Goetz, Matthew P.

The therapeutic options for patients with noninvasive or invasive breast cancer are complex and varied. These NCCN Clinical Practice Guidelines for Breast Cancer include recommendations for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget disease, phyllodes tumor, inflammatory breast cancer, and management of breast cancer during pregnancy. The content featured in this issue focuses on the recommendations for overall management of ductal carcinoma in situ and the workup and locoregional management of early stage invasive breast cancer. For the full version of the NCCN Guidelines for Breast Cancer, visit NCCN.org .

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low overall survival rates and high molecular heterogeneity; therefore, few targeted therapies are available. The luminal androgen receptor (LAR) is the most consistently identified TNBC subtype, but the clinical utility has yet to be established. Here, we constructed a novel genomic classifier, LAR-Sig, that distinguishes the LAR subtype from other TNBC subtypes and provide evidence that it is a clinically distinct disease. A meta-analysis of seven TNBC datasets (n = 1086 samples) from neoadjuvant clinical trials demonstrated that LAR patients have significantly reduced response (pCR) rates than non-LAR TNBC patients (odds ratio = 2.11, 95% CI: 1.33, 2.89). Moreover, deconvolution of the tumor microenvironment confirmed an enrichment of luminal epithelium corresponding with a decrease in basal and myoepithelium in LAR TNBC tumors. Increased immunosuppression in LAR patients may lead to a decreased presence of cycling T-cells and plasma cells. While, an increased presence of myofibroblast-like cancer-associated cells may impede drug delivery and treatment. In summary, the lower levels of tumor infiltrating lymphocytes (TILs), reduced immune activity in the micro-environment, and lower pCR rates after NAC, suggest that new therapeutic strategies for the LAR TNBC subtype need to be developed.

Abstract While epoxyeicosatrienoic acids (EETs) have been implicated in breast cancer growth and progression, less is known about their effects on oncogene transcription. We have previously found that the oncogenic regioisomer (±)14,15-EET drives mitochondrial respiration, ATP synthesis, and proliferation of ER+/HER2- breast cancer cells [Cell Chem Biol. 2017 Oct 19;24(10):1259-1275]. RNAseq analysis was performed (5 replicates per condition) on serum starved (16 hours) MCF-7 cells which were then treated with (±)14,15-EET or vehicle (2 hours; serum and phenol red free medium without estradiol). Using gene set enrichment analysis (GSEA), we found that (±)14,15-EET activated an estrogen receptor alpha (ER) hallmark early response gene set and synchronously activated a MYC hallmark gene set. With activation of the MYC hallmark gene set, c-Myc gene expression was also induced at 2 hours (3.99-fold; P=2.3 x 10-17; FDR=2.8 x 10-14). These data suggest an alternative pathway for activation of estrogen and MYC regulated genes in the absence of estradiol. The 15 genes most transcriptionally activated by (±)14,15-EET at 2 hours were: VMP1, ZFP36, JUNB, FOS, IER3, EGR1, IER5L, ELF3, JUN, NR4A1, HES1, DUSP1, MYC, TOB1, and CITED2 [fold change range: 3.25 (CITED2) to 90.5 (FOS); all P< 2.86 x 10-17; all FDR < 3.03 x 10-14]. The ER regulated genes most transcriptionally activated (>1.5 fold) were: IER3, TOB1, AREG, CISH, KCNMB3, and PDK4 (fold change range: 1.77 to 4.35; P= 1.74 x 10-18 to 1.35 x 10-5; FDR=5.54 x 10-15 to 6.4 x 10-4). The MYC regulated genes most transcriptionally activated (> 1.5-fold change) were EIF4A1 (fold change= 2.37; P=1.0 x 10-7; FDR=1.33 x 10-5), IRF9 (fold change=1.58; P=0.0044; FDR= 0.026), and FOSL1 (fold change=1.51; P=0.008; FDR=0.04). Supporting the hypothesis of (±)14,15-EET activation of ER-regulated transcription, (±)14,15-EET promoted nuclear translocation of ER at 1 hour measured by DAPI normalized immunofluorescence [MCF-7 nuclear ER increase of 1.66-fold (P=0.031); ZR75-1 nuclear ER increase of 1.77-fold (P=0.015)]. Supporting the hypothesis of (±)14,15-EET activation of MYC-regulated transcription, (±)14,15-EET treatment promoted nuclear translocation of c-Myc with MCF-7 cells exhibiting a 1.22-fold increase at 2 hours (P=0.002). (±)14,15-EET also promoted nuclear translocation of FITC-70 kDa dextran with MCF-7 cells exhibiting an increase of 1.35-fold at 1 hour (P=0.029). These data suggest that (±)14,15-EET can induce an estradiol-like immediate early gene response in ER+/HER2- breast cancer cells correlating with c-Myc activation. In summary, while the effect of (±)14,15-EET on nuclear translocation may be partially cargo agnostic, (±)14,15-EET promotes ER and c-Myc nuclear translocation and associated transcription, mimicking a tandem hormonal and growth factor response. Citation Format: Jianxun Lei, Zhijun Guo, Julissa Molina-Vega, Paloma Cervantes, Swaathi Jayaraman, John R. Hawse, Carlos Perez, Juan Abrahante, Xiaojia Tang, Krishna Kalari, Jinhua Wang, John R. Falck, Carol Lange, Matthew P. Goetz, David Potter. (±) 14,15-epoxyeicosatrienoic acid induces hallmark ER and MYC gene expression and associated ER and c-Myc nuclear translocation in ER+/HER2- breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB564.

TPS610 Background: Several studies demonstrated worsening disease-free survival in patients who failed to achieve complete pathological response after neoadjuvant chemotherapy (NAC), particularly in HER2-positive (HER2+) breast cancer. Despite the recent approval of trastuzumab emtansine (T-DM1) in patients with residual disease after NAC approximately 12% of patients still develop recurrent and metastatic disease. TPIV100 is a multi-epitope vaccine that includes a pool of 4 degenerate HER2-derived HLA-DR epitopes, which activate CD4 helper T cells, admixed with GM-CSF. In our previous phase I trial this vaccine was shown to be safe in combination with trastuzumab in stage II-III HER2+ breast cancer after completion of standard of care chemotherapy. Furthermore, this vaccine also generated robust long-lasting T-cell immune responses and antibody immunity against HER2. Methods: This trial is a multi-center, randomized, placebo-controlled, phase II trial of TPIV100 in combination with T-DM1 in stage II-III HER2+ breast cancer patients with residual disease after NAC. This trial is currently opened through the ACCRU consortium. Eligible patients include those with stage II-III HER2+ with residual disease, in the breast and/or lymph nodes, after trastuzumab ± pertuzumab-based NAC, with ECOG PS ≤ 2 and adequate organ function. Patients with baseline left ventricular ejection fraction < 50%, history of trastuzumab-related cardiac toxicity, myocardial infarction or stroke < 6 months, history of congestive heart failure, autoimmune disease, immunocompromised patients with known HIV or those on chronic steroid, hypersensitivity to GM-CSF, and other active malignancy < 3 years are excluded. TPIV100 or placebo, in combination with GM-CSF, will be given concurrently with T-DM1. To ensure that TPIV100 in combination with T-DM1 is safe, there is a run-in phase with 20 patients treated with the combination. If there is no significant dose-limiting toxicity observed in the run-in phase, the trial will be expanded to the randomized phase II portion, which will include 240 patients. Eligible patients will be randomized in a 2:1 fashion with ER/PR as a stratification factor. The primary endpoint is invasive disease-free survival, and the secondary endpoint includes immunogenicity of TPIV100 as assessed by IFN-g ELIspot analysis. Correlative studies include assessment of helper T-cell response distribution (including Th1, Th2, Th17, Tfh), HER2-specific antibody immunity, and HLA genotype. Currently, 20 patients in the run-in phase have been enrolled. Enrollment to the randomized phase II portion is expected to begin in March 2022. Clinical trial information: NCT04197687.

555 Background: Women with early-stage breast cancer (BCa) are living longer, and survivorship issues including second primary cancers (SPCs) are therefore increasingly important. This study seeks to determine the risk of SPCs, including contralateral breast cancer (CBCa), in women with early-stage invasive lobular carcinoma (ILC). Methods: The study population consisted of women with BCa diagnosis between 2000-2018 within the NCI Surveillance, Epidemiology, and End Results (SEER v 3.2.9) registry and between 2000-2021 in the Mayo Clinic Cancer Registry. In the SEER registry, the standardized incidence ratios (SIR) for SPCs were estimated for adult women with histologically confirmed early stage (I-III) ILC compared to the development of any primary cancer in the general population. For reference, SIRs for SPCs for a similar patient population with invasive ductal carcinoma (IDC) of the breast were also estimated. For comparison of CBCa between ILC and IDC, follow up records from the Mayo Clinic Cancer Registry were evaluated among adult women who underwent lumpectomy or unilateral mastectomy for early stage ILC or IDC. A multivariable Cox proportional hazards regression was performed to compare the risk of CBCa between ILC and IDC while adjusting for the age at diagnosis, race/ethnicity and estrogen receptor status of the primary tumor. Results: An increased incidence (p<0.05) of any SPCs and second BCa was noted for both ILC and IDC compared to the expected rate of primary cancer in the general population (Table). Interestingly, women with ILC were observed to have a significantly increased incidence of subsequent gastric cancer which was not seen for women with IDC. An SIR >1.5 was noted for subsequent risk of sarcoma, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) for both ILC and IDC (Table). In the Mayo Clinic Cancer Registry of 2,218 women with ILC and 14,629 women with IDC meeting the inclusion criteria, the 5-year risk of CBCa was 2.6% and 1.5% (log rank statistic p-value =0.001) for ILC and IDC respectively. A significantly increased risk of CBCa was noted in women with ILC (Hazard Ratio 1.82, 95% CI 1.34-2.47, p<0.001) compared to women with IDC in the multivariable analysis. Conclusions: Women with ILC may have an increased risk of SPC and second BCa compared to the risk of any primary cancer in the general population. The increased incidence of gastric cancer and the higher risk of CBCa in ILC survivors compared to women with IDC is intriguing and needs to be investigated further including evaluation of the role of germline genetic factors. [Table: see text]

525 Background: Homologous recombination deficiency (HRD) status can be used to identify patients who are eligible for treatment with DNA damaging agents. Using a 3-biomarker Genomic Instability Score (GIS) threshold of ≥42, studies have previously examined the association between HRD status and outcomes in patients with triple negative breast cancer (TNBC). However, evidence suggests that a GIS threshold of ≥33 may be more appropriate. Here, we conducted an exploratory analysis evaluating the ability of ≥33 and ≥42 GIS thresholds to predict response to platinum-based treatment in patients with TNBC. Methods: Patients across 5 cohorts (TBCRC0301, TBCRC0082, NCT013725793, PrECOG 01054, combined cisplatin cohort4) were included in this analysis if they had a primary TNBC diagnosis, received neoadjuvant platinum-based treatment, had a valid GIS, and had known pathologic complete response (pCR) status. GIS was determined by a combination of loss of heterozygosity, telomeric-allelic imbalance, and large-scale state transitions.4,5 BRCA mutation status was defined by loss of function resulting from a pathogenic variant in BRCA1 or BRCA2. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), were calculated by comparing binary threshold status and binary pCR status. Results: A total of 204 tumors (158 BRCAwt; 33 BRCAm; 13 unknown) were included; pCR to platinum-based treatment occurred in 55 cases (39 BRCAwt; 14 BRCAm; 2 unknown). Sensitivity, specificity, PPV, and NPV were comparable between the ≥33 and ≥42 GIS thresholds, with the ≥33 threshold producing higher sensitivity values. This was true when thresholds were applied to all samples and to BRCAwt samples only (Table). Among patients who achieved pCR in response to platinum-based treatment, 5.5% of patients in the full cohort and 7.7% of those in the BRCAwt cohort had a GIS between 33-41. Conclusions: To ensure that the majority of patients likely to benefit from treatment are identified, a GIS of ≥33 may be the most appropriate threshold to predict response to platinum-based treatment in patients with TNBC; however, a prospective trial will be needed to confirm these findings. Additional studies will be important to determine whether this threshold may be appropriate to determine eligibility for other DNA-damaging agents such as PARP inhibitors. 1. Ann Oncol. 2020;31(11):1518-25 2. J Nucl Med. 2015;56(1):31-7. 3. Breast Cancer Res Treat. 2015;151(3):629-38. 4. Clin Cancer Res. 2016;22(15):3764-73. 5. Breast Cancer Res Treat. 2014;16(6):1-9. [Table: see text]

Abstract Purpose Cyclin dependent kinase 4/6 inhibitors (CDK4/6is), in combination with endocrine therapy (ET), are standard either in the first (1L) or second line (2L) setting for the treatment of hormone receptor (HR) positive, HER2-negative metastatic breast cancer (MBC). However, the optimal sequencing of treatments after progression on CDK4/6i remains unknown. We performed a single institution analysis to identify treatments and outcomes after progression on a CDK4/6i. Methods We identified patients with HR-positive, HER2-negative MBC prescribed a CDK4/6i in the 1L or 2L settings from December 2014-February 2018 at Mayo Clinic in Rochester, Minnesota. Outcomes were collected through September 30, 2020. Results Palbociclib, in combination with letrozole or fulvestrant, was the most prescribed CDK4/6i. The 1L and 2L CDK4/6i cohorts exhibited comparable overall survival (OS), but progression free survival (PFS) was longer in the 1L than the 2L cohort [28.2 months (95% CI 19.6–34.9) vs 19.8 months (95% CI 15.7–29.6)]. The most common post-CDK4/6i treatments were PI3K/mTOR inhibitors (PI3K/mTORi), single agent ET, or chemotherapy. PFS in the 1L CDK4/6i cohort following PI3K/mTORi was 8.5 months (95% CI 5.5 months – NE), single-agent ET was 6.0 months (95% CI 3.3–14.0 months), and chemotherapy PFS was 5.4 months (95% CI 3.3 months- NE). Conclusions Following progression on a CDK 4/6i, mPFS was short, with similar PFS times comparing chemotherapy and ET, with slightly longer PFS for targeted strategies (PI3K/mTOR). These results highlight a major need to better understand the mechanisms of CDK4/6i resistance and identify new therapeutic strategies for these patients.

AbstractThe purpose of this study was to develop and validate a population pharmacokinetic model for Z‐endoxifen in patients with advanced solid tumors and to identify clinical variables that influence pharmacokinetic parameters. Z‐endoxifen‐HCl was administered orally once a day on a 28‐day cycle (±3 days) over 11 dose levels ranging from 20 to 360 mg. A total of 1256 Z‐endoxifen plasma concentration samples from 80 patients were analyzed using nonlinear mixed‐effects modeling to develop a population pharmacokinetic model for Z‐endoxifen. A 2‐compartment model with oral depot and linear elimination adequately described the data. The estimated apparent total clearance, apparent central volume of distribution, and apparent peripheral volume of distribution were 4.89 L/h, 323 L, and 39.7 L, respectively, with weight‐effect exponents of 0.75, 1, and 1, respectively. This model was used to explore the effects of clinical and demographic variables on Z‐endoxifen pharmacokinetics. Weight, race on clearance, and aspartate aminotransferase on the absorption rate constant were identified as significant covariates in the final model. This novel population pharmacokinetic model provides insight regarding factors that may affect the pharmacokinetics of Z‐endoxifen and may assist in the design of future clinical trials.

Abstract Background: Patients with homologous recombin ation (HR) deficient tumors may benefit from treatment with DNA damaging agents. Markers of genomic instability can be used to identify HR deficiency, including a 3-biomarker Genomic Instability Score (GIS). For patients with ovarian cancer (OC), the FDA-approved GIS threshold for identifying HR deficiency is 42, set as the 5th percentile for BRCA deficient tumors. Recently, a lower 1st percentile cutoff of 33 was explored in OC; this threshold was significantly associated with improved outcome after platinum-based treatment.1,2 Determining an optimal GIS threshold for different types of tumors is crucial, as the GIS distribution may vary between different cancers and even between different cancer subtypes. We propose GIS thresholds for breast cancer separately for triple-negative breast cancer (TNBC) and estrogen receptor-positive (ER+) breast cancer, using the exploratory threshold of 33 for OC as a comparator. Methods: GISs in BRCA deficient tumors were determined for patients newly diagnosed with varying stages of OC, TNBC, or ER+ breast cancer across 5 cohorts (Timms et al,3 TCGA,4 Abkevich et al,5 TBCRC008,6 the OlympiaD trial7). GIS was determined as a combination of loss of heterozygosity, telomeric-allelic imbalance, and large-scale state transitions. BRCA deficiency was defined by loss of function resulting from a pathogenic variant in BRCA1 or BRCA2 or by methylation of the BRCA1 promoter region, with loss of heterozygosity in the affected gene. GIS distributions in different cancer types and subtypes were compared using the Kolmogorov-Smirnov test. A normal distribution was fit to GISs in BRCA deficient ER+ breast tumors. The 1st percentile of the fitted distribution was chosen as the threshold. Results: A total of 561 OC tumors (190 BRCA deficient), 99 TNBC tumors (44 BRCA deficient), and 406 ER+ breast tumors (76 BRCA deficient) were included across the 5 cohorts. When score distributions were evaluated for BRCA deficient tumors, the GIS distribution within ER+ breast cancer was significantly different than for OC (p=9.6x10-5) and TNBC (p=3.2x10-4). This indicates that different GIS thresholds are appropriate for breast cancer subtypes and that the GIS threshold developed for OC is not appropriate for ER+ breast cancer. The 1st percentile of a normal distribution fit in BRCA deficient ER+ breast cancer tumors yields a threshold of 24. Using this threshold, 45.1% (183/406; 75 BRCA deficient, 108 BRCA intact) of ER+ breast tumors were HR deficient. In contrast, the GIS distribution for TNBC was not significantly different than for OC (p=0.77). Using the exploratory threshold of 33, 63.6% (63/99; 44 BRCA deficient, 19 BRCA intact) of TNBC tumors were HR deficient. Conclusions: When compared to OC, the distribution of GIS in BRCA deficient tumors was different for ER+ breast cancer, but not for TNBC. These findings are consistent with the fact that OC and TNBC are known to have similar molecular signatures.8 Exploratory thresholds of 24 for ER+ breast cancer, and 33 for TNBC and OC could be examined to determine if these cutoffs are associated with a benefit from treatment with DNA targeting agents. Clinical validity and utility of these more inclusive 1% thresholds would require demonstration of correlation with clinical outcomes. The threshold difference observed between these cancer subtypes also suggests that cancer or cancer subtype specific thresholds may be needed as evaluations of HR deficiency expands beyond OC to identify candidates for PARP inhibitors. References: 1 Mol Cancer Res. 2018;16(7):1103-11. 2 Cancers. 2021;13(5):946. 3 Br J Cancer. 2012;107(10):1776-82. 4 Nature. 2012;490(7418):61-70. 5 Breast Cancer Res. 2014;16(145):1-9. 6 J Nucl Med. 2015;56(1):31-7. 7 NEJM. 2017;377(17):1700. 8 Int J Mol Sci. 2016;17(5):759. Citation Format: Kirsten M Timms, Lauren Lenz, Chris Neff, Cara Solimeno, Darl Flake, Judy C Boughey, Matthew P Goetz, Andrea Richardson, Anna Maria Storniolo, Alexander Gutin, Roisin M Connolly, Vered Stearns, Jerry S Lanchbury. Identifying homologous recombination deficiency in breast cancer: Genomic instability score thresholds differ in breast cancer subtypes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-09.

Abstract Background: Cyclin-dependent kinase 4/6 inhibitors (CKD4/6i) have demonstrated clinical utility extending progression-free survival (PFS) and overall survival (OS) for advanced hormone receptor positive and HER2 negative (HR+/HER2-) breast cancer patients. The efficacy in early-stage breast cancer (eBC) is unclear, with conflicting results from adjuvant CDK4/6i trials on invasive disease-free survival. Thus, there is a critical need to identify biomarkers of response (BoR) to determine which, if any, eBC patients could benefit from this treatment. This BoR could also stratify advanced BC patients for likelihood to respond to CDK4/6i. Metabolism is influenced by both genome and environment, and changes in the metabolome can be correlated with drug responsiveness. Thus, metabolite BoRs may serve to identify eBC patients for which CDK4/6i would offer a therapeutic benefit.Methods: Plasma and serum samples from 50 early-stage ER+/HER2- breast cancer patients, treated with neoadjuvant CDK4/6 inhibitor palbociclib (palbo) and aromatase inhibitor (AI) anastrozole on NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774), were collected from treatment-naïve patients (BL) and 3 consecutive time points: anastrozole,1 mg daily for 4 weeks (C1D1), anastrozole plus palbo,125 mg daily, for 15 days (C1D15), and for 4-5 months before surgery (SURG). Metabolites were extracted from all samples via methanol and chloroform precipitation and quantified using an unbiased, non-destructive, nuclear magnetic resonance (NMR)-based profiling platform (Olaris®, Inc., Waltham, MA). Statistical analysis and machine learning was used to identify differential metabolites and generate predictive models. A separate validation set of samples was collected from a subset of patients (N=6) who received an additional cycle of palbo treatment prior to surgery to assess model accuracy. Results: Non-parametric differential expression analysis of BL/C1D1, BL/C1D15, and C1D1/C1D15 identified 53 ,97, and 90 differential NMR resonances in plasma (p&lt;0.05) and 36, 34, and 25 differential NMR resonances in serum (p&lt;0.05), respectively. Based on the proliferative marker Ki67 levels at C1D15, 37 patients were classified as responders (Ki67≤2.7%) and 6 patients as non-responders (Ki67&gt;2.7%). Analysis of the responder (R) and non-responder (NR) groups identified that 13 plasma and 14 serum resonances (21 unique resonances and 6 overlapping) were differentially expressed (p&lt;0.05) at C1D1. Many of the differential resonances could be mapped back to amino acid metabolites including several branched chain amino acids such as leucine, valine, and isoleucine, and positively charged amino acids such as lysine. A Olaris® BoR score was generated using 5 differential resonances that had an AUC of 0.931 (training set) and 100% accuracy when predicting palbo-response in a blinded test set (N=6).Conclusion: The differential metabolites identified from matching plasma and serum samples suggest that, compared to serum, plasma has a better representation of the metabolic changes associated with palbo treatment-response. While comparing samples from R and NR patients, amino acids were found to be consistently altered in both serum and plasma before palbo treatment. In addition, a BoR model based on select metabolites could precisely stratify palbo-response in a blinded dataset. A larger independent validation cohort is ongoing. Citation Format: Chen Dong, Shana Thomas, Chandrashekhar Honrao, Leonardo O. Rodrigues, Nathalie Tessier, Bo Zhang, Souzan Sanati, Kiran Vij, Brenda J. Ernst, Karen S. Anderson, Mateusz Opyrchal, Foluso Ademuyiwa, Lindsay L. Peterson, Matthew P. Goetz, Donald Northfelt, Elizabeth O'Day, Cynthia Ma. Identifying a metabolite signature that correlates with tumor proliferation in early-stage breast cancer patients treated with CDK4/6 inhibitors from matched plasma and serum samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-20.