Pritchard, Kathleen I.

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.

LBA4 Background: Lapatinib (L) is a HER1-HER2 tyrosine kinase inhibitor. The Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO) Trial is a randomised, phase III trial comparing 3 oral L-containing regimens with T, each given for 1 year. Methods: From June 2007 to July 2011, 8381 patients (pts) were randomised from 946 sites in 44 countries to receive either L+T, T→L, L, or T. Anti-HER2 therapy was initiated after completing all chemotherapy (N=4613), concurrently with a taxane following anthracycline (N=3337), or concurrently with a non-anthracycline, platinum-containing regimen (N=431). The L arm was closed in Aug 2011 for futility and is not presented. The primary endpoint is invasive disease-free survival (DFS). L+T vs. T is tested for superiority and T→L vs. T is tested for non-inferiority at 1.11 margin. P≤0.025 is required for statistical significance. 850 DFS events in the L+T vs. T comparison would provide 80% power to detect a true hazard ratio (HR) of 0.80 with experiment-wide alpha=0.05. The current analysis was planned to occur either after observing these 850 events or at 4.5 yrs median follow up (MFU). Results: Pt and disease characteristics were well balanced. 40% were node-negative and 57% were hormone receptor positive. Only 555 DFS events for the L+T vs. T comparison were observed at 4.5 years MFU. HR for DFS was 0.84 (97.5% CI, 0.70-1.02; P=0.048; 4-yr DFS%=88% vs. 86%) for L+T vs. T and 0.93 (97.5% CI, 0.76-1.13; non-inferiority P=0.044; 4-yr DFS%=87% vs. 86%) for T→L vs. T. Diarrhoea (75% vs. 20%), rash (55% vs. 20%) and hepatobiliary (23% vs. 16%) adverse events were more frequent in L+T vs. T. Primary cardiac endpoints were infrequent (<1%) in all arms. Conclusions: L+T has lower risk of a DFS event compared with T, and T→L appeared non-inferior to T, but neither finding was statistically significant. The first DFS results of dual HER2 blockade in the adjuvant ALTTO at 4.5 years MFU are unexpected considering the effect shown by doubling the pCR rate with L+T vs. T in the NeoALLTO trial. Follow up continues. Clinical trial information: NCT00490139.

Abstract Genetic risks in breast cancer remain only partly understood. Here, we report the results of a genome-wide association study of germline DNA from 4,658 women, including 252 women experiencing a breast cancer recurrence, who were entered on the MA.27 adjuvant trial comparing the aromatase inhibitors (AI) anastrozole and exemestane. Single-nucleotide polymorphisms (SNP) of top significance were identified in the gene encoding MIR2052HG, a long noncoding RNA of unknown function. Heterozygous or homozygous individuals for variant alleles exhibited a ∼40% or ∼63% decrease, respectively, in the hazard of breast cancer recurrence relative to homozygous wild-type individuals. Functional genomic studies in lymphoblastoid cell lines and ERα-positive breast cancer cell lines showed that expression from MIR2052HG and the ESR1 gene encoding estrogen receptor-α (ERα) was induced by estrogen and AI in a SNP-dependent manner. Variant SNP genotypes exhibited increased ERα binding to estrogen response elements, relative to wild-type genotypes, a pattern that was reversed by AI treatment. Further, variant SNPs were associated with lower expression of MIR2052HG and ERα. RNAi-mediated silencing of MIR2052HG in breast cancer cell lines decreased ERα expression, cell proliferation, and anchorage-independent colony formation. Mechanistic investigations revealed that MIR2052HG sustained ERα levels both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated, proteasome-dependent degradation of ERα. Taken together, our results define MIR2052HS as a functionally polymorphic gene that affects risks of breast cancer recurrence in women treated with AI. More broadly, our results offer a pharmacogenomic basis to understand differences in the response of breast cancer patients to AI therapy. Cancer Res; 76(23); 7012–23. ©2016 AACR.

Abstract BACKGROUND: In SWOG-8814A, pts with ER+ node+ breast cancer and low 21 gene recurrence scores (RS) had good prognosis and no CAF benefit, but high RS predicted longer survival from CAF followed by T (CAF-T) vs T (Albain, Lancet Oncol 2010). The aims of SWOG-8814B were to identify novel genes and networks for 1) prognosis of early and late relapse and 2) prediction of CAF benefit, using whole transcriptome expression analysis with next generation RNA sequencing (NGS). METHODS: Stored RNA previously extracted for SWOG-8814A (T, CAF-T arms; T, 5 yrs) was analyzed for RNA/library yield (see companion abstract Cherbavaz et al. for methods). Genes were sequenced and expression of mRNA species was related to disease-free survival (DFS) using Cox proportional hazards. Discovery analyses controlled false discovery rate (FDR) at 10%. Genes were identified for prognosis on T and prediction on CAF-T vs T. Networks of genes/pathways were explored. Early (0-5 yrs) and late (5-13+ yrs) time periods were studied. Gene Ontology, Cytoscape, pathway and hierarchical clustering were used for functional gene and metagene analyses. RESULTS: Of 367 samples, 354 (96%; 142 T, 212 CAF-T; 141 DFS events) had sufficient RNA/library yield, with 20,101 genes sequenced. For prognosis on T, there were 2327 and 568 genes discovered in early and all-yrs follow-up, with only 9 genes prognostic after 5 yrs. Prognosis analyses for residual risk after CAF-T were uninformative. Functional mapping found that genes prognostic for worse DFS were enriched for proliferation (G2M, M-phase), cellular metabolism, DNA repair, stress response and EMT; whereas, those with better DFS involved transcription regulation/repression via zinc finger proteins. Hierarchical clustering (T arm) found significant DFS prognostic metagene signatures for ER-related genes, immune response, ECM/stroma, chromatin remodeling-transcription factor activity and TGFb pathway. All varied for early vs late DFS events. For example, low ER/high stroma expression signatures correlated with high proliferation gene expression and were strongly associated with early events (standardized [st] HR 2.94, p&lt;0.001). Late recurrence was associated with high proliferation, both individually (stHR 1.51, p=.035) and in combination with higher ER expression (stHR 1.51, p=0.09). Fifteen genes predicted CAF benefit (9 better DFS, 6 worse), or 129 genes if FDR relaxed to 20%. Cluster analysis for CAF prediction is ongoing. CONCLUSIONS: Unique genes, clusters and pathways were identified by NGS of archival material in ER+ N+ breast cancer, including previously unreported signatures. While ER, stroma and proliferation-related signatures were associated with early prognosis, proliferation best predicted worse DFS after 5 yrs. NGS of the primary tumor is most informative for early events in pts with only 5 years of T, with few genes selecting only for late relapse. If validated, these signatures may identify pts with excellent DFS despite positive nodes for endocrine therapy alone as well as others for whom chemotherapy and/or biologics are also required . SUPPORT: NCI CA 180888, 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. Molecular predictors of outcome on adjuvant CAF plus tamoxifen (T) vs T in postmenopausal patients (pts) with ER+, node+ breast cancer – Transcriptome expression analysis of the phase III trial SWOG-8814. [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 S3-02.

Abstract Background: We have previously shown in a phase III neoadjuvant trial that early development of lapatinib-induced rash (i.e. within 6 weeks after lapatinib initiation) is independently associated with a higher chance of obtaining a pathological complete response (Azim et al; JCO 2013). In the current study, we aimed to investigate whether early lapatinib-induced rash is associated with improved survival in the context of a large phase III adjuvant trial. Methods: This analysis is based on the ALTTO trial (BIG 2-06, Alliance N063D), in which patients with HER2-positive early breast cancer were randomized to adjuvant trastuzumab, lapatinib, their sequence or their combination for a total duration of 1 year. In this sub-study, we evaluated whether the development of rash (any grade) within 6 weeks of lapatinib initiation was associated with disease-free (DFS) and overall survival (OS). All analyses were tested in a multivariate model adjusted for treatment arm, treatment completion and trial stratification factors. Results: A total of 6,098 lapatinib-treated patients were included in the current analysis; of whom 2,006 patients (32.9%) developed early lapatinib-induced rash, 1,025 (16.8%) developed rash after 6 weeks and 3,067 (50.3%) did not develop rash. No differences in patient characteristics were observed between the three groups apart from a higher frequency of younger patients (≤ 50) in the early rash group (54% vs. 47% and 44%, p&lt;0.0001). At a median follow-up of 4.5 years, 876 (14.37%) and 377 (6.18%) patients in the lapatinib containing arms experienced a DFS and OS event, respectively. In a multivariate analysis confined to patients randomized to the lapatinib containing arms, the development of early rash was associated with improved DFS (HR: 0.80; 95%CI: 0.69-0.93, p=0.004) and OS (HR: 0.61; 95%CI: 0.48 - 0.78, p&lt;0.001) compared to patients who did not develop early rash, with no interaction according to patient's age (p=0.9). No significant association was observed between the development of rash after 6 weeks of lapatinib initiation and survival. Compared to patients randomized to the trastuzumab alone arm (n=2,076), patients who developed early rash in the sequence (n=580) or combination (n=704) arms of trastuzumab/lapatinib had superior DFS (Sequence: HR 0.75 [95% CI: 0.58 – 0.98], p=0.034; Combination: HR 0.69 [95% CI: 0.54 – 0.89], p=0.005) and OS (Sequence: HR 0.57 [95%CI: 0.36 – 0.88], p=0.012; Combination: HR 0.59 [95% CI: 0.39 – 0.89], p=0.011). On the other hand, patients randomized to the lapatinib only arm who developed early rash (n=722) still had inferior DFS (HR 1.28 [95% CI: 1.04 – 1.59], p=0.02) with no difference in OS (HR: 0.95; 95%CI: 0.67 – 1.35, p=0.79) compared to patients randomized to the trastuzumab alone arm. Conclusions: The results support our previous findings in the neoadjuvant setting that early development of skin rash within the first 6 weeks can identify patients who derive superior benefit of lapatinib treatment. Citation Format: Azim Jr HA, Sonnenblick A, Agbor-Tarh D, Bradbury I, Daly F, Huang Y, Dueck AC, Pritchard K, Wolff AC, Jackisch C, Lang I, Untch M, Smith I, Boyle F, Xu B, Gomez H, Perez E, Piccart M, de Azambuja E. The impact of early lapatinib-induced rash on disease-free and overall survival in patients treated within the ALTTO phase III randomized trial. [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 PD5-07.

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 &lt;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.

Abstract Background: The Trial Assigning Individualized Options for Treatment (TAILORx) is a prospective trial evaluating the role of endocrine therapy or chemoendocrine therapy in patients 18-75 years of age with estrogen receptor (ER)-positive, HER2-negative breast cancer, a primary tumor between 0.6-5.0 cm, and negative axillary nodes, a population for whom chemotherapy is typically recommended or least considered based on National Comprehensive Cancer Center Network (NCCN) guidelines. Methods: The trial was designed to demonstrate non-inferiority of endocrine therapy compared with chemoendocrine therapy in the randomized group with an Oncotype DX Recurrence Score (RS) of 11-25. Patients with a low RS &lt; 11 were assigned to endocrine therapy alone and with a high RS &gt; 25 assigned to chemoendocrine therapy, and both groups were followed in a prospective registry. The definition of an intermediate RS differed in this trial (RS 11-25) from the original reports (RS 18-30) in order to reduce the risk of chemotherapy undertreatment in patients with a mid-range or low RS (Sparano & Paik. J Clin Oncol 2008; 26:721-728). Results: The trial enrolled 10,273 patients between April 2006 and October 2010, of whom 6907 patients (67.2%) had a mid-range RS of 11-25, 1737 (16.9%) had a high RS &gt; 25, and 1639 (15.9%) had a low RS of &lt; 11. At the fourth planned interim analysis, the ECOG-ACRIN data monitoring committee recommended that the study continue as planned for the randomized group with a RS 11-25, and that the results be released to the investigators for the low risk group with a RS &lt;11. The characteristics of the low risk registry population are as follows: age 50 or less (27%), 51-60 (35%), &gt; 60 (39%); tumor size &lt; 1 cm (8%), 1-2 cm (61%), &gt; 2 cm (31%); histologic grade low (34%), intermediate (59%), high (7%); breast conservation (68%) or mastectomy (32%). Initial endocrine therapy included tamoxifen in 35%, aromatase inhibitors in 59%, ovarian function suppression in 3%, and unspecified therapy in 3%; 5 patients received adjuvant chemotherapy (1 of whom relapsed). Five-year rates (and 95% confidence intervals [CI]) for low RS group were 99.2% (98.5, 99.6%) for distant relapse free interval, 98.5% (97.7, 99.1%) for relapse-free interval, 93.7% (92.2, 94.9%) for invasive disease free survival, and 98.2% (97.3, 98.7%) for overall survival. Information regarding ER, PR, and HER2 RNA expression will be presented. Conclusions: Despite meeting guidelines for recommending or at least considering adjuvant chemotherapy based on classical clinicopathologic features, the risk of recurrence was very low at 5 years in patients with ER-positive, HER2-negative, axillary node-negative breast cancer and a low RS of &lt; 11 treated with endocrine therapy alone without chemotherapy. Citation Format: Sparano JA, Gray RJ, Makower DF, Pritchard KI, Albain KS, Hayes DF, Geyer Jr CE, Dees EC, Perez EA, Olson Jr JA, Zujweski J, Keane MM, Gomez Moreno HL, Reddi RP, Goggins TF, Mayer IA, Brufsky AM, Toppmeyer DL, Kaklamani VG, Atkins JN, Berenberg JL, Sledge Jr GW. Prospective trial of endocrine therapy alone in patients with estrogen receptor positive, HER2-negative, node-negative breast cancer: Results of the TAILORx low risk registry. [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 P2-08-01.

Background Lapatinib (L) plus trastuzumab (T) improves outcomes for metastatic human epidermal growth factor 2–positive breast cancer and increases the pathologic complete response in the neoadjuvant setting, but their role as adjuvant therapy remains uncertain. Methods In the Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization trial, patients with centrally confirmed human epidermal growth factor 2–positive early breast cancer were randomly assigned to 1 year of adjuvant therapy with T, L, their sequence (T→L), or their combination (L+T). The primary end point was disease-free survival (DFS), with 850 events required for 80% power to detect a hazard ratio (HR) of 0.8 for L+T versus T. Results Between June 2007 and July 2011, 8,381 patients were enrolled. In 2011, due to futility to demonstrate noninferiority of L versus T, the L arm was closed, and patients free of disease were offered adjuvant T. A protocol modification required P ≤ .025 for the two remaining pairwise comparisons. At a protocol-specified analysis with a median follow-up of 4.5 years, a 16% reduction in the DFS hazard rate was observed with L+T compared with T (555 DFS events; HR, 0.84; 97.5% CI, 0.70 to 1.02; P = .048), and a 4% reduction was observed with T→L compared with T (HR, 0.96; 97.5% CI, 0.80 to 1.15; P = .61). L-treated patients experienced more diarrhea, cutaneous rash, and hepatic toxicity compared with T-treated patients. The incidence of cardiac toxicity was low in all treatment arms. Conclusion Adjuvant treatment that includes L did not significantly improve DFS compared with T alone and added toxicity. One year of adjuvant T remains standard of care.

Abstract Background: CTCs are associated with clinical outcomes in metastatic breast cancer irrespective of ER/PR/HER2 status. Some data support the prognostic relevance of serial CTC enumeration relative to adjuvant chemotherapy in early stage breast cancer. However, data from a large scale study focused on HER2 directed therapy for HER2+ disease have been lacking. We therefore sought to prospectively evaluate the effect of trastuzumab +/- lapatinib on CTCs and assess the prognostic/predictive value of CTC monitoring in HER2+ early stage breast cancer patients (pts). Methods: The Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO; NCT00490139) Trial is an international, randomized, open-label phase III study of two targeted agents for HER2+ breast cancer. From June 2007 to July 2011, 8381 pts were randomised from 946 sites in 44 countries to 1 of 4 arms with sequential or concurrent chemotherapy: (i) 52 wks of trastuzumab (T); (ii) 52 wks of oral lapatinib (L); (iii) 12 or 18 wks of T followed by a washout and then 34 or 38 wks of L; or (iv) 52 wks of L+T. 540 (6%) pts provided optional informed consent and up to 30 mL peripheral blood suitable for CTC analyses at baseline with additional collections at 13 or 19 wks, 52 wks, 18 mos, 24 mos, and recurrence. CTC analyses are being conducted in three laboratories (Mayo Clinic Rochester, n=431; Institut Jules Bordet and University of Munich, n=109). 2-3 x 10 mL CellSave™ samples are pooled and processed at each time point for CTC enumeration and HER2 expression using the immunomagnetic/immunofluorescence assay (CellSearch™). A round-robin concordance project was done between Mayo Clinic Rochester and Institut Jules Bordet before embarking on the primary correlative work. Results: At baseline, 20% pts had detectable (i.e., ≥1) EpCAM+/CK+/DAPI+/CD45- CTCs, and 16% pts had detectable EpCAM+/CK+/DAPI+/CD45-/HER2+ CTCs. Correlative analyses with clinical outcome are ongoing with plans for completion by Fall 2014. Conclusions: CTCs were detected in 20% of pts with HER2+ early stage breast cancer. This is similar to the frequency of detection in mixed early stage breast cancer populations relative to ER/PR and HER2 status. Concordance of enumeration and HER2 assessments between the two experienced laboratories, and correlation between disease free survival and CTC findings (from serial samples collected at baseline, during the course of HER2 directed therapy, and at set intervals of follow-up) will be reported. Citation Format: Minetta C Liu, Brigitte Rack, Amylou C Dueck, David W Hillman, Michael B Campion, Monica M Reinholz, Kevin C Halling, Christos Sotiriou, Françoise Rothé, Marion Maetens, Ghizlane Rouas, Wolfgang Janni, Antonio C Wolff, Lyndsay N Harris, Julie R Gralow, Kathleen I Pritchard, Susan Ellard, Nguyet A Le-Lindqwister, Frances Boyle, Evandro De Azambuja, Martine J Piccart-Gebhart, Michail Ignatiadis, Edith A Perez. Circulating tumor cell (CTC) enumeration and HER2 assessment as predictors of breast cancer outcomes in the ALTTO (BIG 2-06, Alliance N063D) Trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-01.

Abstract Background: ASP is hypothesized to decrease the risk of breast cancer (BRCA) and BRCA recurrences. Thus we performed an exploratory analysis of ASP and CC use within the NCIC CTG MA.27 adjuvant trial comparing exemestane (E;N=3789) to anastrozole (A; N=3787) with a factorial second randomization to CC or placebo (P). Neither A or E was superior in breast cancer outcomes. Baseline low-dose ASP use is an accepted surrogate of cardiovascular risk factors and was used as a stratification factor across all 4 arms. Randomization to CC-P was discontinued after 18 months (n = 1622) due to concerns of cardiac toxicity. Methods: Patients taking &gt;81mg of ASP daily at baseline were ineligible for randomization and use of &gt;81mg of ASP daily was not allowed. Women enrolled during CC randomization were included in the comparison of E and A, stratified by whether they had been randomized to CC [yes, no;N=1622] and concomitant low-dose ASP [≤81 mg/day (yes, no); N=2209]. Other stratification factors included: lymph-nodes (negative, positive, or unknown); prior adjuvant chemotherapy (yes, no). The primary endpoint, event-free-survival (EFS), was defined as time from randomization to time of locoregional or distant disease recurrence, new primary breast cancer, or death from any cause. Secondary endpoints included overall survival (OS) defined as time from randomization to time of death from any cause and distant disease-free-survival (DDFS), defined as time from randomization to time of distant disease recurrence. Univariate (uni) assessment of CC and ASP use was assessed with stratified log-rank test, adjusting for lymph-node status and adjuvant chemotherapy and applied by intention-to-treat. Exploratory multivariate (multi) analyses (N = 1622) had forced inclusion of treatment and used step-wise forward stratified Cox modeling to examine the effects of CC, ASP use and baseline patient characteristics on outcomes; a factor was added with Wald test statistic p ≤ 0.05. Results: At median follow-up of 4.1 years, 186/1622 (11%) patients had an EFS event; 125 (8%) had died from any cause, and 80 (5%) had distant BRCA relapse. CC did not have significant uni association with outcomes: EFS p-value=0.92; OS p-value p = 0.56; DDFS p-value=0.55. ASP use was associated with worse EFS [p = 0.006, HR 1.48 (95% CI 1.12–1.96)], worse OS [p = 0.0002, HR 1.87 (95% CI (1.35–2.61)], and non-significant difference in DDFS (p = 0.72). CC had no multi association with EFS, OS or DDFS. ASP use had no multi association with EFS and DDFS (p &gt; 0.05). However, ASP use had multi prognostic association with worse OS [p = 0.01; HR 1.67 (95% CI 1.13–2.49)]. Conclusions: Users of either CC or low dose ASP had similar DDFS to non-users in MA.27. As expected, low-dose ASP users (with presumptive cardiovascular risk factors) had worse OS than non-users. Inadequate numbers of patients randomized to CC and lack of randomization to aspirin leaves inadequate evidence from MA.27 to determine whether anti-inflammatories influence breast cancer outcomes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-13-02.