Goetz, Matthew P.

Abstract Background: With few targeted therapies available for triple-negative breast cancer (TNBC), there is a critical need to identify biomarkers that can predict chemotherapy-response. Copy number amplifications (CNAmps) have been implicated in tumorigenesis, progression, and treatment response. Previously, we performed a prospective neoadjuvant chemotherapy study (BEAUTY) and identified a 5 Mb CNAmp in TNBC patients associated with pathological complete response rates to taxane- and anthracycline-based chemotherapy. Here we further interrogated this amplicon and its role in chemotherapy response. Methods: Using siRNA screening of genes identified in the 5 Mb CNAmp, followed by cytotoxicity assays, we identified RDH10 significantly associated with chemo-sensitivity in TNBC cell lines. Functional studies were performed using RDH10 knockdown or overexpression followed by cytotoxicity, cell proliferation, luciferase reporter assays, HPLC or western blot. Results: RDH10 is a rate-limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite of retinoid metabolism that is frequently aberrant in cancer. Previous studies demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways often amplified in TNBC. We discovered that genetic manipulation of RDH10 affected ATRA intracellular concentrations, contributing to PIN1 degradation and taxane-response. Furthermore, co-treatment of TNBC cells with ATRA significantly increased taxane-sensitivity. Conclusion: RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting that RDH10 may be a novel biomarker of taxane response and the addition of ATRA to standard chemotherapy may improve chemo-sensitivity.

TPS624 Background: Abemaciclib (LY2835219), a cell cycle inhibitor of CDK4/CDK6, demonstrated a clinically manageable safety profile and single-agent anti-tumor activity in MBC; all tumor responses were observed in hormone receptor positive (HR+) disease (Tolaney SM, Rosen LS, Beeram M, et al. San Antonio Breast Cancer Symposium, 2014, Abstract P5-19-13). Non-steroidal aromatase inhibitors (NSAI, letrozole and anastrazole), approved in the first-line setting for postmenopausal women with HR+ MBC, are being evaluated in combination with abemaciclib for safety and tolerability in a phase Ib study (NCT02057133) and in the present study (NCT02246621) to assess clinical efficacy. Methods: MONARCH 3 is a randomized, double-blind, placebo-controlled, phase III study of abemaciclib + NSAI vs placebo + NSAI in locoregionally recurrent (not amenable to curative treatment) breast cancer or MBC, with no prior systemic therapy in this disease setting. Patients will be randomized 2:1, and stratified by nature of disease (visceral vs bone-only metastases vs other) and prior (neo)adjuvant endocrine therapy (aromatase inhibitor vs other vs none). Abemaciclib 150 mg or placebo will be given continuously PO every 12 hours until progression, along with anastrozole 1 mg or letrozole 2.5 mg once daily at the investigator’s discretion, and assessments will occur every 28 days. Postmenopausal women with HR+, HER2- disease, a disease-free interval > 12 mos after completion of (neo)adjuvant endocrine therapy, ECOG PS ≤ 1, adequate organ function, and measurable disease or nonmeasurable bone-only disease (RECIST v1.1) are eligible. The primary endpoint is progression-free survival (PFS); a key secondary endpoint is overall survival (OS). The study has 80% power to detect an increase in PFS of approximately 40% (hazard ratio = 0.714). Assuming a median PFS of 10 mos in the control arm, this corresponds to a 4-mo increase in the median PFS. PFS and OS will be hierarchically tested to maintain an overall type I error rate of 2.5%. Enrollment began November 2014; planned enrollment is 450 patients. Clinical trial information: NCT02246621.

Abstract Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

Abstract Background: With few targeted therapies available for triple-negative breast cancer (TNBC), there is a critical need to identify biomarkers that can predict chemotherapy-response. Copy number amplifications (CNAmps) have been implicated in tumorigenesis, progression, and treatment response. Previously, we performed a prospective neoadjuvant chemotherapy study (BEAUTY) and identified a 5 Mb CNAmp in TNBC patients associated with pathological complete response rates to taxane- and anthracycline-based chemotherapy. Here we further interrogated this amplicon and its role in chemotherapy response. Methods: Using siRNA screening of genes identified in the 5 Mb CNAmp, followed by cytotoxicity assays, we identified RDH10 significantly associated with chemo-sensitivity in TNBC cell lines. Functional studies were performed using RDH10 knockdown or overexpression followed by cytotoxicity, cell proliferation, luciferase reporter assays, HPLC or western blot.Results: RDH10 is a rate-limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite of retinoid metabolism that is frequently aberrant in cancer. Previous studies demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways often amplified in TNBC. We discovered that genetic manipulation of RDH10 affected ATRA intracellular concentrations, contributing to PIN1 degradation and taxane-response. Furthermore, co-treatment of TNBC cells with ATRA significantly increased taxane-sensitivity.Conclusion: RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting that RDH10 may be a novel biomarker of taxane response and the addition of ATRA to standard chemotherapy may improve chemo-sensitivity.

Abstract Background Abemaciclib demonstrated efficacy in hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. Here we provide a comprehensive summary of the most common adverse events (AEs), their management, and whether AEs or dose reductions influenced progression-free survival (PFS), in the MONARCH 2 and 3 trials. Materials and Methods Incidence of the most clinically relevant AEs, management, and outcomes were summarized. Time-dependent covariate analyses examined the impact of dose reductions on PFS. PFS was estimated for patients with and without early onset of diarrhea or neutropenia. Results The most frequently reported AE was diarrhea, with clinically significant diarrhea (grade ≥2) reported for 42.8% of patients taking abemaciclib. Median time to onset was 1 week, and duration ranged from 6 to 12 days, depending on grade and study. Diarrhea was adequately managed by antidiarrheal medication (72.8%), dose omissions (17.3%), and reductions (16.7%). The highest rates of grade ≥2 diarrhea were observed in the first cycles and decreased in subsequent cycles. Neutropenia (grade ≥3) occurred in 25.4% of abemaciclib-treated patients. Neutropenia resolved with dose omissions (16.8%) and/or dose reductions (11.2%). Incidence of febrile neutropenia (0.7%) or other relevant grade ≥3 hematological events (<9%) was low. Venous thromboembolic events (5.3%) were primarily treated with anticoagulants. Interstitial lung disease/pneumonitis (3.4%) was treated with corticosteroids and/or antibiotics. PFS benefit of abemaciclib was not impacted by dose reductions or early onset of toxicities. Conclusion Abemaciclib was generally well tolerated. The most common AEs were effectively managed by supportive medications, and/or dose adjustments, with no detriment to PFS. Implications for Practice Treatment with abemaciclib plus fulvestrant or nonsteroidal aromatase inhibitors is generally well tolerated in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. In MONARCH 2 and MONARCH 3, any-grade diarrhea and grade ≥3 neutropenia were effectively managed with supportive medication and/or dose adjustment. Venous thromboembolic events were treated with anticoagulants and did not often require treatment discontinuation. Interstitial lung disease/pneumonitis was infrequent and treated with corticosteroids and/or antibiotics. Clinicians should be aware of and implement management strategies, including dose adjustments according to local labels, for commonly occurring and serious adverse events to ensure continued treatment and optimize clinical benefit/risk ratio.

Abstract Background Black race is associated with worse outcomes in early breast cancer. We evaluated clinicopathologic characteristics, the 21-gene recurrence score (RS), treatment delivered, and clinical outcomes by race and ethnicity among women who participated in the Trial Assigning Individualized Options for Treatment. Methods The association between clinical outcomes and race (White, Black, Asian, other or unknown) and ethnicity (Hispanic vs non-Hispanic) was examined using proportional hazards models. All P values are 2-sided. Results Of 9719 eligible women with hormone receptor–positive, HER2-negative, node-negative breast cancer, there were 8189 (84.3%) Whites, 693 (7.1%) Blacks, 405 (4.2%) Asians, and 432 (4.4%) with other or unknown race. Regarding ethnicity, 889 (9.1%) were Hispanic. There were no substantial differences in RS or ESR1, PGR, or HER2 RNA expression by race or ethnicity. After adjustment for other covariates, compared with White race, Black race was associated with higher distant recurrence rates (hazard ratio [HR] = 1.60, 95% confidence intervals [CI] = 1.07 to 2.41) and worse overall survival in the RS 11-25 cohort (HR = 1.51, 95% CI = 1.06 to 2.15) and entire population (HR = 1.41, 95% CI = 1.05 to 1.90). Hispanic ethnicity and Asian race were associated with better outcomes. There was no evidence of chemotherapy benefit for any racial or ethnic group in those with a RS of 11-25. Conclusions Black women had worse clinical outcomes despite similar 21-gene assay RS results and comparable systemic therapy in the Trial Assigning Individualized Options for Treatment. Similar to Whites, Black women did not benefit from adjuvant chemotherapy if the 21-gene RS was 11-25. Further research is required to elucidate the basis for this racial disparity in prognosis.

Abstract Purpose: To determine genetic variants that could differentiate aromatase inhibitors (AIs) efficacy in early stage breast cancer. Experimental Design: We performed a stratified cox-proportional hazards analysis utilizing stratification factors and other covariates examining Breast Cancer Free Interval (BCFI) SNP-treatment interaction in Caucasian patients entered on the MA.27 trial. This analysis involved 4465 patients (2226 on anastrozole and 2239 on exemestane arm) including 245 (121 on anastrozole and 124 events on exemestane arm) with a breast event. Preclinical laboratory studies included luciferase activity, chromatin immunoprecipitation (ChIP) assay, and cell migration assays. Results: We identified 887 SNPs with a p-value <1E-4 that could differentiate anastrozole from exemestane efficacy. We next used GTEx databases to determine whether these SNPs might be cis-eQTLs with nearby genes, and found that 95 SNPs were eQTL with 14 genes. Functional validation of SNP effect in these 14 genes on response to anastrozole and exemestane revealed that 3 SNPs showed genotype-dependent differences between anastrozole and exemestane. Two of the three SNPs, rs1877193 and rs6735923 located upstream of LY75 gene, were associated with higher LY75 gene expression. Our drug-SNP interaction GWAS showed that both SNPs were associated with better BCFI for exemestane compared to anastrozole (HR= 0.447, 0.458). The SNP rs62293499 located downstream of the GPR160 gene was also associated with better BCFI for exemestane compared to anastrozole (HR=0.433). Interestingly, all 3 SNPs were associated with worse outcome (shorter BCFI) in anastrozole treated patients (HR=1.39~1.58), but with longer BCFI in the exemestane treated patients (HR=0.58~0.71) based on our GWAS results in MA.27 trial. However, all 3 SNPs showed no association with BCFI if the two treatment arms, anastrozole and exemestane, were combined (HR=0.92~1.08). Consistently, cells with Ly75 or GPR160 SNP variant genotypes were more sensitive to exemestane compared to anastrozole. Mechanistically, the 3 SNPs regulated estrogen receptor-dependant LY75 and GPR160 expression. LY75 suppression induced epithelial-to-mesenchymal transition (EMT) in breast cancer cell lines, accompanied by increased migratory capacity in vitro. LY75 knockdown also resulted in predominant downregulation of functional pathways such as cell proliferation, while pathways associated with mesenchymal stimulation were generally increased. GPR160 knockdown also resulted in downregulation of cell proliferation pathways. Conclusions: This SNP genotype and AI treatment interaction clinical study revealed unique genetic variants that differentiate anastrozole and exemestane efficacy. The signals were lost in the GWAS analysis when anastrozole and exemestane were combined. Preclinical laboratory studies revealed novel functions of LY75 and GPR160 in breast cancer. These findings represent potential steps towards individualized AI therapy. Citation Format: Junmei Cairns, James N. Ingle, Krishna R. Kalari, Lois E. Shepherd, Matthew J. Ellis, Paul E. Goss, Poulami Barman, Erin E. Carlson, Matthew P. Goetz, Richard M. Weinshilboum, Liewei Wang. The interaction between SNP genotype and aromatase inhibitor treatment response in early breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-103.

Abstract Purpose: Blood-based candidate biomarkers of disease can be monitored by analyzing CTCs and/or circulating cfDNA isolated from the peripheral blood. Our primary objective is to understand the relative contributions of these circulating factors (i.e., CTCs and cfDNA) to the overall disease profile in MBC. Methods: Clinically archived FFPE tumor tissue and prospective blood samples are collected through a minimal risk protocol approved by the Mayo Clinic IRB (#16-001540) from patients with MBC and objective evidence of disease progression. Blood samples include 20 mL whole blood in Streck blood collection tubes (BCTs) for platelet poor plasma (PPP); 20 mL whole blood in AccuCyte BCTs for CTCs, WBCs, and PPP; and 10 mL whole blood in EDTA BCTs for PPP. Nucleated, EpCAM+/cytokeratin+/CD45- CTCs are identified, assessed for ER/HER2 status, and isolated using a centrifugation and direct imaging platform that allows for single cell retrieval (RareCyte). DNA is extracted from PPP, CTCs, WBCs, and FFPE tumor tissue using established methods. Targeted sequencing for SNVs/indels is performed on paired WBCs and CTC-DNA, AccuCyte-cfDNA, Streck-cfDNA, EDTA-cfDNA, and tumor tissue derived DNA using the same NGS panel and informatics pipeline (65 genes; CleanPlex OncoZoom; Paragon Genomics). Results: Tissue and blood samples were collected from 40 patients with metastatic breast cancer. 10 cases were selected for initial analyses on the basis of CTC yield (range 3-113 per 3.75 mL blood); up to 5 CTCs per subject were isolated and pooled for DNA extraction. Plasma cfDNA yields and variant allele frequencies were highly comparable between AccuCyte and Streck collected blood samples. Mutations (range 1-3) were identified in CTC-DNA and/or cfDNA in 9 of 10 cases for a total of 18 detected mutations: 10 in CTC-DNA and cfDNA (BRCA2 N372H, PIK3CA E542K, PIK3CA E545K (x3), PIK3CA H1047R, PTEN R130P, RET G691S, TP53 C135W, TP53 Q192*); 5 in CTC-DNA only (EGFR R521K, EGFR T790M, PIK3CA H1047R, SMAD4 C363Y, TP53 N263D); and 3 in cfDNA only (DNMT3A W893S, DNMT3A S714C, TP53 Q136E). Parallel analyses of samples from 10 more subjects are in progress. Analysis of tumor tissue for all 20 subjects and of EDTA-cfDNA and single CTCs for a subset of cases is ongoing. Updated results will be presented at the meeting. Conclusions: It is feasible to isolate high quality CTCs and cfDNA from the same blood collection tube to perform targeted sequencing; this streamlines specimen processing, decreases overall costs, and minimizes required blood volumes. Importantly, there is overlap in the majority of mutations identified in CTC-DNA and cfDNA, but actionable mutations (e.g., PIK3CA, EGFR) were detected in CTC-DNA only. The clinical and theranostic relevance of these findings is unclear and warrants further investigation. Citation Format: Minetta C. Liu, Karthik V. Giridhar, Roberto A. Leon Ferre, Jamie L. Carroll, Matthew P. Goetz, Tufia C. Haddad, Deanne R. Smith, Siddhartha Yadav, Vidushi Kapoor, Guoying Liu, Tad George, Nolan Ericson, Arturo B. Ramirez, Eric Kaldjian, Keegan E. Haselkorn. Comparison of circulating tumor cell (CTC) derived DNA and circulating cell-free DNA (cfDNA) from simultaneous blood sampling of patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3119.

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. With currently no targeted therapies for TNBC, identification of novel biomarkers to improve chemo-response is vital. Recently, copy number amplifications (CNAs) have been implicated in treatment response in several diseases including breast cancer. Our clinical studies have associated a 5 Mb CNA in TNBC patients with chemo-response, which may elucidate novel biomarkers and treatment strategies for TNBC. Analysis of RNAseq and Whole Exome Sequencing data from 42 TNBC patient tumors enrolled in the BEAUTY Study, identified a 5 Mb CNA on chromosome 8q which associated with pathological complete response following taxane and anthracycline chemotherapy. By performing siRNA knockdown cytotoxicity studies in TNBC cell lines for genes within this amplified region, we identified RDH10 as a potential novel biomarker for increasing chemo-sensitivity. RDH10 is the rate limiting step in the synthesis of all-trans retinoic acid (ATRA). Previous studies have demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways, amplified in TNBC. We discovered overexpression of RDH10 to increase endogenous levels of ATRA, contributing to the degradation of PIN1 and taxane sensitivity. A rescue experiment with PIN1 was able to restore the chemo-resistance phenotype. Similarly, we found treating TNBC cells with ATRA phenocopied RDH10's effect on PIN1 and improved chemo-sensitivity. Furthermore, analyses of chemo-responsive PDX tumors containing the 5 Mb CNA, overexpressed RDH10 and exhibited lower levels of PIN1 when compared to non-responders. In conclusion, RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting RDH10 as a novel biomarker for chemo-response and ATRA as a novel treatment to improve chemo-sensitivity. Citation Format: Jordan Mark Grainger, Jia Yu, Krishna R. Kalari, Peter Vedell, Kevin Thompson, Matthew P. Goetz, Vera J. Suman, Judy C. Boughey, Liewei Wang. Study of copy number amplification and chemo-response in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1928.

550 Background: Ductal carcinoma in situ (DCIS) is a heterogeneous group of non-invasive cancers with varying propensity for recurrence. There are no validated markers that identify patients at risk for invasive recurrence following lumpectomy. HOXB13/IL17BR (H/I) is expressed in both DCIS and invasive cancer (Ma Cancer Cell. 2004) and associated with recurrence in stage I cancer. A 5-gene molecular grade index (MGI) distinguishes outcome in grade II disease (Ma Clin Cancer Res. 2008). We sought to determine whether H/I or MGI was associated with invasive recurrence in resected DCIS. Methods: We identified patients at Mayo Rochester who underwent lumpectomy ± radiation for DCIS between 1988 and 2001 and performed a nested case/control study. Cases were patients with ipsilateral invasive recurrence, matched to controls based on age, surgery date, length of follow-up, and adjuvant radiation. DCIS was macrodissected from paraffin sections and RNA extracted to obtain H/I and MGI RT-PCR profiles. The association of H/I and MGI with case-control status was assessed with conditional logistic regression; results reported are odds ratios (OR) with 95% confidence intervals (CI). The association of H/I and MGI as continuous variables with nuclear grade (low, intermediate, high) was estimated with Spearman's rank correlation. Results: 427 patients underwent lumpectomy for DCIS, and 33 were identified as possible cases. Histologic review excluded 8 (non-sufficient tissue or DCIS recurrence without invasion). 25 cases were matched to 48 controls (2 controls for 23 cases, 1 control for 2 cases). Both H/I (r = 0.43, p = 0.0001) and MGI (r = 0.41, p = 0.0004) were significantly correlated with nuclear grade. Positive H/I was associated with trend towards higher recurrence (OR 1.9; p = 0.22) that was most pronounced in cases/controls (15/28) treated with lumpectomy alone (OR 3.3; CI: 0.8–13.2, p = 0.09). Similarly, positive MGI was associated with a trend towards higher recurrence risk in patients treated with surgery alone (OR 2.0; CI: 0.6–6.4, p = 0.27). Conclusions: These data suggest that both HOXB13/IL17BR and MGI can identify a subset of patients with surgically resected DCIS who are at risk for invasive recurrence. Further studies in larger cohorts are needed to confirm these findings. [Table: see text]