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Background: Combination chemotherapy (CT) remains a standard of care in advanced breast cancer (ABC) with aggressive disease features (rapidly progressing or highly symptomatic disease, including life-threatening visceral crisis). To date, no data have been published on a head-to-head comparison of CDK4/6 inhibitor (CDK4/6i) + endocrine therapy (ET) vs combination CT in this patient (pt) population. RIGHT Choice, a randomized, open-label, multi-national, Phase II trial, investigated the efficacy and safety of first-line ribociclib (RIB) + ET vs combination CT in pre/perimenopausal pts with HR+/HER2− ABC with aggressive disease (where combination CT is clinically indicated by physician’s judgment). Here we report the results of the primary endpoint of progression-free survival (PFS) and key secondary endpoints from this study. Methods: Pre/perimenopausal pts with HR+/HER2− ABC (>10% estrogen receptor–positive [ER+]) and no prior systemic therapy for ABC were randomized 1:1 to receive either RIB (600 mg daily, 3 weeks on/1 week off) with letrozole/anastrozole + goserelin or investigator’s choice of CT (docetaxel + capecitabine, paclitaxel + gemcitabine, or capecitabine + vinorelbine). Randomization was stratified by presence of liver metastases and whether the disease-free interval (duration from date of complete tumor resection for primary breast cancer lesion to the date of documented disease recurrence) was less than two years. Pts included in the trial had ABC not amenable to curative therapy for which combination CT was clinically indicated by physician’s judgment (i.e., symptomatic visceral metastases, rapid progression of disease or impending visceral compromise, or markedly symptomatic non-visceral disease). Median PFS (mPFS) and median time to treatment failure (mTTF) were evaluated by Kaplan-Meier methods. Overall response rate (ORR) was also analyzed, with quality of life and biomarker analyses planned. RIGHT Choice is registered at ClinicalTrials.gov (NCT03839823). Results: A total of 222 pts (112 RIB + ET; 110 CT) were enrolled from Feb 2019 to Nov 2021. Pts with symptomatic visceral metastases (n=150; 67.6%), rapid disease progression (n=41; 18.5%), and markedly symptomatic non-visceral metastases (n=31; 14.0%) were included. Overall, 116 pts (52.3%) had visceral crisis based on guideline definitions. A majority of pts (n=190; 85.6%) had tumors that were ≥50% ER+. At data cutoff (Apr 12, 2022), median follow-up was 24.1 mo; 45.5% and 23.6% of pts remained on treatment in the RIB + ET arm and combination CT arm, respectively. The primary endpoint was met, with a statistically significant PFS benefit of ≈1 year for RIB + ET vs combination CT (mPFS, 24.0 vs 12.3 mo; hazard ratio, 0.54; 95% CI, 0.36-0.79; P=.0007). OS data were immature at data cutoff. The mTTF was ≈10 mo longer for RIB + ET vs CT (18.6 vs 8.5 mo; hazard ratio, 0.45; 95% CI, 0.32-0.63). The ORR was similar for RIB + ET vs CT (65.2% vs 60.0%). No new safety signals were observed in pts on RIB. Lower rates of treatment-related serious adverse events (AEs; 1.8% vs 8.0%) and lower rates of discontinuation due to treatment-related AEs (7.1% vs 23.0%) were seen with RIB + ET vs CT, respectively. AEs observed with combination CT were consistent with the published data. Conclusions: This analysis demonstrated a statistically significant and clinically meaningful PFS benefit with RIB + ET over combination CT in the first-line pre/perimenopausal pt population with aggressive HR+/HER2− ABC disease. This is the first study comparing a CDK4/6i + ET vs combination CT and demonstrating the superiority of RIB + ET in aggressive HR+/HER2− ABC. This evidence supports RIB+ ET use as a preferred option for this pt population. Citation Format: Yen-Shen Lu, Eznal Izwadi Bin Mohd Mahidin, Hamdy Azim, Yeşim ERALP, Yoon-Sim Yap, Seock-Ah Im, Julie Rihani, James Bowles, Teresa Delgar Alfaro, Jiwen Wu, Melissa Gao, Khemaies Slimane, Nagi El Saghir. Primary results from the randomized Phase II RIGHT Choice trial of premenopausal patients with aggressive HR+/HER2− advanced breast cancer treated with ribociclib + endocrine therapy vs physician’s choice combination chemotherapy [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 GS1-10.

Background: Mucinous carcinoma is the third most common histological subtype of breast cancer after ductal and lobular carcinomas, accounting for approximately 3% of invasive breast cancers. Although considered a favourable subtype with de-escalation of treatment recommended in the National Comprehensive Cancer Network guidelines, recurrence can occur and supporting data is limited. We thus examined prognostic factors of pure mucinous breast cancer (PMBC) in an international multicentre cohort study. Methods: Patients diagnosed between January 2000 to December 2015 with hormone receptor-positive HER2-negative stage I to III PMBC, invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) at 6 centers in Singapore, Taiwan, Korea and Japan were evaluated. Cox proportional hazards regression analyses were used to compare relapse-free survival (RFS), distant relapse-free survival (DRFS) and overall survival (OS) by histological subtypes, and to identify prognostic factors for PMBC. Results: Of 23,105 women eligible for analysis, 20,684 had IDC, 1,475 had ILC and 946 had PMBC. The median follow-up was 6.6 years; 5-year RFS, DRFS and OS for PMBC were 94.6%, 96.5% and 98.1% respectively. On multivariable cox regression analyses, PMBC demonstrated superior RFS (hazard ratio [HR] = 0.70, 95% CI: 0.56 - 0.88), DRFS (HR = 0.69, 95% CI: 0.53 - 0.89) and OS (HR = 0.70, 95% CI: 0.52 - 0.93) compared to IDC, while ILC had comparable survival outcomes as IDC. When restricted to only PMBC, significant independent prognostic factors for RFS included ethnicity (vs Chinese, “Others” [non-Chinese/Japanese/Korean, mainly Malay and Indian]: HR = 2.62, 95% CI 1.23 – 5.57), older age (vs < 40 years, >70 years: HR = 3.53, 95% CI 1.67 – 7.46), tumor size (vs T1, T3-4: HR = 2.79, 95% CI 1.45 – 5.37), positive lymph nodes (HR = 2.04, 95% CI: 1.10 – 3.77), use of radiotherapy (HR = 0.54, 95% CI 0.33 – 0.91) and endocrine therapy (HR = 0.31, 95% CI 0.12 – 0.77). On further analysis, the inferior RFS, DRFS and OS in older patients (>70 years) were driven largely by non-breast cancer deaths rather than relapses. Use of endocrine therapy was also associated with superior DRFS (HR = 0.26, 95% CI 0.09 – 0.73) but not OS. In a subgroup analysis, use of chemotherapy was associated with improved DRFS (HR = 0.25, 95% CI 0.08 – 0.82) and OS (HR = 0.07, 95% CI 0.01 – 0.37) with a trend in RFS (HR = 0.41, 95% CI 0.14 – 1.24) for lymph node-positive PMBC; no differences in outcomes were observed for the lymph node-negative subgroup. Conclusions: Larger tumor size, lymph node positivity and ethnicity were significant factors for RFS in PMBC. Use of endocrine therapy was associated with superior RFS and DRFS, while chemotherapy was associated with better DRFS and OS for lymph-node positive PMBC. Citation Format: Ryan Tan, Whee Sze Ong, Kyung-Hun Lee, Seri Park, Jabed Iqbal, Yeon Hee Park, Jeong-Eon Lee, Jong Han Yu, Ching-Hung Lin, Yen-Shen Lu, Makiko Ono, Takayuki Ueno, Yoichi Naito, Tatsuya Onishi, Geok hoon Lim, Su-Ming Tan, Han-Byoel Lee, Jiwon Koh, Han Suk Ryu, Wonshik Han, Veronique Kiak Mien Tan, Fuh-Yong Wong, Seock-Ah Im, Puay Hoon Tan, Yoon-Sim Yap. Prognostic factors in non-metastatic hormone receptor-positive HER2-negative mucinous breast cancer: an international multicentre cohort 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 P3-05-36.

Purpose:Natural killer (NK) cells exert antibody-dependent cell cytotoxicity (ADCC). We infused expanded, activated autologous NK cells to potentiate trastuzumab-mediated ADCC in patients with HER2-positive malignancies.Patients and Methods:In a phase I trial, patients with treatment-refractory HER2-positive solid tumors received trastuzumab, with or without bevacizumab, and autologous NK cells expanded by 10-day coculture with K562-mb15-41BBL cells. Primary objectives included safety and recommended phase II dose determination; secondary objectives included monitoring NK-cell activity and RECIST antitumor efficacy.Results:In 60 cultures with cells from 31 subjects, median NK-cell expansion from peripheral blood was 340-fold (range, 91–603). NK cells expressed high levels of CD16, the mediator of ADCC, and exerted powerful killing of trastuzumab-targeted cells. In the 22 subjects enrolled in phase I dose escalation, trastuzumab plus NK cells were well tolerated; MTD was not reached. Phase IB (n = 9) included multiple cycles of NK cells (1 × 107/kg) and addition of bevacizumab. Although no objective response was observed, 6 of 19 subjects who received at least 1 × 107/kg NK cells at cycle 1 had stable disease for ≥6 months (median, 8.8 months; range 6.0–12.0). One patient, the only one with the high-affinity F158V CD16 variant, had a partial response. Peripheral blood NK cells progressively downregulated CD16 postinfusion; paired tumor biopsies showed increased NK cells, lymphocytic infiltrates, and apoptosis posttreatment.Conclusions:NK-cell therapy in combination with trastuzumab was well tolerated, with target engagement and preliminary antitumor activity, supporting continued assessment of this approach in phase II trials.

Supplementary data contains list of genes and SNP details

It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the JAK2 SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the NOTCH3 SNP rs200504060 and the HIF1A SNP rs142179458 with breast cancer risk. In silico analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. Cancer Res; 77(19); 5428–37. ©2017 AACR.

Supplementary Materials, Figures

Background: In addition to inhibiting cell cycle progression, CDK4/6 inhibitors have been found to modulate anti-cancer immune response (Goel S, et al. Nature. 2017;548:471-475). Data from preclinical studies suggested multiple mechanisms—involving changes in both cancer cells and immune cells—by which CDK4/6 inhibition can enhance anti-cancer immunity. These mechanisms include increased antigen presentation, interferon response triggering, and regulatory T-cell suppression. Clinical correlates to preclinical observations have so far been obtained using paired biopsies from neoadjuvant breast cancer trials (Johnston S, et al. J Clin Oncol. 2019;37:178-189; Hurvitz S, et al. Clin Cancer Res. 2020; ;26:566-80). Here, we describe a gene expression analysis of paired pre- and on-treatment biopsies from CLEE011A2115C and CLEE011X2107, two phase I clinical trials evaluating ribociclib in combination with ET in the metastatic breast cancer (MBC) setting. Methods: The nCounter PanCancer IO 360 Panel (NanoString) was used to quantify expression of 770 genes in 7 pairs of tumor samples (baseline vs. cycle 1 day 15) from two phase I trials in the MBC setting (5 patients from CLEE011A2115C and 2 from CLEE011X2107). All patients were treated with ribociclib and an ET. Pairwise differential gene expression analysis of individual genes and previously published immune-related gene signatures was conducted (Bedognetti D, et al. Curr Opin Oncol. 2015;27:433-444). Results: Many genes that were markedly suppressed with treatment (eg, MKI67, MYC, CDK2, CCNB1, TYMS, and DNMT1) were related to proliferation, DNA replication, and G1/S transition of the cell cycle, as expected. Interestingly, expression of numerous genes involved in immune response was increased. These were mostly interferon-regulated genes; particularly, a gene signature indicative of a T cell-inflamed tumor microenvironment (Ayers M, et al. J Clin Invest. 2017;127:2930-2940) was upregulated. Immune-related genes tended to preferentially increase with treatment in patients who later experienced a clinical response. Since bulk RNA from tumor biopsies was analyzed, we could not distinguish whether the observed expression changes reflected increased infiltration by immune cells or an endogenous interferon response in cancer cells. The latter could be triggered by DNMT1 suppression (as previously described), which then promotes T cell-mediated immunity by increasing antigen presentation and chemokine production in cancer cells. Conclusions: This analysis was conducted with a small sample size and is considered hypothesis generating; further investigation is needed. Cell cycle and proliferation markers showed robust suppression by ribociclib-based treatment regimens, as expected. Conversely, a notable increase in immune-related genes was detected that tended to occur preferentially in patients who later experienced more favorable clinical outcomes. Both changes occurred early (cycle 1 day 15). To our knowledge, this is the first clinical biomarker report of immune activation mediated by CDK4/6 inhibition in MBC. Citation Format: Dejan Juric, Chong Ma, Ralph Tiedt, Yoon-Sim Yap, Joanne Chiu, Pamela Munster, Roohi Ismail-Khan, Laura Garcia-Estevez, Ingrid A. Mayer, Carlos Becerra, Nadia Solovieff, Agnes Lteif, Faye Su, Yen-Shen Lu. Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies [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 P5-13-18.

Background: The Phase III MONALEESA (ML)-2, -3, and -7 trials showed significant improvement in progression-free survival (PFS) and overall survival (OS) with ribociclib (RIB) + endocrine therapy (ET) over placebo (PBO) + ET in patients (pts) with HR+/HER2− advanced breast cancer (ABC); improvement in OS with cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) has been observed in some, but not all clinical trials. Gene expression analyses for each separate ML study were reported previously. Given the differences in CDK4 vs CDK6 inhibition between RIB and other CDK4/6i, we evaluated the association between cell cycle (CC)–related genes and outcomes based on pooled analysis of gene expression using tumor samples from the ML-2, -3, and -7 trials. Methods: Gene expression data were generated from pre-treatment archival tumor samples (primary, 73%; metastatic, 27%) with a customized NanoString nCounter panel (781 genes) including genes involved in CC, other signaling pathways, and breast cancer biology. Samples were pooled from 1139 pre- and postmenopausal pts with HR+/HER2− ABC across the 3 ML studies, which included pts on first- and second-line therapy. Data were categorized into training (80%) and test (20%) datasets. The training dataset was used to analyze each gene (modeled continuously) individually for an association with PFS, and genes with a gene × treatment (tx) interaction P value Results: This report focused on CC-related genes and signatures. Gene expression levels of CDKN2B and the expression ratio of CCND1/CDKN2A showed a predictive relationship with benefit from RIB in both training and test sets (Table). PFS benefit with RIB was consistent regardless of the CDK4/CDK6 expression ratio or level of expression of CCNE1, CDK2, RB1, combined CC-related genes, E2F gene signatures, RB gene signature, combined DNA-replication genes, or combined proliferation-related genes. A machine learning approach identified a clinico-genomic signature that was prognostic for PFS benefit with RIB. Selected variables included gene expression levels of FXBO5, PGR, RBBP8, and STC2 and several clinical features (tx arm, de novo disease, prior ET, and visceral disease). Pts with a low signature score had a longer mPFS vs pts with a high signature score, in the RIB (HR, 0.37; 95% CI, 0.22-0.62) and PBO (HR, 0.30; 95% CI, 0.15-0.59) arms. Conclusion: In the largest pooled analysis of the association of gene expression profile data with CDK4/6i tx response in pts with HR+/HER2− ABC, the PFS benefit with RIB + ET over ET alone was consistent irrespective of expression levels of most CC genes. Variation in magnitude of RIB benefit was observed, depending on CDKN2B expression levels, CCND1/CDKN2A expression ratio, and machine learning–derived signature scores. The clinico-genomic CDK4/6i signature requires validation in additional datasets. Table 1: Progression-Free Survival by Gene Expression Subgroup Citation Format: Aditya Bardia, Faye Su, Nadia Solovieff, Fabrice Andre, Carlos Arteaga, Patrick Neven, Yoon-Sim Yap, Yen-Shen Lu, Stephen K. Chia, Dennis Slamon, Seock-Ah Im, Arunava Chakravartty, Agnes Lteif, Tetiana Taran, Debu Tripathy. Pooled gene expression analysis and association with treatment response in patients with HR+/HER2− advanced breast cancer in the MONALEESA-2, -3, and -7 trials [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 PD17-08.

Background: Genetic alterations that contribute to resistance to therapy may be acquired during treatment (tx) for hormone receptor−positive/human epidermal growth factor receptor−negative (HR+/HER2−) advanced breast cancer (ABC). A previous pooled analysis of circulating tumor DNA (ctDNA) in MONALEESA (ML)-2, -3, and -7 identified potential predictive biomarkers for response and resistance to ribociclib (RIB) at baseline (BL). Here, we describe an analysis of paired BL and end of treatment (EOT) samples from ML-2, -3, and -7 to identify acquired mechanisms that may impact resistance to RIB + endocrine therapy (ET) vs placebo (PBO) + ET. Methods: ML-2 (NCT01958021), ML-3 (NCT02422615), and ML-7 (NCT02278120) evaluated efficacy and safety of RIB + ET vs PBO + ET in pre- and postmenopausal patients (pts) with HR+/HER2− ABC treated in first-line (1L) and second-line (2L) settings. Plasma samples were collected at cycle 1 day 1 (C1D1; prior to first therapy exposure) and at EOT (± 28 days of recorded progression). ctDNA was sequenced using a targeted next-generation sequencing panel of ≈550 genes. Genes with an alteration frequency of >5% at EOT, regardless of their frequency at BL, were included. Tumor mutational burden (TMB) was assessed by tx arm; a TMB cutoff of 10 mutations/MB was used to categorize pts as TMB high vs low. To assess differences in the presence of alterations, a McNemar test was performed on paired samples and adjusted (adj) for multiple testing using the false discovery rate (FDR). A Bayesian mixed effects model was used to account for ctDNA fraction and trial and to test for tx-specific resistance by including a tx × visit interaction term. Results: A total of 905 paired samples from ML-2, -3, and -7 were included in this analysis, 441 and 464 samples from pts treated with RIB + ET and PBO + ET, respectively. Overall, 17 genes had an alteration frequency of >5% at EOT. The ctDNA fraction was higher at EOT vs C1D1 in both the RIB (P=.037) and PBO (P=.033) arms. The frequency of alterations in RB1 (10.4% vs 2.0%), ATM (11.3% vs 8.4%), FAT1 (4.8% vs 3.0%), and FAT3 (5.0% vs 2.5%) was higher at EOT vs C1D1 in the RIB arm (FDR-adj P10) at EOT increased from 1.1% to 5.7% in the RIB arm and from 1.7% to 3% in the PBO arm. After accounting for ctDNA fraction and trial, a larger numerical increase in TMB was observed for RIB (odds ratio [OR], 9.0; 95% CI, 2.9-32.7) vs PBO (OR, 2.1; 95% CI, 0.7-6.5); however, the model did not support a differential tx effect. Conclusions: This comprehensive analysis of pooled samples from ML-2, -3, and -7 identified acquired gene alterations in pts with HR+/HER2− ABC treated with 1L or 2L RIB + ET or PBO + ET. The frequency of several genes known to contribute to resistance (ESR1, RB1, ATM, FAT1, and FAT3) was higher at EOT vs C1D1 in pts treated with RIB + ET, while ESR1 and GATA3 alterations were higher at EOT vs C1D1 in pts treated with PBO + ET. This paired dataset of BL and EOT samples from pts with HR+/HER2− ABC treated with a CDK4/6 inhibitor and ET is the largest to date and could be used to validate and confirm acquired resistance mechanisms with low alteration frequency. Citation Format: Fabrice Andre, Nadia Solovieff, Faye Su, Aditya Bardia, Patrick Neven, Yoon-Sim Yap, Debu Tripathy, Yen-Shen Lu, Dennis Slamon, Stephen K. Chia, Mukta Joshi, Arunava Chakravartty, Agnes Lteif, Tetiana Taran, Carlos Arteaga. Identification of mechanisms of acquired resistance to ribociclib plus endocrine therapy using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials [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 P5-02-14.

Background: LSZ102 is an orally bioavailable selective estrogen receptor degrader (SERD) that inhibits estrogen receptor (ER) mediated gene transcription, induces receptor degradation, and blocks ER-dependent cell growth in preclinical models. This first-in-human study of LSZ102 is evaluating LSZ102 as a single agent (SA) and in combination with the CDK4/6 inhibitor ribociclib or the PI3K inhibitor alpelisib (BYL719) in patients (pts) with locally advanced/metastatic ER-positive (ER+) breast cancer (BC). Here, we present the immunohistochemistry (IHC) and circulating tumor DNA (ctDNA) data obtained from a subset of pts treated with either LSZ102 SA or the combination of LSZ102 and ribociclib. Methods: ER IHC was performed with an antirabbit antibody (clone SP1) on FFPE samples collected at baseline and cycle 1 day 15 (C1D15), and the ER value was reported using the H-score method. A Novartis exploratory PanCancer next-generation sequencing ~600 gene panel for cell-free DNA (cfDNA) was used on plasma samples collected from pts treated with SA or the combination at baseline, on treatment (C1D1, C2D1/C3D1, C5D1, CXD1) at radiological assessments and at the end of treatment (EOT). Results: One hundred fifty-three pts with MBC (median number of prior lines of therapy in metastatic setting = 4; range, 0-10) were treated with LSZ102 SA or the combination of LSZ102 and ribociclib (data cutoff, November 30, 2018). ER protein levels, assessed by IHC H-score, are downregulated in about 75% of C1D15 samples in both SA and combination-treated pts. In this cohort of heavily pretreated pts, cfDNA sequencing was performed on approximately 400 samples from 135 pts, and the genomic landscape is described both at baseline and EOT for pts with detectable ctDNA (~77% of pts). At baseline, approximately 40% of pts had mutations in the ESR1 ligand-binding domain (LBD), and approximately 40% of pts had PIK3CA activating mutations. LBD ESR1 mutations were mainly subclonal with several pts harboring multiple mutations at baseline. The frequency of ESR1 Y537S mutation was no higher in EOT samples after LSZ102 treatment compared with baseline. Moreover, the tumor reduction of target lesions > 30% was observed in pts harboring different baseline ESR1 mutations, including Y537S, treated with either SA or the combination. Furthermore, the longitudinal cfDNA analysis showed a consistent reduction of the overall circulating tumor fraction (ctDNA fraction), from baseline for the majority of patients who achieved either partial response or stable disease with either SA or the combination. Conclusions: Consistent with being a SERD, LSZ102 treatment leads to the degradation of ER in tumor biopsies. The addition of ribociclib does not alter LSZ102-induced ER downregulation. The most common genetic alterations found in cfDNA collected from enrolled ER+ MBC patients before the study treatment were ESR1 and PIK3CA mutations. Importantly, the antitumor activity was observed in pts harboring a variety of ESR1 LBD mutations. Furthermore, enrichment of ESR1 Y537S mutations was not observed after LSZ102 treatment. Finally, the on-treatment longitudinal assessment of cfDNA measuring ctDNA fraction and mutation dynamics is maturing as a useful tool for monitoring the treatment efficacy. Citation Format: Komal Jhaveri, Dejan Juric, Sara Cresta, Yoon-Sim Yap, Francois P. Duhoux, Catherine Terret, Rachel Layman, Alejandro Balbin, Qing Sheng, Serena Liao, Adam Crystal, Giuseppe Curigliano. Tumor ER protein modulation, molecular characterization and monitoring of cfDNA in phase 1 study of LSZ102 and LSZ102 + ribociclib in patients with ER+ MBC [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-07.

Background: The combination of ribociclib (an oral CDK4/6 inhibitor) and ET improved survival outcomes in patients (pts) with ER+ ABC in comparison to single-agent ET. In preclinical models, LSZ102 (an experimental oral SERD) demonstrated synergistic activity with ribociclib. This phase 1/1b, open-label study evaluates single-agent LSZ102 (Arm A) and LSZ102 in combination with ribociclib (Arm B) or alpelisib (Arm C) in pts with ER+ ABC who had progressed after ET. Here we present preliminary data from dose escalation of Arm B. Methods: Dose-escalation evaluated daily (QD) LSZ102 at different doses plus ribociclib starting at 300 mg QD either in a 3-week on/1-week (3w/1w) off or in continuous regimen. Pts (aged ≥18 years; ECOG PS 0-1) with histologically confirmed ER+ ABC and progression after ET were enrolled. Prior fulvestrant and/or CDK4/6 inhibitor therapy were permitted. The primary objective was to characterize the safety and tolerability of LSZ102 and ribociclib in combination and identify a recommended dose for expansion (RDE). Secondary objectives included preliminary antitumor activity and characterization of pharmacokinetics. Results: As of 30-Nov-2018, 76 pts were enrolled. The median age of pts was 59 years, 78% (n=59) were Caucasian, 75% (n=57) had an ECOG PS of 0, 63% (n=48) had received prior fulvestrant, and 43% (n=33) a prior CDK4/6 inhibitor; the median number of prior lines of therapy (metastatic setting) was 4 (range 0-10). Pts received LSZ102 QD in combination with ribociclib QD either in a 3w/1w (n=42) or continuous (n=24) regimen, fasted, fed or without regard to food. LSZ102 twice daily (BID) was also explored with a ribociclib BID continuous regimen (n=10). Doses of LSZ102 ranged from 200-600 mg QD and 200-300 mg BID. Doses of ribociclib ranged from 200-600 mg and 200-400 mg in the 3w/1w and continuous regimens, respectively. At data cut-off, 47 pts had discontinued treatment, 42 due to disease progression. Three dose-limiting toxicities were reported: decreased appetite (1 pt) and febrile neutropenia and sepsis (1 pt), all in the 3w/1w regimen. The most common treatment-related adverse events (AEs) were nausea (48.7%), diarrhea (31.6%), fatigue (26.3%), and neutropenia (25.0%). The most common treatment-related grade 3 or 4 AE was neutropenia (10.5%). Four pts (5.3%) required dose reduction due to an AE. Median PFS was 6.0 months. Of 71 evaluable pts, 11% (n=8) had a PR, 39% (n=28) had a best overall response of SD and 16% (n=11) had non-CR/non-PD. ORR, DCR and CBR ≥6 months (includes non-CR/non-PD) were 11% (n=8), 66% (n=47) and 21% (n=15), respectively. The RDE is 450 mg QD LSZ102 with 400 mg QD ribociclib for both ribociclib regimens. Preliminary PK showed the exposure of LSZ102 and ribociclib in combination at the RDE were largely comparable with exposure of the corresponding single agents. Conclusion: The combination of LSZ102 and ribociclib was well-tolerated and demonstrated antitumor activity in heavily pre-treated pts with ER+ ABC many of whom had progressed after fulvestrant and/or CDK4/6 inhibitor therapy. Citation Format: Komal Jhaveri, Dejan Juric, Sara Cresta, Yoon-Sim Yap, Francois P Duhoux, Catherine Terret, Rachel Layman, Nicole Kundamal, Serena Liao, Yan Ji, Adam Crystal, Giuseppe Curigliano. A phase 1/1b study of LSZ102, an oral selective estrogen receptor degrader (SERD), in combination with ribociclib in patients with estrogen receptor-positive (ER+) advanced breast cancer (ABC) who had progressed after endocrine therapy (ET) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-09.

Background: The MONALEESA (ML)-2, -3, and -7 trials have shown a significant benefit in overall survival (OS) with ribociclib (RIB) + endocrine therapy (ET) over placebo (PBO) + ET in HR+/HER2− advanced breast cancer. HR+ breast cancer is a clinically and biologically heterogeneous disease, with identified intrinsic subtypes that vary in incidence, survival rate, and response to treatment. In a pooled analysis of the ML studies, patients with both luminal and HER2-enriched (HER2E) subtypes exhibited a consistent progression-free survival benefit with RIB + ET. The HER2E subtype (RIB, 14%; PBO, 11%), which is associated with ET resistance and poor outcomes, exhibited the greatest relative reduction in risk of progression or death (61%) with RIB + ET. Here, we report a pooled analysis of the ML-2, -3, and -7 trials, presenting OS by intrinsic subtype.. Methods: Tumor samples from patients enrolled in the ML-2, -3, and -7 trials underwent PAM50-based subtyping (blinded from clinical data), and the correlation between intrinsic subtype and OS was analyzed. Gene expression profiling of formalin-fixed, paraffin-embedded tumor samples was performed using a customized NanoString nCounter GX 800-gene panel. The prognostic and/or predictive relationship between PAM50-based subtypes and OS was evaluated using univariable and multivariable Cox proportional hazards models. Multivariable models were adjusted for known clinical prognostic factors, including age, prior chemotherapy, prior ET, ECOG performance status, visceral disease (presence of liver/lung metastases), bone-only metastases, histological grade, number of metastatic sites, tumor type, and de novo metastatic disease.. Results: From the pooled patient population (N = 2066), 997 tumor (71% primary) samples from the RIB (n = 585) and PBO (n = 412) arms of the ML trials (ML-2, n = 318; ML-3, n = 414; ML-7, n = 265) were profiled. Subtype distribution was consistent across treatment arms (Table). A similar benefit with RIB vs PBO was observed in the ITT (hazard ratio [HR], 0.76; 95% CI, 0.67-0.86) and biomarker (HR, 0.75; 95% CI, 0.63-0.89) populations. In both univariable and multivariable analyses, intrinsic subtype was prognostic for OS in both the RIB and PBO arms (P < .0001 for both arms); patients with luminal A subtype had the best OS outcomes in both arms, whereas patients with basal-like subtype had the worst OS outcomes. Intrinsic subtype was also predictive of OS (subtype-treatment interaction: P = .016 [univariable], P = .007 [multivariable]) with a consistent OS benefit with RIB treatment in all subtypes except for basal-like (Table). Patients with HER2E (HR, 0.60; P = .018), luminal B (HR, 0.69; P = .023), and luminal A (HR, 0.75; P = .021) subtypes all derived benefit from RIB. In patients with the basal-like subtype (n = 30), the HR was 1.89 (P = .148); these results should be interpreted with caution due to the small sample size (3% in each arm) and exploratory nature of this analysis.Conclusions: This pooled analysis of the ML trials confirmed the prognostic and predictive value of intrinsic subtype (PAM50 based) for OS. The addition of RIB to ET resulted in consistent OS benefit across all subtypes except for basal-like. The. consistent survival benefit in the HER2E subtype, which is associated with endocrine resistance and a very poor prognosis compared with luminal disease, warrants further investigation. SubtypeTreatment ArmDistribution, n (%)OS, median (95% CI), monthsHR (95% CI)P ValueaLuminal ARIB320 (55)68.0 (61.5-NR)0.75 (0.58-0.96).021PBO222 (54)54.6 (48.3-66.2)Luminal BRIB154 (26)58.8 (48.3-79.2)0.69 (0.50-0.95).023PBO124 (30)44.9 (35.5-52.6)HER2-enrichedRIB95 (16)40.3 (33.4-49.0)0.60 (0.40-0.92).018PBO52 (13)29.4 (23.9-42.0)Basal-likeRIB16 (3)19.4 (10.7-33.2)1.89 (0.80-4.47).148PBO14 (3)21.2 (12.8-NR)aP values are all two-sided. NR, not reached. Citation Format: Lisa Carey, Nadia Solovieff, Fabrice André, Joyce O'Shaughnessy, David A Cameron, Wolfgang Janni, Gabe S Sonke, Yoon-Sim Yap, Denise A Yardley, Juan Pablo Zarate, Tetiana Taran, Faye Su, Agnes Lteif, Aleix Prat. Correlative analysis of overall survival by intrinsic subtype across the MONALEESA-2, -3, and -7 studies of ribociclib + endocrine therapy in patients with HR+/HER2− advanced breast cancer [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 GS2-00.

Background: MONALEESA-2 (ML-2) recently reported a statistically significant overall survival (OS) benefit with first-line ribociclib (RIB) + letrozole (LET) over placebo (PBO) + LET in postmenopausal patients with HR+/HER2- advanced breast cancer (ABC) (median, 63.9 vs 51.4 months; hazard ratio, 0.76; 95% CI, 0.63-0.93; P = .004). Understanding OS outcomes in clinically relevant subgroups of patients is important for improving personalized care and prognosis. Here, we report the results of a prespecified exploratory OS analysis in select patient subgroups by baseline location and number of metastatic sites.. Methods: Postmenopausal patients with HR+/HER2− ABC were randomized 1:1 to receive first-line RIB or PBO with LET. Prespecified exploratory OS analyses were performed for subgroups of special interest by baseline location (bone only [yes or no], liver involvement [yes or no], liver or lung involvement [yes or no]) and number of metastatic sites (< 3 or ≥ 3). The data are hypothesis generating since this analysis was exploratory and not powered for statistical significance.. Results: A total of 668 patients were included in the analysis. A consistent improvement in OS was observed with RIB + LET vs PBO + LET in all subgroups regardless of baseline metastatic site (Table). RIB + LET demonstrated an OS benefit over PBO + LET in clinically relevant subgroups by baseline location and number of metastatic sites, including subgroups of patients with liver metastases, liver or lung metastases, and ≥ 3 metastatic sites, who generally have a worse prognosis.. Conclusion: Consistent with the intent-to-treat population of ML-2, the results of this prespecified exploratory analysis demonstrated an OS benefit with RIB + LET independent of the site and number of metastatic lesions. TableTreatment Arm (n)HR (95% CI)Bone-only metastasisYesRIB + LET (69)0.78 (0.50-1.21)PBO + LET (79)NoRIB + LET (265)0.77(0.61-0.96)PBO + LET (255)Liver involvementYesRIB + LET (59)0.81 (0.54-1.24)PBO + LET (72)NoRIB + LET (275)0.77 (0.62-0.97)PBO + LET (262)Liver or lung involvementYesRIB + LET (182)0.81(0.62-1.05)PBO + LET (190)NoRIB + LET (152)0.71 (0.53-0.96)PBO + LET (144)No. of metastatic sites< 3RIB + LET (220)0.78(0.61-1.00)PBO + LET (222)≥ 3RIB + LET (114)0.71(0.51-0.98)PBO + LET (112) Citation Format: Joyce O'Shaughnessy, Salomon M Stemmer, Howard A Burris, Yoon-Sim Yap, Gabe Sonke, Lowell Hart, Mario Campone, Katarina Petrakova, Eric P Winer, Wolfgang Janni, Pierfranco Conte, David A Cameron, Fabrice André, Carlos Arteaga, Juan Pablo Zarate, Arunava Chakravartty, Tetiana Taran, Fabienne Le Gac, Paolo Serra, Gabriel N Hortobagyi. Overall survival subgroup analysis by metastatic site from the phase 3 MONALEESA-2 study of first-line ribociclib + letrozole in postmenopausal patients with advanced HR+/HER2− breast cancer [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 GS2-01.

Background: First-line ribociclib (RIB; a selective cyclin-dependent kinase 4/6 inhibitor) + letrozole (LET) significantly prolonged progression-free survival vs placebo + LET in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC). The Phase Ib MONALEESASIA dose-escalation/-expansion study (NCT02333370) is investigating RIB + LET in Asian pts with HR+, HER2– ABC; here we report results in Asian non-Japanese pts. Methods: Asian, non-Japanese postmenopausal women with HR+, HER2– ABC, with no prior systemic treatment for ABC, received RIB (starting dose 400 mg/day; 3-weeks-on/1-week-off; option to escalate or de-escalate) + LET (2.5 mg/day; continuous) until disease progression or discontinuation for other reasons. Dose escalation was guided by a Bayesian Logistic Regression Model (BLRM) with overdose control. The primary objective of the dose-escalation part was to estimate the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D), based on the BLRM, safety data, and pharmacokinetic analyses; secondary/exploratory objectives included safety and tolerability, and antitumor activity. Results: At data cut-off (January 16, 2017), 26 pts were enrolled; 13 in the dose escalation (RIB 400 mg: n=6; RIB 600 mg: n=7) and 13 in the 600 mg dose expansion. Treatment was ongoing in 18 pts (69%; 400 mg: n=3; 600 mg: n=15); the most common reason for discontinuation was disease progression (400 mg: n=3; 600 mg: n=4). One dose-limiting toxicity occurred at 600 mg (Grade 3 increased alanine transaminase [ALT]). The MTD/RP2D was declared as 600 mg/day RIB (3-weeks-on/1-week-off) + 2.5 mg/day LET (continuous). The most common all-grade, all-cause adverse events (AEs; ≥35% in either cohort; 400 mg vs 600 mg) were neutropenia (n=1 [17%] vs n=8 [40%]), increased ALT (n=1 [17%] vs n=7 [35%]), decreased neutrophil count (n=3 [50%] vs n=7 [35%]), and alopecia (n=3 [50%] vs n=2 [10%]). All-cause Grade 3/4 AEs occurred in 4 pts (67%) at 400 mg and 18 pts (90%) at 600 mg; the most common (≥25% in either cohort; 400 mg vs 600 mg) were decreased neutrophil count (n=3 [50%] vs n=7 [35%]) and neutropenia (n=1 [17%] vs n=6 [30%]). At least one RIB dose reduction due to AEs occurred in 2 pts at 400 mg and 10 pts at 600 mg. At least one RIB dose interruption due to AEs occurred in 4 pts at 400 mg and 13 pts at 600 mg. Post-baseline QTcF >480 ms occurred in 0 pts at 400 mg and 4 pts (20%) at 600 mg. No pt had a post-baseline QTcF >500 ms. The overall response rate was 50% (n=3) at 400 mg and 35% (n=7) at 600 mg (all partial responses). Stable disease was reported in 1 pt (17%) at 400 mg and 8 pts (40%) at 600 mg. Conclusions: RIB + LET demonstrated a manageable safety profile with preliminary signs of antitumor activity in Asian non-Japanese pts with HR+, HER2– ABC; the safety profile was broadly consistent with that seen in non-Asian pts. The MTD/RP2D was declared as 600 mg/day RIB (3-weeks-on/1-week-off) + 2.5 mg/day LET (continuous). Further safety and efficacy data for RIB + LET will be reported in this pt population after the dose expansion is completed. Citation Format: Yap YS, Ito Y, Suarez-Vizcarra J, Liu X, Han Y, Germa C, Chiu J. Phase Ib study of ribociclib + letrozole in Asian non-Japanese patients with hormone receptor-positive, HER2-negative advanced breast cancer: MONALEESASIA [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-28.

It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the JAK2 SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the NOTCH3 SNP rs200504060 and the HIF1A SNP rs142179458 with breast cancer risk. In silico analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. Cancer Res; 77(19); 5428–37. ©2017 AACR.

Background: MONALEESASIA (NCT02333370) is an ongoing Phase Ib study in which Asian patients (pts) with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) ABC are treated with ribociclib (a cyclin-dependent kinase 4/6 inhibitor) and either letrozole (all pts), fulvestrant, or tamoxifen (Japanese pts). Tumor biomarker analysis in MONALEESASIA using NanoString technology for gene expression was presented previously (Yap YS, et al. ESMO Asia 2018 [Abstract 400]). Here we report additional biomarker analysis of baseline ctDNA. Methods: Plasma samples were collected at baseline and end of treatment; ctDNA was extracted and used to generate next-generation sequencing libraries. The DNA libraries were then enriched for a specific 2.9 Mb of the human genome containing ≈550 oncogenes and tumor suppressor genes. Captured libraries were then combined in equimolar pools and sequenced, targeting ≥70 million sequencing reads per sample to ensure that unique coverage of the genes was >1000× or approached the maximal complexity of the library. Results: ctDNA was analyzed in baseline plasma samples collected from 77 pts (49 Japanese and 28 non-Japanese). Similar gene alteration frequencies were observed in Japanese and non-Japanese pts except for ESR1 alterations, which were more common in Japanese pts (Table). TP53 alterations were more common in pts with progressive disease, and the frequencies of TP53, ESR1, and FAT1 were slightly lower in pre- vs postmenopausal pts; however, these differences were not statistically significant. Response rates by genetic alteration are shown in the table. Conclusion: There appeared to be a trend toward a higher likelihood of alterations in TP53 and PIK3CA in pts with poorer responses, although the sample size was small. These data are hypothesis generating, and additional analysis is ongoing. Gene AlterationsPIK3CATP53CCND1FAT1ESR1FGFR1CDH1Frequency, n (%)Japanesen=4917 (35)11 (22)5 (10)3 (6)7 (14)5 (10)4 (8)Non-Japanesen=288 (29)7 (25)3 (11)5 (18)02 (7)0Best Overall Response, n of Patients with Alteration (% Response Type)PRn=359 (26)6 (17)5 (14)3 (9)2 (6)4 (11)0SDn=299 (31)7 (24)1 (3)3 (10)3 (10)03 (10)NCRNPDn=63 (50)1 (17)002 (33)1 (17)1 (17)PDn=74 (57)4 (57)2 (29)2 (29)02 (29)0CCND1, cyclin D1; CDH1, cadherin 1; ESR1, estrogen receptor 1; FAT1, FAT atypical cadherin 1; FGFR1, fibroblast growth factor receptor 1; NCRNPD, not complete response nor progressive disease; PD, progressive disease; PIK3CA, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α; PR, partial response; SD, stable disease; TP53, tumor protein p53. Citation Format: Yoon-Sim Yap, Faye Su, Nadia Solovieff, Yoshinori Ito, Norikaza Masuda, Takashi Ishikawa, Tomoyuki Aruga, Seung Jin Kim, Wei He, Mihaela Gazdoiu, Joanne Chiu. Baseline circulating tumor DNA (ctDNA) analysis in Asian women with HR+/HER2- advanced breast cancer (ABC) receiving ribociclib + endocrine therapy in the Phase Ib MONALEESASIA trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT143.

Background Understanding the genetic heterogeneity of circulating immune and tumors cells at single-cell resolution will enable one to decipher the interplay between cancer and immune cells, and ultimately lead to better design of immunotherapy assays. Marker-free methods for isolation of cells are attractive because they provide an opportunity to analyze a larger set of these cells that may otherwise be missed due to variable or no expression of protein (marker) markers. Integration of the Biolidics ClearCell® FX System and the Fluidigm® Polaris™ system enabled us not only to develop a marker-free workflow to isolate immune and circulating tumor cells (CTCs) but also to seamlessly prepare amplified cDNA from immune and CTCs for full-length mRNA-seq analysis. Method and Results The ClearCell FX System processes blood samples and isolates CTCs in a marker-free manner. During the CTC isolation process the immune cells are separated from CTCs on the microfluidic device. To differentiate larger blood cells from CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Different enrichment strategies on Polaris allowed us to either specifically select for CTCs (live+, universal cell marker+, CD45-, CD31-) or immune cells (live+, universal cell marker+, CD45+). The enriched cells were then lysed, mRNA were reverse-transcribed, and cDNA were preamplified on a Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated (off-IFC) using the Nextera® XT DNA library prep protocol and sequenced on Illumina® MiSeq™ systems. We successfully profiled full-length mRNA of 81 CTCs from stage III and IV subjects. Based on hormone receptor status, these six subjects were categorized as three types: (1) ER-/PR-/HER2- (triple-negative breast cancer; n=1), (2) ER+/PR+/HER2- (n=3), and (3) ER+/PR+/HER2- (n=2). For immune cell comparative analysis, we included CD45+ (FACS-sorted) from healthy donor. Unsupervised hierarchal clustering of gene expression data showed clustering by subject with a unique set of genes expressed by triple-negative breast cancer only. Heterogeneity was noted in immune cells from subjects at baseline (pretreatment), under treatment with drugs and a healthy subject without cancer. Using TraCeR computational method, we successfully reconstructed full-length, paired T cell receptor (TCR) sequences from CD45+ single-cell RNA sequence data from breast cancer subjects. Conclusion We present the feasibility of integrating two microfluidics platforms to isolate circulating immune and tumor cells for transcriptome and functional study. Our data suggests that the heterogeneity of immune and cancer cells can be elucidated from single-cell mRNA sequencing data and full-length, paired T cell receptor sequences can be reconstructed from immune cells of breast cancer subjects. Citation Format: Naveen Ramalingam, Yifang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Ninez Delos Angeles, Chad Sanada, Andrew Wu, Yoon Sim Yap, Jay West, Ali Asgar Bhagat, Kyle Hukari, Mark Lynch, David King. Full-Length mRNA transcriptome analysis of matched circulating tumor and immune cells from breast cancer subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-326.

Triple-negative breast cancer (TNBC), as immunohistochemically defined by its estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and human epidermal growth factor receptor-2 (HER2)-negative status, is an important subtype due to its biologically aggressive behavior and limited treatment options available. TNBC is associated with an overall poorer prognosis, with higher risk of disease recurrence/progression and shorter duration of treatment response, i.e., treatment resistance. Treatment resistance may be largely attributed to cancer stem cells (CSCs), which are intrinsically treatment resistant and continually self-renew, proliferate, and differentiate into different phenotypes. Activation of the cell biologic program, epithelial-mesenchymal transition (EMT), has been demonstrated to promote the dedifferentiation of heterogeneous subpopulations of cancer cells towards CSC phenotypes. We hypothesized that induction of the mesenchymal-epithelial transition (MET) program might disrupt CSC function, drive differentiation, and render greater susceptibility to conventional chemotherapy. In this study, we utilized high-throughput chemical-genetic screens to uncover a potent class of MET mediators. With the use of in vitro and in vivo models of TNBC, we showed that changing the malignant cell state to a differentiated phenotype by inducing MET reduced mammosphere formation, increased chemosensitivity, and decreased the tumor burden in NSG mice. Delving into the mechanisms of tumor differentiation via ChIP-seq, RNA-seq, and Gene Ontology analysis revealed differences in metabolic status between cell states, which might be exploited in the treatment of TNBC. We also assessed combinations of MET mediators, in order to increase the potency and durability of differentiation. Hence, this study assessed the role of differentiation in the treatment of TNBC and the efficacy of various MET mediators, singly and in combination, in inducing differentiation. Citation Format: Ser Yue Loo, Liping Toh, Elina Pathak, Wilson Tan, Siming Ma, Ju Yuan, Giridharan Periyasamy, Federico Torta, Jack Chan, Tira Tan, Yi Rong Sim, Veronique Tan, Benita Tan, Preetha Madhukumar, Wei Sean Yong, Kong Wee Ong, Chow Yin Wong, Markus R. Wenk, Roger Foo, Yoon-Sim Yap, Elaine Lim, Wai Leong Tam. Inducing cell state transitions in triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B22.

Background: Cyclin D-cyclin-dependent kinase (CDK) 4/6 complexes promote cell proliferation through phosphorylation of retinoblastoma protein (Rb). In breast cancer, cyclin D-CDK4/6 activity can be increased through cyclin D gene (CCND1) amplification or loss of the CDK4/6 negative regulator p16. Here we present efficacy data from the Phase III MONALEESA-2 study of ribociclib (CDK4/6 inhibitor) + letrozole vs. placebo + letrozole for first-line treatment of HR+, HER2- ABC, assessed in baseline tumors by protein levels of Rb, p16, the cell proliferation marker Ki67, and by gene expression levels of CDKN2A (p16) and CCND1. Methods: Postmenopausal women with HR+, HER2- ABC with no prior systemic therapy for advanced disease were randomized 1:1 to receive ribociclib or placebo (600 mg/day 3-weeks-on/1-week-off) + letrozole (2.5 mg/day continuous). The primary endpoint was investigator-assessed progression-free survival (PFS). Provision of a representative baseline tumor biopsy or archival tissue at screening was mandatory if available. Baseline tumor tissue was evaluated for protein biomarkers (immunohistochemistry) and gene expression (NanoString nCounter® Human Cancer Reference panel). Results: Of 668 patients randomized, 479 were evaluable for total Rb, and 416 (87%) displayed high levels (H-score ≥100). p16 protein levels were evaluable in 405 patients; 165 (41%) had low (H-score 14% of cells in 247 (53%) patients. The median messenger RNA expression level was used as the cut-off to define patients with low or high baseline CDKN2A and CCND1 gene expression. An improved PFS was observed by the addition of ribociclib to letrozole in all the above patient subgroups, with hazard ratios ranging from 0.40 (high p16 by H-score; 95% confidence interval [CI] 0.16-1.0; p=0.06) to 0.64 (≤14% Ki67-positive cells; 95% CI 0.39-1.0; p=0.07). Patients with less or greater than 14% Ki67-positive cells, lower or higher p16 levels, Rb levels, or CDKN2A or CCND1 gene expression benefitted from the addition of ribociclib to letrozole to a similar extent. Conclusions: A consistent benefit from ribociclib + letrozole vs. placebo + letrozole was observed irrespective of baseline Rb, p16, and Ki67 levels or CDKN2A and CCND1 gene expression levels. Hormone receptor positivity remains the only established biomarker of response to CDK4/6 inhibitors. Citation Format: Fabrice Andre, Salomon M. Stemmer, Mario Campone, Katarina Petrakova, Shani Paluch-Shimon, Yoon-Sim Yap, Norbert Marschner, Arlene Chan, Cristian Villanueva, Lowell L. Hart, Carlos L. Arteaga, Gabe S. Sonke, Eva-Maria Grischke, Emilio Alba, Arnd Nusch, Denise A. Yardley, Erik Jakobsen, Sibel Blau, Sara M. Tolaney, Faye Su, Wei He, Caroline Germa, Gabriel N. Hortobagyi. Ribociclib + letrozole for first-line treatment of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC): efficacy by baseline tumor markers [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 CT045. doi:10.1158/1538-7445.AM2017-CT045

Background Label-free methods for isolating circulating tumor cells (CTCs) are attractive because they provide an opportunity to analyze a larger set of CTCs that may otherwise be missed due to variable or no expression of protein (label) markers. Understanding genetic and functional heterogeneity in CTCs allows us to gain insight into the mechanisms underscoring metastasis, drug resistance, and tumor aggressiveness. Currently, a simple workflow for isolation and molecular characterization of single CTCs by mRNA sequencing is lacking. In order to address this challenge, we developed a label-free workflow to isolate CTCs from breast cancer patients for full-length mRNA sequencing analysis by integrating the ClearCell® FX System with the Polaris™ system. The ClearCell FX system processes blood samples from cancer patients and enriches for CTCs in a label-free antibody-independent manner. The low level of nonspecifically isolated white blood cells from ClearCell FX is further depleted on the Polaris system by negative enrichment of viable CTCs. This unique integration of systems will enable researchers to perturb single CTCs in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. Method and Results CTCs from 7.5 mL of peripheral blood sample from breast cancer patients were enriched using ClearCell FX. To differentiate larger blood cells from putative CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Single CTCs were selected on Polaris (Fluidigm) system, lysed and reverse-transcribed, and cDNA were preamplified on the Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated using the Nextera® kit and sequenced on Illumina® MiSeq™ and NextSeq™ systems. We successfully processed blood samples from four patients. Sequenced data showed high-quality metrics, with read depth of up to 2.5 million reads (MiSeq) or 60 million reads (NextSeq), with a low percentage of mapped reads to ribosomal RNA and mitochondrial RNA. Unsupervised hierarchical clustering of gene expression data showed clustering by patient, but considerable heterogeneity was also observed among the CTCs from the same patient. We will provide insights into full-length mRNA transcriptome of single CTCs from triple negative breast cancer patient. Conclusion We present the feasibility of integrating two microfluidics platforms to capture single CTCs for transcriptome and functional study. Our data suggests that the heterogeneity of tumor sample and characterization of metastatic processes can be elucidated from single-cell mRNA sequencing of CTCs. Citation Format: Naveen Ramalingam, Yi Fang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Jovina Tan, Chong Tracy Lu, Ninez Delos Angeles, Chad Sanada, Cassandra Greene, Kyle Hukari, Andrew Wu, Yoon-Sim Yap, Jay West, Ali Asgar Bhagat. Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients [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 2923. doi:10.1158/1538-7445.AM2017-2923

Background: HER2-positive tumors are often associated with poor prognosis, chemo-resistances and some patients eventually develop refractory disease during HER2 targeted therapy. While different mechanisms of trastuzumab resistance are being identified in recent years, contribution of confounding factors such as inherent genomic heterogeneity, equivocal HER2 amplifications and presence of chromosome 17 polysomy has been less understood thus far. In this pilot study, we aim to examine HER2 heterogeneity in CTCs obtained from breast cancer patients in the Asian population setting. Methods: CTCs were enriched from blood samples using a label-free spiral microfluidics-based ClearCell® FX system. A total of 26 samples were collected from patients diagnosed with HER2 positive (17/26) and HER2 negative (9/26) breast cancer. The enriched CTCs were analyzed using conventional diagnostic modalities (fluorescence in-situ hybridisation (FISH) and immunocytochemistry) to examine HER2 status. Concordance rate between CTCs and matched primary tumor was evaluated. Results: HER2-positive CTCs were successfully identified in 14 out of 17 HER2+ patients (82.4%); HER2 gene amplification and chromosome 17 polysomy were observed in 10(58.8%) and 13(76.5%) patients respectively. HER2+ CTC counts ranged from 2 to 30 cells from 7.5ml blood (median: 4 HER2+ CTCs/7.5ml). HER2 amplification was not observed in any of the 9 patients with HER2-negative tumors, though 5 out of 9 patients (55.6%) were identified with CTCs harbouring gain in chromosome 17 (median: 2 HER2+ CTCs/7.5ml). A “false positive” cut-off of more than 2 cells/7.5ml blood were established using receiver operating characteristic (ROC) curve analysis and a concordance rate of approximately 70% between paired tumor tissue and CTC among the 26 patients. Immunofluorescence labelling of CTCs with cytokeratin, CD45 and HER2 antibodies further revealed heterogeneity in HER2 expression on CTCs. Conclusion: CTCs capture the heterogeneity of breast cancer, and could potentially overcome limitations of tissue biopsy which are site specific. HER2- patients, as confirmed by tissue biopsy, with HER2+ CTCs pose interesting questions while determining treatment regime. Citation Format: Man Chun Leong, Tse Hui Lim, Chye Ling Tan, Yong Wei Chua, Elaine Hsuen Lim, Kiley Wei Jen Loh, Guek Eng Lee, Rebecca Dent, Raymond Chee Hui Ng, Andrew Wu, Wan-Teck Lim, Alvin Soon-Tiong Lim, Yoon-Sim Yap. Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5008.

INTRODUCTION Although mutations in BRCA1 and BRCA2 are associated with familial hereditary breast and ovarian cancer, mutations are only found in 20 to 25% of these patients. This suggests that additional genes may be mutated. The Casitas B-lineage lymphoma (CBL) gene encodes a RING finger E3 ubiquitin ligase. Recent studies have shown that mutations in the CBL gene occurs in human acute myeloid leukemia and lung cancers. The relationship of the CBL gene with breast cancer, however, is not well characterized. The aim of this study was to identify mutations in the CBL gene in Asian patients with familial or early-onset breast cancer. METHODS All patients (n=75) were accrued at the risk assessment clinic at the National Cancer Centre Singapore, and presented with a family history of breast and/or ovarian cancer or early-onset breast cancer. Screening for mutations in the BRCA1 and BRCA2 genes was performed using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Next generation sequencing was done using the Agilent SureSelect target-enrichment technology on the Illumina MiSeq platform, followed by validation using Sanger sequencing. RESULTS Two mis-sense variants in the CBL gene were identified. They are CBL c. 560C>T (p. A187V) and c. 1858C>T (p.L620F) occurring in 2/75 (3%) and 8/75 (11%) of the patients, respectively. Both mis-sense mutations were predicted to be damaging using Polyphen-2 and SIFT. The p.A187V alteration was predicted to interrupt the N-terminal tyrosine kinase binding domain (TKB) that is important for ligand-induced ubiquitination of receptor tyrosine kinases (RTKs). The CBL c. 1858C>T variant has previously been reported in the NCBI database (rs2227988). Three individuals, who carried this mutation, had early-onset breast cancer. In addition, the p.L620F alteration within the proline-rich region, may disrupt the interactions of binding partners to the c-CBL protein, which are required to recruit c-CBL to RTKs. All patients, except one, with germline CBL mutations were negative for BRCA1 and BRCA2 mutations. CONCLUSION This preliminary study has identified damaging CBL mutations in an Asian population with breast cancer and warrants further investigations in other breast cancer populations. Citation Format: Edward Wong, Yoon Sim Yap, Ying Ying Cheng, Delia Chua, Lewis Zuocheng Hong, William F. Burkholder, Gay Hui Ho, Min Han Tan, Peter Ang, Ann S.G. Lee. Mutations in the CBL gene among breast cancer patients in an Asian clinic-based population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1287. doi:10.1158/1538-7445.AM2014-1287

Tamoxifen (TAM) is a prodrug with a complex metabolic pathway involving several metabolic enzymes. Although TAM is mainly metabolised by cytochrome P450 (CYP) 2D6 and CYP3A4/5 enzymes, other CYP enzymes such as CYP1A2, CYP2B6, CYP2C9, and CYP2C19, also catalysed the metabolism of TAM to N-desmethyltamoxifen (NDM), 4-hydroxytamoxifen (4OHT) and endoxifen (END) with 4OHT and END being the active metabolites of TAM. The aim of this study was to investigate the impact of CYP2C19 polymorphisms on the plasma concentrations and metabolic ratios of TAM and its three metabolites, NDM, 4OHT and END. The exons and intron-boundaries of CYP2C19 gene as well as the upstream and downstream regions (about 14 kilobases) were sequenced in 240 Asian healthy subjects (Chinese, Malay, Indians, N=80 each) to identify the polymorphisms present in Asian population. Linkage disequilibrium between identified polymorphisms was examined via Haploview (version 4.0) and tag-SNPs were identified via TAGGER. These tag-SNPs were subsequently genotyped in 164 Asian breast cancer patients using direct sequencing. Plasma levels of TAM and its metabolites were determined at steady state using HPLC with fluorescence detection. Genotypic-phenotypic associations were performed using non-parametric Kruskal-Wallis test and Mann-Whitney U-test. A moderate linkage pattern was observed across the CYP2C19 polymorphisms in the three Asian healthy populations. A total of 13 tag-SNPs and one reported functional SNP were analyzed in Asian breast cancer patients. Patients carrying the AC and CC genotypes of the exonic polymorphism 1251A>C (rs17886522) was associated with 2.3-fold reduction in the median (range) MREND-NDM compared to patients with the reference genotype [AA vs AC+CC: 5.14 (0.92 − 27.81) vs 2.25 (1.26 − 10.72), P = 0.026]. In contrast, median (range) MREND-4-OHT was found to be 1.3-fold higher in patients carrying one or two copies of the −3219T>G variant allele compared to patients carrying the two copies of wild-type allele [TT vs TG + GG: 6.57 (1.99 − 13.18) vs 8.76 (3.78 − 12.80), P = 0.001]. Modest increases in the plasma concentrations of TAM and NDM were associated with 1251A>C (rs17886522). In addition to CYP2D6 polymorphisms, polymorphic variants present in the 5α upstream region of CYP2C19 were found to influence the metabolic ratios of TAM and its metabolites but not the plasma concentrations of the analytes in this exploratory study. The combinative effect of genetic variants in various phase I pharmacogenes on plasma levels of tamoxifen warrants further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2668. doi:1538-7445.AM2012-2668

Genome-wide association studies (GWAS) have identified several breast cancer susceptibility loci associated with European populations, some of which were also found to be associated with breast cancer among women in China. Women in Singapore are predominantly of Chinese ancestry with an urbanized lifestyle, with breast cancer rates approaching those of the West. To explore the role of these breast cancer susceptibility variants, a Taqman-based microfluidics strategy was established to carry out fast track replication of 22 SNPs for 1,146 cases and 1,534 controls to identify significant risk variants for breast cancer. Interestingly, the most significant SNP [rs2046210; odds ratio (OR), 1.4; 95% confidence interval, 1.2-1.6; P=7x10−6], residing in 6q25.1, had been initially identified in a Chinese population, suggesting a strong and consistent association of this SNP with Chinese ancestry. SNPs in FGFR2 (rs2981579, rs1219648, rs2981582, rs112000014), 8q24 (rs13281615), and MAP3K1 (rs16886165) showed significant associations similar to those observed in women of European ancestry, with ORs ranging from 1.1 to 1.7 (P=5x10−5 to 0.04). Ranking of the SNPs in terms of their ORs for Singaporean Chinese women was compared to that from Western and Chinese studies. The ranking for Singaporean Chinese women resembled, but was not identical to, that of women in China, reflecting the effect of ethnicity. Comprehensive annotation of the genotyping data with associated clinicopathological features enabled the identification of significant SNPs associated with tumour size, lymph node metastasis, and estrogen/progesterone receptor status. Findings from this study implicate 6q25.1 and FGFR2 as the most important susceptibility loci for Asian women of Chinese ancestry and may guide future fine-mapping studies to identify causal variants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1647. doi:1538-7445.AM2012-1647