer Database 2010-2019 for pts with cN+ ER+/HER2- BC treated with NAC and surgery. Breast pCR was defined as ypT0/ypTis and nodal pCR as ypN0/ypN0i+. Ki-67 was available in 2018 & 2019 only and was used to evaluate Ki-67 and nodal pCR. 2010-2018 data were used to evaluate nodal pCR and OS. OS was analyzed using multivariable Cox proportional hazards regression. Results: In 2018-2019, 4,801 pts were identified and 2,473 (51.5%) had Ki-67 available. Nodal pCR was 23.7% and was higher in pts < 50 years old (28.1% vs 21.1%) and in those with Ki67 ≥ 20% (28.4% vs 12.7%), both p < 0.001. Pts < 50 with Ki67 ≥ 20% had the highest nodal pCR at 31.7%, followed by age ≥ 50 with Ki67 ≥ 20% at 26.3%. With Ki67 < 20%, nodal pCR was 15.4% (in age < 50) and 11.3% (in age ≥ 50). From 2010-2018, we identified 20,084 cN+ ER+/HER2- BC pts treated with NAC. Total pCR was 7.4%, 14.3% had nodal pCR only, 3.8% had breast pCR only, and 74.5% had residual disease in breast and nodes. OS at 5 years was 79.1% and varied by NAC response: 90.8% with total pCR, 83.8% with nodal pCR only, 80.7% with breast pCR only, and 76.9% with residual disease in breast and nodes. Specifically nodal pCR (with or without breast pCR) was seen in 22.0% and was associated with 5-year OS rate of 86.4% compared to 77.1% without nodal pCR, p < 0.001. On multivariable analysis adjusted for other clinical and treatment factors, nodal pCR was associated with better OS (adjusted HR 0.56, 95% CI: 0.50-0.61, p < 0.001) in all ages combined and within both the age < 50 and age ≥ 50 subgroups (see Table). In a subgroup of pts approximating RxPonder entry criteria (defined as cT1-3, N1, Grade I or II, ER+/PR+), results were consistent with the overall cohort: nodal pCR varied by both age (17.5% in age < 50 and 13.6% in age ≥ 50, p < 0.001) and by Ki67 ≥ 20% vs < 20% (16.8% vs 7.9%, p < 0.001) and nodal pCR remained prognostic for OS with adjusted HR 0.63 (95% CI: 0.50-0.81, p < 0.001). Conclusion: In cN+ ER+/HER2- BC treated with NAC, nodal pCR is more common in pts< 50 and those with high Ki-67 and is highly prognostic for OS. These data strongly suggest that NAC chemotherapy benefit should not be evaluated using total pCR rates in isolation, but for N+ pts to also consider nodal response. Given that nodal pCR is highly prognostic for OS, future neoadjuvant strategies should consider nodal pCR as a potential intermediate biomarker for long term survival. Multivariable analysis of factors associated with overall survival, including the adjusted effect of nodal pCR Citation Format: Dan Moldoveanu, Matthew P. Goetz, Tanya L. Hoskin, Courtney N. Day, Judy C. Boughey. Age, Ki-67, Nodal pCR and overall survival following Neoadjuvant Chemotherapy for Node Positive ER+/Her2- Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-22.

Abstract Background: Triple-negative breast cancers (TNBC) exhibit high rates of recurrence and mortality. However, recent studies suggest that a subset of patients (pts) with early-stage TNBC enriched in tumor-infiltrating lymphocytes (TILs) have excellent clinical outcomes even in the absence of systemic therapy. Additional histological biomarkers that could identify pts for future systemic therapy escalation/de-escalation strategies are of great interest. TNBC are frequently highly proliferative with abundant mitoses. However, classic markers of proliferation (manual mitosis counting and Ki-67) appear to offer no prognostic value. Here, we evaluated the prognostic effects of automated mitotic spindle hotspot (AMSH) counting on RFS in independent cohorts of systemically untreated early-stage TNBC. Methods: AMSH counting was conducted with a state-of-the-art deep learning algorithm trained on the detection of mitoses within 2 mm2 areas with the highest mitotic density (i.e. hotspots) in digital H&E images. Details of the development, training and validation of the algorithm were published previously [1] in a cohort of unselected TNBC. We obtained AMSH counts in a centrally confirmed TNBC cohort from Mayo Clinic [2] and focused our analysis on pts who received locoregional therapy but no systemic therapy. Using a fractional polynomial analysis with a multivariable proportional hazards regression model, we confirmed the assumption of linearity in the log hazard for the continuous variable AMSH and evaluated whether AMSH counts were prognostic of RFS. We corroborated our findings in an independent cohort of systemically untreated TNBC pts from the Radboud University Medical Center in the Netherlands (Radboud Cohort). Results are reported at a median follow-up of 8.1 and 6.7 years for the Mayo and Netherlands cohorts, respectively. Results: Among 182 pts with who did not receive systemic therapy in the Mayo Cohort, 140 (77%) with available AMSH counts were included. The mean age was 61 (range: 31-94), 71% were postmenopausal, 67% had tumors ≤ 2cm, and 83% were node-negative. As expected, most tumors were Nottingham grade 3 (84%) and had a high Ki-67 proliferation index (54% with Ki-67 >30%). Most tumors (73%) had stromal TILs ≤ 30%. The median AMSH count was 18 (IQR: 8, 42). AMSH counts were linearly associated with grade and tumor size, with the proportion of pts with grade 3 tumors and size > 2 cm increasing as the AMSH counts increased (p=0.007 and p=0.059, respectively). In a multivariate model controlling for nodal status, tumor size, and stromal TILs, AMSH counts were independently associated with RFS (p< 0.0001). For every 10-point increase in the AMSH count, we observed a 17% increase in the risk of experiencing an RFS event (HR 1.17, 95% CI 1.08-1.26). We corroborated our findings in the Radboud Cohort (n=126). The mean age was 68 (range: 40-96), and 81% were node-negative. While the median AMSH count was 36 (IQR: 16-63), higher than in the Mayo Cohort (p=0.004), the prognostic impact was similar, with a significant association between AMSH count and RFS (p=0.028) in a multivariate model corrected for nodal status, tumor size, and stromal TILs. For every 10-point increase in the AMSH count in the Netherlands cohort, we observed a 9% increase in the risk of experiencing an RFS event (HR 1.09, 95% CI 1.01-1.17). RFS rates according to AMSH counts for both cohorts are shown in the Table. Conclusions: AMSH counting is a new proliferation biomarker that provides prognostic value independent of nodal status, tumor size, and stromal TILs in systemically untreated early-stage TNBC. Plans are underway to evaluate AMSH counts in additional cohorts of systemically untreated TNBC, and in other disease settings such as prior to neoadjuvant systemic therapy. If validated, this biomarker should be prospectively evaluated as a potential selection biomarker in clinical trials of systemic therapy de-escalation. References: 1. PMID: 29994086 2. PMID: 28913760 Table RFS according to AMSH counts in the Mayo and Radboud Cohorts Citation Format: Roberto A. Leon-Ferre, Jodi M. Carter, David Zahrieh, Jason P. Sinnwell, Roberto Salgado, Vera Suman, David Hillman, Judy C. Boughey, Krishna R. Kalari, Fergus J. Couch, James N. Ingle, Maschenka Balkenkohl, Francesco Ciompi, Jeroen van der Laak, Matthew P. Goetz. Mitotic spindle hotspot counting using deep learning networks is highly associated with clinical outcomes in patients with early-stage triple-negative breast cancer who did not receive systemic therapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-34.

Abstract Background: Adjuvant endocrine therapy (ET) in ER+ breast cancer (BC) reduces local, distant, and contralateral BC events and improves overall survival (OS). Furthermore, decreased adherence or omission of ET increases the risk of death. However, in ER+ pts with early-stage BC treated with NAC who have a pathologic complete response (pCR), the importance of adjuvant ET may be called into question. We sought to examine the impact of ET omission on the survival of pts with ER+ BC treated with NAC, according to pCR vs residual disease. Methods: We queried the National Cancer Database (NCDB) 2010-2018 for female pts with stage I-III ER+ BC treated with NAC followed by surgery. pCR was defined as ypT0/ypTis, ypN0. The percent receiving adjuvant ET and the impact of adjuvant ET omission on overall survival (OS) in patients with and without pCR were assessed separately based on HER2 expression. OS was analyzed with adjuvant ET as a time-dependent covariate using Cox proportional hazards regression. Results: We identified 34,394 pts treated with NAC for ER+ BC (28,434 ER+/HER2-, 5960 ER+/HER2+). Pts with ER+/HER2+ BC were less likely than pts with ER+/HER2- BC to have received adjuvant ET (61.6% vs 88.8%, p< 0.001). Overall, 4505 (13.1%) had pCR (9.1% of ER+/HER2- and 32.0% of ER+/HER2+). Within each subtype, pts with pCR were significantly less likely to start adjuvant ET after surgery than pts with residual disease (78.4% vs 89.8% for ER+/HER2- and 46.5% vs 68.7% for ER+/HER2+, each p< 0.001), Table 1. Regarding those with residual disease, pts with ER+/HER2+ BC were less likely than ER+/HER2- BC to receive adjuvant ET (68.7% vs 89.8%, p< 0.001). Median follow-up was 4.4 years. Among pts with pCR, 5-year OS was 93.2% (95% CI: 92.1-94.4%) for ER+/HER2- BC and 94.3% (95% CI: 93.1-95.5%) for ER+/HER2+ BC (p=0.08), while among patients with residual disease 5-year OS was 81.7% (95% CI: 81.1-82.2%) and 85.7% (95% CI: 84.5-86.9%) for the two subtypes respectively (p< 0.001). On multivariable analysis, omission of adjuvant ET was significantly associated with poorer OS in patients with residual disease for both ER+/HER2- BC (adjusted HR 1.72, p< 0.001) and ER+/HER2+ BC (adjusted HR 1.63, p< 0.001). In contrast, omission of adjuvant ET was not significantly associated with OS in patients with pCR, regardless of HER2 status (ER+/HER2- adjusted HR 1.28, p=0.20; ER+/HER2+ adjusted HR 1.13, p=0.54), Table 1. Conclusions: In pts receiving NAC for ER+ BC, those with ER+/HER2+ disease were less likely to have received adjuvant ET compared to ER+/HER2- patients, regardless of pCR. In pts with residual disease after NAC, omission of adjuvant ET was associated with significantly higher risk of death. These data provide strong support for interventions to increase utilization of ET, especially for patients with residual disease following NAC. The observation that ET omission did not impact OS in pts with ER+ BC who achieve pCR following NAC is hypothesis generating and may have implications for future de-escalation trials for this subset of patients. Table 1. Differential use of adjuvant endocrine therapy by subtype and pCR and the impact on overall survival Citation Format: Grace M. Choong, Judy C. Boughey, Tanya L. Hoskin, Courtney N. Day, Matthew P. Goetz. Impact of adjuvant endocrine therapy (ET) omission in ER+ breast cancer (BC) treated with neoadjuvant chemotherapy (NAC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-12.

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Abstract Introduction: We performed matched case-control studies utilizing cohorts of postmenopausal women with ER+ breast cancer receiving adjuvant aromatase inhibitors (AI) on MA.27 [anastrozole, exemestane] or PreFace [letrozole] to assess the association between estrogen suppression after 6 months of treatment and an early breast cancer (EBC) event within 5 years of AI initiation (Clin Cancer Res 2020;26:2986-98). We found a significant 3.0-fold increase in risk of an EBC event for those taking anastrozole with levels of estrone (E1) ≥1.3 pg/mL and estradiol (E2) ≥0.5 pg/mL, but not for exemestane or letrozole. Given these findings we designed a prospective pharmacodynamic (PD) study to evaluate the impact of anastrozole (1 mg/day: ANA1) on E1 and E2 levels, and among those with inadequate estrogen suppression (IES: E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL), to evaluate the safety and PD efficacy of high-dose anastrozole (10 mg/day: ANA10), which has been found to be safe in prior clinical trials (Cancer 1998;83:1142-52). Methods: Post-menopausal women with stage I-III, ER ≥1% positive/HER2-negative breast cancer who were candidates for anastrozole were eligible after completion of locoregional therapy and chemotherapy, as clinically indicated. Women who were pre-menopausal at diagnosis were not eligible. All patients received 8-10 weeks of ANA1, after which those with adequate estrogen suppression (AES: E1< 1.3 pg/mL or E2< 0.5 pg/mL) came off study. Those with IES went on to receive ANA10 for 8-10 weeks, followed by letrozole (2.5 mg/day: LET) for 8-10 weeks. All patients were managed at their treating oncologist’s discretion following study discontinuation. E1 and E2 blood levels were measured pre-treatment and after completion of each treatment cycle by a CLIA-approved liquid chromatography with tandem mass spectrometry in the Immunochemical Core Laboratory at Mayo Clinic. With a sample size of 29 patients with IES after ANA1, a one-sided binomial test of proportions with a significance level of 0.05 will have an 87% chance of rejecting the proportion with AES after ANA10 is at most 25% (Ho) when the true proportion is at least 50%. Specifically, the null hypothesis is rejected if the number of women with AES after ANA10 is 12 or more. Data lock was July 6, 2022. Results: Of the 161 women enrolled from April 2020 through May 2022, 3 withdrew consent prior to start of ANA1 and 2 were ineligible; thus, 156 women comprised the study cohort. Median patient age was 64 years (range 44-86), 10% of patients were of Hispanic ethnicity and/or non-white race, and 15% received chemotherapy. Six patients remain on ANA1, and 10 discontinued ANA1 due to refusal (7), adverse event (AE) (2), or COVID-19 (1). Forty-one of the remaining 140 patients (29.3%; 95%CI: 21.9-37.6%) had IES with ANA1. Nine of these 41 patients did not go on to ANA10 due to refusal (6) or AE (3). Of the 32 patients who started ANA10, 8 remain on treatment, 5 discontinued due to refusal (3) or AE (1-grade 2 urinary tract infection; 1-grade 1 palpitations), and 19 had a blood draw 45 days or more after starting ANA10. No grade 3-5 AEs or grade 2 hot flashes or arthralgias were reported. Of these 19 patients, 14 achieved AES with ANA10 (73.7%; 95%CI: 48.8-90.9%). All 19 patients switched to LET of which 3 remain on treatment, 1 is missing E1/E2 data, and 15 had a blood draw 45 days or more after starting LET. Of these 15 patients, 10 maintained AES, 2 acquired AES with LET, and 3 no longer had AES. Anastrozole and letrozole drug levels will be reported at the meeting. Conclusions: Approximately 29% of postmenopausal women with ER+/HER2- BC receiving adjuvant anastrozole 1 mg/daily had IES. A majority of these patients achieved AES with dose escalation to ANA10 without tolerability issues. E1 and E2 levels are logical biomarkers given the mechanism of action of anastrozole, and further study utilizing them to determine the optimal dose of anastrozole for a given patient should be performed. Citation Format: Tufia C. Haddad, Vera Suman, Karthik V. Giridhar, Alvaro Moreno-Aspitia, Donald Northfelt, Brenda Ernst, Kostandinos Sideras, Ciara C. O’Sullivan, Ravinder Singh, Zeruesenay Desta, Jodi Taraba, Barbara Goodnature, Matthew P. Goetz, Liewei Wang, James N. Ingle. Anastrozole dose escalation for optimal estrogen suppression in postmenopausal early stage breast cancer: A prospective trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-04-02.