Shak, S.

Abstract Funding: Supported by National Cancer Institute grants U10CA180888, U10CA180819, U10CA180820, U10CA180821, U10CA180868, U10CA180863; and in part by Susan G. Komen for the Cure® Research Program, The Hope Foundation for Cancer Research, Breast Cancer Research Foundation, and Genomic Health, Inc. Acknowledgement: The authors wish to thank Dr. Ana M. Gonzalez-Angulo, MD, for her invaluable contributions to the design and implementation of this study. Background: The clinical utility of the RS to determine CT benefit is well established in pts with HR+, HER2-, axillary lymph node (LN)-negative BC. Retrospective analyses from SWOG S8814 support the potential prognostic and predictive role of RS for CT benefit in postmenopausal pts with LN+ BC. SWOG S1007 is a prospective, randomized trial of endocrine therapy (ET) vs. chemoendocrine therapy (CET) in women with 1-3 +LN and a RS < 25 (NCT01272037). Methods: Eligibility criteria included women > 18 years of age with HR+, HER2- BC and 1-3 +LN and no contraindications to taxane and/or anthracycline based CT. Women with a RS < 25 were randomized to receive ET or CET in 1:1 randomization using 3 stratification factors: (1) RS (0-13 vs.14-25); (2) menopausal status; and (3) axillary nodal dissection vs. sentinel node biopsy. The primary objective was to determine the effect of CT on invasive disease-free survival (IDFS) and whether the effect depended on the RS. The primary analysis was to test for a significant interaction of the treatment arm and continuous RS using a Cox regression model for IDFS, adjusting for treatment, RS, and menopausal status. A total of 832 IDFS events were expected for the final analysis. Secondary objectives included overall survival (OS). The protocol specified that interaction between treatment and the stratification variables was to be tested and, if significant, separate analyses performed by stratum. Annual interim analyses were planned starting at 24% of events. At the third interim analysis with 410 IDFS events, the Data and Safety Monitoring Committee recommended reporting results, with a decision by the NCI’s Cancer Therapy Evaluation Program, the study sponsor. Results: Of the 9,383 women screened from 2/28/11-9/29/17, 5,083 pts (54.2%) were randomized. With a median follow-up of 5.1 years, 447 IDFS events have been observed. For the primary analysis, the interaction test for CT benefit and continuous RS was not statistically significant, p=0.30. In a model with CT, RS, and menopausal status (no interaction term), higher continuous RS was associated with worse IDFS [HR 1.06, 2-sided p<0.001, 95% Confidence Interval (CI) 1.04-1.07], and CT was associated with an improvement in IDFS (HR 0.81, p=0.026, 95% CI 0.67-0.98). In a pre-specified analysis, a significant interaction was identified between CT and menopausal status (p=0.004), necessitating separate analyses by menopausal status. In postmenopausal pts (N=3350, 67%), adjusting for continuous RS, the HR for CET vs. ET was not significant (HR=0.97, p=0.82, 95% CI 0.78-1.22; 5-year IDFS 91.6% vs. 91.9%) indicating no benefit from CT. In premenopausal pts (N=1665, 33%), the HR (0.54) was statistically significant (p=0.0004, 95% CI 0.38-0.76; 5-year IDFS 94.2% vs. 89.0%), indicating CT benefit. In premenopausal pts, ovarian suppression was performed in 15.9% vs. 3.7% (ET vs. CET), and 47.9% vs. 26.4% reported menstruation after 6 months of treatment. Although the number of events is limited, the HR for treatment adjusted by RS for OS in premenopausal pts was 0.47 (p=0.032, 95% CI 0.24-0.94). At this time, there is no differential effect with CT in regard to other stratification factors. Conclusions: There is a significant differential treatment effect of CT benefit based on RS for premenopausal vs. postmenopausal women requiring separate analyses. While only 54% of the protocol specified events are recorded and pts will be followed for 15 years, the current data show that adjuvant therapy can be de-escalated to ET alone in postmenopausal pts with a RS < 25 and 1-3 +LN. However, there is a strong IDFS benefit for CET in premenopausal pts, with an early indication of an OS improvement. Citation Format: Kevin Kalinsky, William E Barlow, Funda Meric-Bernstam, Julie R Gralow, Kathy S Albain, Daniel Hayes, Nancy Lin, Edith A Perez, Lori J Goldstein, Stephen Chia, Subkhbinder Dhesy-Thind, Priya Rastogi, Emilio Alba, Suzette Delaloge, Miguel Martín, Miguel Gil Gil, Claudia Arce-Salinas, Etienne Brain, In Hae Park, Jean-Yves Pierga, Ana Hernandez Lluch, Manuel Ramos Vasquez, Manuel Ruiz Borrego, Kyung Hae Jung, Jean-Marc Ferrero, Anne Schott, Steve Shak, Priyanka Sharma, Danika L Lew, Jieling Miao, Debu Tripathy, Gabriel Hortobagyi, Lajos Pusztai. First results from a phase III randomized clinical trial of standard adjuvant endocrine therapy (ET) +/- chemotherapy (CT) in patients (pts) with 1-3 positive nodes, hormone receptor-positive (HR+) and HER2-negative (HER2-) breast cancer (BC) with recurrence score (RS) < 25: SWOG S1007 (RxPonder) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-00.

Abstract BACKGROUND: Unique genes and pathways were identified for prognosis on tamoxifen (T, 5 yrs) and prediction on CAF-T vs T in S8814 using whole transcriptome RNA-Seq from archival FFPE tissue. (Albain, et al; Cherbavaz, et al; SABCS 2015) Discovery was robust for early DFS events but sparse for late. The aims of this new analysis were to 1) utilize a new endpoint BCSE for gene discovery of late events, prognosis and prediction and 2) add intronic counts to the previous exonic results to define whole genes impacting on late BCSE. METHODS: Charts of patients (pts) on CAF-T (212) vs T (142) were reviewed to define the BCSE endpoint (local/regional, contralateral, distant). Deaths without BC were treated as competing risks. BCSE models (including metagenes) of late prognosis and prediction used cumulative incidence functions. Consolidated intronic regions counts within genes were added to exonic regions counts. Using these “whole gene” (WG) counts, association of gene expression with time to BCSE was assessed by Cox regression. A multiple WG score (MWGS) for BCSE prognosis beyond 5 yrs (to 12.5 yrs) was constructed and evaluated for 1-3 and 4+ node (N) groups. False discovery rate was controlled at 10%. RESULTS: More exons and WG were discovered for prognosis on T alone over 12.5 yrs with the BCSE endpoint than DFS. For prognosis of late BCSE after 5 yrs, more genes were discovered using WG (n=111) than by exons (n=9). There were significantly fewer genes for late BCSE on CAF-T (8, WG; 0, exons). The functions of WG prognostic for late BCSE were: cell cycle/proliferation-26 genes, chromosome segregation/mitotic spindle-22, DNA repair/maintenance-10, transcriptional/translational control-5, cell adhesion/migration-4, immune-3, diverse/unknown-32 and growth factor/hormone receptor signaling-9 (this group was only found by WGs, not exons). Of these 111 WG, a MWGS prognostic for late BCSE on T used 57 previously discovered genes pre-specified for this analysis. Probability of BCSE beyond 5 yrs for low vs high MWGS was 8% vs 21% in N1-3+ and 17% vs 42% in N4+. Late prognosis on T differed by low vs high risk defined in a metagene model: cumulative BCSE at year 10 was 0% vs 47% (low vs high risk, p=0.001). Prediction of 10-yr incidence of BCSE varied by risk level by treatment in a metagene model: low risk- CAF-T=47%, T=0% (p=0.045); high risk- CAF-T=35%, T=45% (p=0.027). CONCLUSIONS: Gene discovery for prognosis of late BCSE is enhanced with a novel WG transcriptome expression approach. Use of chemotherapy (CT) before T significantly attenuated gene discovery, so that molecular tools for decisions on extending endocrine therapy (ET) may not be reliable in a setting of prior CT. Some pts on ET for 5 yrs may not require either longer ET or CT, given a N+ cohort was defined with no BCSE observed over 12.5 yrs. For prediction of CT benefit, CAF-T appeared to be inferior to T in a low risk metagene model for BCSE. In sum, these results add more evidence that ET alone may be sufficient (perhaps better) in select N+ settings. Validation in SWOG S1007 (RxPONDER) is planned. SUPPORT: NCI CA180888, 180819, 180821, 180820, 180863; in part, Genomic Health, Inc. Citation Format: Albain KS, Crager MR, Barlow WE, Baehner FL, Bergamaschi A, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Cherbavaz DB, Sing AP, Shak S, Hortobagyi GN, Hayes DF. Discovery of molecular predictors of late breast cancer specific events (BCSE) in ER+, node+ breast cancer – new transcriptome expression whole gene analysis of the phase III adjuvant trial SWOG S8814 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD7-07.

Abstract BACKGROUND: We previously reported that low 21 gene Recurrence Scores (RS) identify patients with ER-positive, lymph node-positive breast cancer who may not benefit from anthracycline-based adjuvant chemotherapy added to tamoxifen (SWOG-8814A NCI correlative science study; Albain et al. Lancet Oncol 2010). New exploratory and comprehensive quantitative analyses now permit whole transcriptome NGS on residual RNA extracted from FFPE blocks 12-18 years post-fixation. Herein, we report methodology details and feasibility results (see companion abstract, Albain et al., for clinical outcomes correlations). METHODS and RESULTS: Sequencing was carried out in Illumina HiSeq 2000 instruments, yielding 4.2 trillion data points. Messenger RNA expression was quantified using 3rd quartile normalization. Both Library (RNA) and Sequencing Standards showed high quality coverage as measured by median uniquely mapped reads over a 13 month window (168M and 182M, respectively, including duplicate reads). The median absolute deviation (MAD) of the relative log expression (RLE) of mapped reads for the Library and Sequencing Standards was 0.22 and 0.05, respectively. The Library Standard variation was greater than the Sequencing Standard, as library preparation was manual. Of 360 patient samples with sufficient RNA (≥ 100 ng total RNA), 354 (98.3%) were successfully sequenced and included in the final analysis data set. Average library yield was 39 ng/μL. Only 5 libraries failed yield requirements and one library failed expression quality metrics. The median insert length was 120 bp with the first and third quartiles 93 and 152 bp, respectively. After removal of duplicate reads, 82% of reads were uniquely mapped, and the median library size was 8.95M (number of unique mapped reads). Sequences with counts <10 for all 354 patients were excluded. The medians of the 1st, 2nd and 3rd quartiles for exons mapped to the RefSeq database were 20, 40 and 78 counts, and for introns, 72, 134 and 244 counts, respectively. The majority of exonic (86.7%) and intronic (95%) sequences were mapped. There were 20,101 RefSeq mapped exons with counts ≥10. Of these exons, 988 passed additional filtering criteria and were subjected to hierarchical clustering, with Gene Ontology and pathway analysis performed on selected gene expression patterns (for results, see companion abstract of Albain et al.). CONCLUSIONS: High quality whole transcriptome NGS is feasible from decades-old clinical trial FFPE specimens that have not been stored in any special fashion. Controlled laboratory, bioinformatics and biostatistics methods, with inclusion of appropriate process controls, ensure robustness and reliability of the NGS process. This in turn results in the discovery and validation of biologically and clinically relevant variations from prior landmark clinical trials. SUPPORT: NCI CA 180888, CA180819, CA180821, CA180820, CA180863; in part, Genomic Health, Inc. Citation Format: Cherbavaz DB, Hayes DF, Qu K, Crager MR, Barlow WR, Goddard AD, Beasley EM, Jeong J, Collin F, Liu M-L, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Sing AP, Baehner FL, Hortobagyi GN, Shak S, Albain KS. Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-01.