Search

The following file contains Tables S1-12. The contents of these tables are referenced through the manuscript and contain the basis for the results summarized in the paper. The list of tables is as follows: S1. CNV. This table contains all the copy number gains and losses identified in the CTCs. S2. SNV. This table contains all of the somatic SNVs identified in the CTCs. S3. Indel. This table contains all of the somatic insertions and deletions identified in the CTCs. S4. NA12877 somatic. This table contains all of the somatic SNVs identified in NA12877. S5. Allele fraction. This table contains allele fraction calculations and compartment number filtering calculations. S6. Sanger and Miseq. This table contains all of the Sanger sequencing and Miseq validation for the 77 somatic SNVs validated in this study. S7. Mutational spectrum. This table contains the mutation spectrum analysis and the comparison of the CTC mutation spectrum to other tumor types. S8. Non-coding annotation. This table contains a list of potentially important somatic noncoding variants. S9. Splicing. This table contains somatic splicing variants. S10. MHC-II. This table contains the potential somatic coding changes which would result in MHC-II binding antigens. S11. Two hit. This table contains genes with two potential inactivating variants, this can include inherited and somatic variants. S12. Phasing. This table contains phased somatic and/or inherited variants that potentially inactivate a gene through a compound heterozygous mechanism.

Purpose: Recent developments in rare-cell technology have led to improved blood-based assays that allow for the reliable detection, enumeration, and more recently, genomic profiling of circulating tumor cells (CTC). We evaluated two different approaches for enumeration of CTCs in a prospective therapeutic study of patients with metastatic triple-negative breast cancer (TNBC).Experimental Design: The CellSearch system, a commercially available and U.S. Food and Drug Administration (FDA)–cleared assay for CTC enumeration, and IE/FC, an alternative method using EPCAM-based immunomagnetic enrichment and flow cytometry that maintains cell viability, were used to enumerate CTCs in the blood of patients with metastatic TNBC. CTC numbers were assessed at baseline and 7 to 14 days after initiation of therapy with cetuximab ± carboplatin in a phase II multicenter clinical trial (TBCRC 001).Results: CTC numbers from two methods were significantly correlated at baseline (r = 0.62) and at 7 to 14 days (r = 0.53). Baseline CTCs showed no association with time-to-progression (TTP), whereas CTCs at 7 to 14 days were significantly correlated with TTP (CellSearch P = 0.02; IE/FC P = 0.03). CTCs at both time points were significantly associated with overall survival (OS) [CellSearch: baseline (P = 0.0001) and 7 to 14 days (P < 0.0001); IE/FC: baseline (P = 0.0009) and 7 to 14 days (P = 0.0086)].Conclusions: Our findings demonstrate that CTC enumeration by two different assays was highly concordant. In addition, results of both assays were significantly correlated with TTP and OS in patients with TNBC. The IE/FC method is also easily adapted to isolation of pure populations of CTCs for genomic profiling. Clin Cancer Res; 21(5); 1098–105. ©2014 AACR.

Distribution of allele fraction of inherited and somatic variants before and after filtering. This figure is similar to Fig. 4a, but includes a plotting of all somatic variants before filtering (black), which contains a large number of false positive errors.

Supplementary Figure 1. Development, optimization and performance of the gene expression assay. Supplementary Figure 2. Evaluation of assay performance using cancer cell line spike-in models. Supplementary Figure 3. Sample processing and quality control measures Supplementary Figure 4. Expression analysis of CTCs and matched normal blood samples. Supplementary Figure 5. Subtype and proliferation status. Supplementary Figure 6. Serial analysis. Supplementary Figure 7. Clustering analysis of copy number data. Supplementary Figure 8. ESR1 and ERBB2 status in CTCs and patient outcome Supplementary Figure 9. CTC molecular subtype and patient outcome.

Supplementary Table 3. Proportional hazard Cox multivariate regression models to estimate risk of progression or death for dichotomized groups based on predefined cutoffs for CellSearch and IE/FC circulating tumor cell (CTC) enumeration at different time points. Multivariate Cox models with the dichotomized CellSearch and IE/FC groups using various cutoffs were used to estimate hazard ratios to compare prognostic value with respect to TTP or OS. For TTP assessment, Models #1 and #3 showed that only CTC numbers obtained via CellSearch using the cutoff of {greater than or equal to}1 CTC/mL at 7-14d approached significance (p

Supplementary Table 1. REMARK flow diagram of patients and sample collection and processing for each enumeration method (CellSearch and IE/FC). Blood samples from 102 patients with metastatic triple-negative breast cancer were collected at baseline and 7-14days after initiation of treatment. Circulating tumor cells in patients' blood from the same draw were contemporaneously enumerated by CellSearch and IE/FC.

Supplementary Table 2. Median overall survival (OS) and time-to-progression (TTP) in months for different groups based on CTC status. Patients were assigned to the following groups: favorable (

Purpose: We profiled circulating tumor cells (CTCs) to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).Methods: CTCs were isolated from 105 patients with MBC using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACS), 28 of whom had serial CTC analysis (74 samples, 2–5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (n = 151) and genome-wide copy-number analysis by array comparative genomic hybridization (aCGH; n = 49).Results: Combined transcriptional and genomic profiling showed that CTCs were 26% ESR1−ERBB2−, 48% ESR1+ERBB2−, and 27% ERBB2+. Serial testing showed that ERBB2 status was more stable over time compared with ESR1 and proliferation (MKI67) status. While cell-to-cell heterogeneity was observed at the single-cell level, with increasingly stable expression in larger pools, patient-specific CTC expression “fingerprints” were also observed. CTC copy-number profiles clustered into three groups based on the extent of genomic aberrations and the presence of large chromosomal imbalances. Comparative analysis showed discordance in ESR1/ER (27%) and ERBB2/HER2 (23%) status between CTCs and matched primary tumors. CTCs in 65% of the patients were considered to have low proliferation potential. Patients who harbored CTCs with high proliferation (MKI67) status had significantly reduced progression-free survival (P = 0.0011) and overall survival (P = 0.0095) compared with patients with low proliferative CTCs.Conclusions: We demonstrate an approach for complete isolation of EPCAM-positive CTCs and downstream comprehensive transcriptional/genomic characterization to examine the biology and assess breast cancer biomarkers in these cells over time. Clin Cancer Res; 24(6); 1486–99. ©2018 AACR.

Supplementary Table 4. Proportional hazard Cox multivariate regression models to estimate risk of progression or death for dichotomized groups based on circulating tumor cell (CTC) status. Patients were assigned to the following groups: favorable (3.8) and IE/FC (p{less than or equal to}0.0049, HR=2.7). Models #1 and #2 revealed borderline significance for patients who were defined as favorable to unfavorable via CellSearch using either cutoff (p=0.0631, HR=2.4 and p=0.0520, HR=7.8).

Purpose:We evaluated the prognostic and predictive value of circulating tumor cells (CTCs) hormone receptor–positive (HR+) metastatic breast cancer (MBC) patients randomized to letrozole alone or letrozole plus bevacizumab in the first-line setting (CALGB 40503).Experimental Design:Blood samples were collected at pretreatment and three additional time points during therapy. The presence of ≥5 CTCs per 7.5 mL of blood was considered CTC positive. Association of CTCs with progression-free survival (PFS) and overall survival (OS) was assessed using Cox regression models.Results:Of 343 patients treated, 294 had CTC data and were included in this analysis. Median follow-up was 39 months. In multivariable analysis, CTC-positive patients at baseline (31%) had significantly reduced PFS [HR, 1.49; 95% confidence interval (CI), 1.12–1.97] and OS (HR, 2.08; 95% CI, 1.49–2.93) compared with CTC negative. Failure to clear CTCs during treatment was associated with significantly increased risk of progression (HR, 2.2; 95% CI, 1.58–3.07) and death (HR, 3.4; 95% CI, 2.36–4.88). CTC-positive patients who received only letrozole had the worse PFS (HR, 2.3; 95% CI, 1.54–3.47) and OS (HR, 2.6; 95% CI, 1.59–4.40). Median PFS in CTC-positive patients was significantly longer (18.0 vs. 7.0 months) in letrozole plus bevacizumab versus letrozole arm (P = 0.0009). Restricted mean survival time analysis further revealed that addition of bevacizumab was associated with PFS benefit in both CTC-positive and CTC-negative patients, but OS benefit was only observed in CTC-positive patients.Conclusions:CTCs were highly prognostic for the addition of bevacizumab to first-line letrozole in patients with HR+ MBC in CALGB 40503. Further research to determine the potential predictive value of CTCs in this setting is warranted.

Purpose:We examined the prognostic impact of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) detected at the time of surgery in 742 untreated patients with early breast cancer.Experimental Design:DTCs in bone marrow were enumerated using the EPCAM-based immunomagnetic enrichment and flow cytometry (IE/FC) assay. CTCs in blood were enumerated either by IE/FC or CellSearch. Median follow-up was 7.1 years for distant recurrence-free survival (DRFS) and 9.1 years for breast cancer–specific survival (BCSS) and overall survival (OS). Cox regressions were used to estimate hazard ratios for DRFS, BCSS, and OS in all patients, as well as in hormone receptor–positive (HR-positive, 87%) and HR-negative (13%) subsets.Results:In multivariate models, CTC positivity by IE/FC was significantly associated with reduced BCSS in both all (n = 288; P = 0.0138) and HR-positive patients (n = 249; P = 0.0454). CTC positivity by CellSearch was significantly associated with reduced DRFS in both all (n = 380; P = 0.0067) and HR-positive patients (n = 328; P = 0.0002). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n = 273), double positivity (CTC+/DTC+, 8%) was significantly associated with reduced DRFS (P = 0.0270), BCSS (P = 0.0205), and OS (P = 0.0168). In HR-positive patients, double positivity (9% of 235) was significantly associated with reduced DRFS (P = 0.0285), BCSS (P = 0.0357), and OS (P = 0.0092).Conclusions:Detection of CTCs in patients with HR-positive early breast cancer was an independent prognostic factor for DRFS (using CellSearch) and BCSS (using IE/FC). Simultaneous detection of DTCs provided additional prognostic power for outcome, including OS.

Supplementary Figures and Tables except Table S7

This file contains descriptions of all of the bioinformatic analyses used in the paper.

Supplementary Figure 1. REMARK flow diagram of patients and sample collection and processing for each enumeration method (CellSearch and IE/FC). Blood samples from 102 patients with metastatic triple-negative breast cancer were collected at baseline and 7-14days after initiation of treatment. Circulating tumor cells in patients' blood from the same draw were contemporaneously enumerated by CellSearch and IE/FC.

Much effort has been dedicated to developing circulating tumor cells (CTC) as a noninvasive cancer biopsy, but with limited success as yet. In this study, we combine a method for isolation of highly pure CTCs using immunomagnetic enrichment/fluorescence-activated cell sorting with advanced whole genome sequencing (WGS), based on long fragment read technology, to illustrate the utility of an accurate, comprehensive, phased, and quantitative genomic analysis platform for CTCs. Whole genomes of 34 CTCs from a patient with metastatic breast cancer were analyzed as 3,072 barcoded subgenomic compartments of long DNA. WGS resulted in a read coverage of 23× per cell and an ensemble call rate of >95%. These barcoded reads enabled accurate detection of somatic mutations present in as few as 12% of CTCs. We found in CTCs a total of 2,766 somatic single-nucleotide variants and 543 indels and multi-base substitutions, 23 of which altered amino acid sequences. Another 16,961 somatic single nucleotide variant and 8,408 indels and multi-base substitutions, 77 of which were nonsynonymous, were detected with varying degrees of prevalence across the 34 CTCs. On the basis of our whole genome data of mutations found in all CTCs, we identified driver mutations and the tissue of origin of these cells, suggesting personalized combination therapies beyond the scope of most gene panels. Taken together, our results show how advanced WGS of CTCs can lead to high-resolution analyses of cancers that can reliably guide personalized therapy. Cancer Res; 77(16); 4530–41. ©2017 AACR.

Supplementary Figure Legends. REMARK flow diagram

TS1-4

PDF file - 965K, FIG S1: Distribution of MAD estimates across all 102 circulating tumor cell samples FIG S2: Visualization of IE/FACS-isolated cells via hematoxylin/eosin staining; FIG S3: Visualization of IE/FACS-isolated cells via fluorescence in-situ hybridization (FISH) analysis; FIG S4: aCGH analysis of spiked BT474 cells at the single cell level; FIG S5: aCGH analysis of spiked MCF7 cells; FIG S6: Study flow chart; FIG S7: Sorting replication in Patient #303; FIG S8: Comparison of circulating tumor cells and matched primary tumor

PDF file - 126K

PDF file - 85K

Background: CTCs are detectable in approximately 50% of pts with metastatic breast cancer, and elevated CTC levels are an independent negative prognostic factor. CTCs have been shown to play a valuable role in predicting outcomes in pts with ABC treated with chemotherapy or endocrine therapy. This phase II trial was conducted to evaluate the clinical benefit (CB) of pazopanib, a VEGF receptor tyrosine kinase inhibitor (TKI) combined with nonsteroidal aromatase inhibitors (NSAIs) in pts with ABC resistant to NSAIs. As an exploratory analysis, we hypothesized that CTC levels might be correlated with response to pazopanib plus endocrine therapy. Methods: Eligibility included postmenopausal women with hormone receptor positive (HR+) ABC and progressive disease after at least one month of NSAIs. Treatment was pazopanib 800 mg/day plus either letrozole or anastrozole. The primary endpoint was clinical benefit rate at 12 weeks (CBR12, wks). Secondary endpoints included progression free survival (PFS), safety, and the impact of pazopanib and NSAI on CTCs. A CBR of 20% was considered a clinically meaningful comparison to the expected CBR of Results: 32 pts were enrolled; 28 are evaluable for study endpoints. The median age was 58 years (range: 41-77). Pts were heavily pre-treated, with a median of 2 prior hormone therapies (range 1-6) and 1 prior chemotherapy (range 0-8). 8 pts (28.6%) stopped treatment due to adverse events, and 6 pts progressed before wk 12. CBR12 was 46.4% (12 SD, 1 PR), and CBR24 was 25% (5 SD, 2 PR). Safety has been presented (ASCO 2020). Median PFS was 20 wks, and median PFS for pts with CBR12 was 24 wks. Five (22%) of 23 pts with CTC data were CTC-positive (5 CTC/7.5 mL). There was no significant association between CBR12 and CTC status at baseline, or between CBR12 and change in CTC status (baseline to 4 wks after initiation of treatment). 7 pts had non-CBR12 and CTC data; 4 pts (n=4/7; 57%) were CTC positive at either baseline, week 4 or both. 11 pts had CBR12 and CTC data; all patients were CTC negative at baseline and remained negative at 4 wks. 7 pts had PFS >6 months (24, 32, 36, 36, 48, 184 and 274 wks), and 6 had CTC data: all were CTC-negative at baseline and 4 wks, then remained negative at 12 wks. Based on baseline values only, CTC-positive pts had a shorter median PFS compared to CTC-negative pts, but the difference was not statistically significant (11 wks vs 14 wks, p=0.18). Persistently CTC negative patients had significantly longer PFS compared to patients who were CTC positive at any time during study treatment (36 wks vs 11 wks, p=0.01). Conclusions: The addition of pazopanib to NSAIs resulted in a CBR12 of 46.4%, and a CBR24 of 25% in pts with heavily pre-treated ABC resistant to NSAIs. These results support clinical efficacy of antiangiogenic TKI in HR+ ABC and suggest benefit in hormone resistant disease. Consistently negative CTCs correlated with response and response duration. PFS was significantly longer in patients who were consistently CTC negative, further defining the prognostic role of CTCs in HR+ ABC. (NCT01466972) Citation Format: Ozge Gumusay, Mark Jesus M. Magbanua, Melanie C. Majure, Travis Deal, Jonathan R. Renslo, Chiara A. Wabl, Michelle E. Melisko, A. Jo Chien, Andrei Goga, Mark M. Moasser, John W. Park, Jimmy Hwang, Hope S. Rugo. Circulating tumor cell (CTC) enumeration in a phase II trial of pazopanib in addition to endocrine therapy in patients with hormone resistant advanced breast cancer (ABC) [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 PS2-19.

Background: Disseminated tumor cells (DTCs) in bone marrow detected after treatment may represent occult residual disease. We enumerated DTCs after neoadjuvant chemotherapy (NACT) in patients (pts) diagnosed with high-risk early stage breast cancer and examined the relationship of these cells with response and survival. Methods: I-SPY SURMOUNT is a sub-study of the I-SPY 2 TRIAL (NCT01042379). Pts enrolled on I-SPY 2, who signed consent for this sub-study, had bone marrow aspirates (BMA) collected after NACT at the time of surgery. DTCs were isolated and enumerated from BMA using immunomagnetic enrichment/flow cytometry (IE/FC). DTCs were defined as EPCAM-positive and CD45-negative nucleated cells. Samples were considered positive using a predetermined threshold of >4 DTCs per mL (Magbanua et al, unpublished data). Pathologic response was assessed using the residual cancer burden (RCB) method at local sites, and pts underwent standard adjuvant therapy if indicated and follow up for recurrence events and death. Relationship of DTCs with clinicopathologic variables was examined using Chi-squared test. Group means were compared using t tests. The log-rank test was used to compare survival curves. Results: A total of 73 patients were enrolled, 51 of whom had successful DTC assessment. The median DTC per mL was 4 (interquartile range 1.2-11.6). 24/51 (47%) were DTC-positive. Clinical characteristics by DTC status are shown in the table. DTC-positive pts were significantly younger (p=0.02) and had larger pretreatment tumors (longest diameter by magnetic resonance imaging) compared to DTC-negative pts (p=0.032). DTCs were not associated with receptor subtype. Thirty pts (41%) achieved a pathologic complete response (pCR). DTCs were not associated with pCR (p= 0.166); however, DTC-positive patients were significantly more likely to have residual cancer (RCB-II/III) after NACT compared to DTC-negative patients (OR 3.3, p=0.037). Median follow up of this cohort was 2.8 years (range: 0.9-4.8). Interim survival analysis showed that DTCs were not significantly correlated with EFS (p=0.6) or DRFS (p=0.41). Conclusions: Detection of DTCs at surgery after NACT is significantly more common in young patients, those with larger tumors, and those with residual disease at surgery. While these associations suggest higher risk for later recurrence, larger studies and longer follow up are necessary to determine if DTCs add prognostic value over pathologic evaluation alone for pts receiving NACT. Citation Format: Mark Jesus M Magbanua, Laura van 't Veer, Amy Clark, A. Jo Chien, Judy Boughey, Heather Han, Anne Wallace, Heather Beckwith, Minetta Liu, Christina Yau, E. Paul Wileyto, Lamorna Brown Swigart, Jane Perlmutter, Lauren Bayne, Shannon Deluca, Stephanie Yee, Erica Carpenter, Laura Esserman, John Park, Lewis Chodosh, Angela DeMichele. Outcomes associated with disseminated tumor cells at surgery after neoadjuvant chemotherapy in high-risk early stage breast cancer: The I-SPY SURMOUNT study [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 PS2-07.

Background: A variety of investigational HER2-inhibitor agents/combinations have been tested in I-SPY 2, including neratinib (N), TDM1 combined with pertuzumab (P) (TDM1/P), and trastuzumab (H) combined with pertuzumab (H/P; prior to this combination becoming standard of care), all with trastuzumab as control (Ctr). All three experimental arms graduated, showing improved efficacy over control in one or more receptor subsets (HR+HER2+, HR-HER2+, or/and HER2+). Here we assess 10 biomarkers in the HER2, ER/PR, and proliferation pathways on multiple levels of resolution (expression, protein, phospho-protein) as predictors of response in these four arms, hypothesizing that highly HER2-activated, proliferative tumors may be more sensitive to HER2-inhibition than those that are more luminal and quiescent. Methods: 192 HER2+ patients were considered in this analysis: (31 Ctr, 65 N, 52 TDM1/P, and 44 H/P). 10 biomarkers relating to HER2, ER, or proliferation were evaluated from pre-treatment biopsies: HER2 IHC (n=145), 3 expression signatures (n=192), BluePrint subtype (n=192), and 5 protein/phospho-protein analytes by RPPA (n=175). Each biomarker was tested for association with pCR in the whole population and within each arm using a logistic model. This analysis was adjusted for HR status and treatment arm as covariates, and performed within receptor subtypes. This analysis does not adjust for multiplicities of other biomarkers. Results: In the population as a whole, HER2 and HER2-signaling biomarkers, evaluated at multiple levels of resolution: IHC, total-/phospho-protein by RPPA, and mRNA (HER2 amplicon module) - are highly correlated (rho=0.8 [0.65-0.92]). Higher HER2 levels and activity are associated with response: HER2 IHC 3+ status (LR p=0.00032), total quantitative ERBB2 protein by RPPA (LR p=5.4E-09), ERBB2 activation levels (LR p=6.58E-06 (pERBB2 (Y1248)) and 9.95E-06 (pEGFR Y1173)), and the ERBB2 amplicon expression signature (LR p=2.38E-08). In contrast, higher average ER/PR expression associates with non-response to HER2-targeted therapy (LR p=4.28E-08). Both HER2 and ER/PR signaling phenotypes are captured by BluePrint subtyping; and consistent with the individual pathway markers, tumors classified Luminal-type had a lower pCR rate relative to those classified as Her2-type (or Basal-type) (LR p=4.84E-11). These associations all retain significance in a model adjusting for HR status and treatment arm, and in the HR+HER2+ subset. In addition, we quantitatively assessed proliferation markers at the total protein (RPPA: Ki67), phospho-protein (pAURKA) and mRNA (proliferation signature Module11_Prolif) levels. All three proliferation biomarkers predict response overall; but this association is strongest within the HR+HER2+ subset (LR p=0.0012 (Module11_prolif), 0.0036 (pAURK), and 0.045 (Ki67)). None of the biomarkers tested were associated with response in the HR-HER2+ subset. Numbers are small within individual arms. Within the HR+HER2+ subtype, higher HER2 and lower ER/PR is observed in responders in all experimental arms; but the proliferation markers Module11_Prolif (LR p=0.0031) and Ki67 total protein (LR p=0.0029) are associated with response to TDM1/P but not H/P or N. Conclusion: High HER2 signaling at the expression, protein, and phospho-protein levels, and low ER signaling, all predict response to HER2-inhibition across treatment arms. Proliferation markers may be useful for prioritizing therapies in the HR+HER2+ subset. Citation Format: Denise M Wolf, Christina Yau, Julia Wulfkuhle, Lamorna Brown-Swigart, Smita M. Asare, Gillian L. Hirst, Laura Sit, Jane Perlmutter, I-SPY 2 TRIAL Consortium, Minetta Liu, John Park, Angela DeMichele, Douglas Yee, Don Berry, Laura Esserman, Emanuel Petricoin, Laura van t' Veer. HER2 signaling, ER, and proliferation biomarkers predict response to multiple HER2-targeted agents/combinations plus standard neoadjuvant therapy in the I-SPY 2 trial [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 P4-10-02.

Background: Previous studies have independently validated the prognostic relevance of residual cancer burden (RCB) after neoadjuvant chemotherapy. We used results from several independent cohorts in a pooled patient-level analysis to evaluate the relationship of RCB with long-term prognosis across different phenotypic subtypes of breast cancer, to assess generalisability in a broad range of practice settings. Methods: In this pooled analysis, 12 institutes and trials in Europe and the USA were identified by personal communications with site investigators. We obtained participant-level RCB results, and data on clinical and pathological stage, tumour subtype and grade, and treatment and follow-up in November, 2019, from patients (aged ≥18 years) with primary stage I–III breast cancer treated with neoadjuvant chemotherapy followed by surgery. We assessed the association between the continuous RCB score and the primary study outcome, event-free survival, using mixed-effects Cox models with the incorporation of random RCB and cohort effects to account for between-study heterogeneity, and stratification to account for differences in baseline hazard across cancer subtypes defined by hormone receptor status and HER2 status. The association was further evaluated within each breast cancer subtype in multivariable analyses incorporating random RCB and cohort effects and adjustments for age and pretreatment clinical T category, nodal status, and tumour grade. Kaplan-Meier estimates of event-free survival at 3, 5, and 10 years were computed for each RCB class within each subtype. Findings: We analysed participant-level data from 5161 patients treated with neoadjuvant chemotherapy between Sept 12, 1994, and Feb 11, 2019. Median age was 49 years (IQR 20–80). 1164 event-free survival events occurred during follow-up (median follow-up 56 months [IQR 0–186]). RCB score was prognostic within each breast cancer subtype, with higher RCB score significantly associated with worse event-free survival. The univariable hazard ratio (HR) associated with one unit increase in RCB ranged from 1·55 (95% CI 1·41–1·71) for hormone receptor-positive, HER2-negative patients to 2·16 (1·79–2·61) for the hormone receptor-negative, HER2-positive group (with or without HER2-targeted therapy; p

We evaluated disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) in patients with stage I-III breast cancer with >4 MM/mL DTC at baseline who received adjuvant zoledronic acid (ZOL). ZOL was administered every 4 weeks for 24 months, and patients underwent bone marrow aspiration at baseline, and 12 and 24 months of ZOL. Complete DTC response ( 30/mL and CTC > 0.8/mL were significantly associated with recurrence and death. Serial reduction in DTCs occurred. Higher baseline DTC > 30/mL and CTC > 0.8/mL correlated with recurrence and death.

Liquid chromatography-mass spectrometry-based metabolomics studies are increasingly applied to large population cohorts, which run for several weeks or even years in data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalisation approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we propose a study design framework with an arrangement for embedding biological sample replicates to quantify variance within and between batches and a workflow that uses these replicates to remove unwanted variation in a hierarchical manner (hRUV). We use this design to produce a dataset of more than 1000 human plasma samples run over an extended period of time. We demonstrate significant improvement of hRUV over existing methods in preserving biological signals whilst removing unwanted variation for large scale metabolomics studies. Our tools not only provide a strategy for large scale data normalisation, but also provides guidance on the design strategy for large omics studies., Mass spectrometry-based metabolomics is a powerful method for profiling large clinical cohorts but batch variations can obscure biologically meaningful differences. Here, the authors develop a computational workflow that removes unwanted data variation while preserving biologically relevant information.

Breast cancer patients with progressing central nervous system (CNS) disease have limited treatment options. Few chemotherapy drugs with activity in breast cancer have well-documented CNS penetration. This phase 2 trial evaluated efficacy and safety of irinotecan 125 mg/m2 on days 1 and 15 with temozolomide 100 mg/m2 days 1–7 and days 15–21 of a 28 day cycle. Breast cancer patients of any biological subtype and progressing brain metastases and/or leptomeningeal disease (LMD) were eligible. The primary endpoint was CNS response rate. Secondary endpoints were clinical benefit rate (CBR), time to progression (TTP), and overall survival (OS). Imaging studies evaluating intracranial and extracranial response were performed every 8 weeks. Thirty patients were evaluable for safety and efficacy. The most common hematologic and non-hematologic adverse events were neutropenia, and nausea and fatigue, respectively. There were two confirmed CNS partial responses (PR) and five patients with stable disease in the CNS ≥ 16 weeks, resulting in a 7% PR and 23% CBR. Median TTP was 2.3 months (range 13–444 days), and median OS from treatment initiation until death was 4.9 months (range 20–1023 days). Excluding patients with LMD, median TTP and OS were 3.1 and 5.6 months, respectively. Only one patient progressed systemically before CNS progression. The combination of irinotecan and temozolomide was well tolerated, demonstrated some clinical activity across multiple breast cancer subtypes with progressing CNS disease, and offers a reasonable option for patients who are not candidates for further radiation or clinical trials.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a high rate of metastatic recurrence and poor prognosis. Molecular mechanism underlying the metastatic behavior of TNBC has not been well elucidated, and newer approaches addressing drivers of metastasis are crucial to improving patient outcomes. PEA15 (Phosphoprotein enriched in astrocytes-15) regulates cell proliferation, apoptosis, and autophagy. In breast cancer, PEA15 expression inhibits invasion by binding to ERK and preventing its nuclear translocation. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the effect of PEA15 phosphorylation status on TNBC remains unknown. In this study, we tested the hypothesis that unphosphorylated PEA15 will prevent metastasis in TNBC through inhibition of the epithelial-to-mesenchymal transition (EMT). Method: We established stable cells overexpressing unphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) PEA15 mutants in MDA-MB-468 cells. To dissect specific Cellular Mechanisms regulated by PEA15 phosphorylation, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to see effects of PEA15 phosphorylation on tumor growth. Results: The clonogenic growth of PEA15-AA–expressing cells was significantly reduced by 80% compared with empty vector-transfected cells (PEA15-V). Anchorage-independent growth, an indicator of in vivo tumorigenicity, was inhibited in cells expressing PEA15-AA by 60% compared with PEA15-V. PEA15-AA upregulated the expression of E-cadherin and decreased the expression of mesenchymal markers, suggesting that PEA15-AA reverses EMT. Compared with PEA15-V, migration and invasion of cells expressing PEA15-AA were reduced by 65% and 72%, respectively. In contrast, PEA15-DD promoted migration, invasion, and expression of mesenchymal markers. To determine the in vivo effect of PEA15-AA, we injected stable PEA15 transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA15-DD–injected group showed greater tumor volumes than PEA15-V and PEA15-AA groups, suggesting that PEA15-AA has antitumor effects both in vitro and in vivo. From the immune and metastasis arrays, we found that expression level of IL-8, which is known to induce EMT, was greatly decreased by PEA15-AA, while IL-8 was highly expressed in PEA15-DD cells. Addition of recombinant IL-8 to the cells expressing PEA15-AA partially rescued mesenchymal characteristics, increasing migration and expression of mesenchymal markers. By contrast, IL-8 knockdown in PEA15-DD–expressing cells decreased the mesenchymal phenotype. These findings indicate that IL-8 may play an important role as a mediator of phosphorylation of PEA-15 in breast cancer cell migration and invasion and suggest that PEA15-AA inhibits the expression of IL-8, thereby reversing EMT. Conclusion: Taken together, our results show that PEA15 phosphorylation serves as an important regulator, having a dual role as an oncogene or tumor suppressor. Further studies are warranted to evaluate the impact of PEA15 phosphorylation status on metastasis in vivo. These findings support the development of PEA15-AA as a potential therapeutic strategy for TNBC. Citation Format: Park J, Chauhan G, Cohen EN, Ueno NT, Battula VL, Tripathy D, Reuben JM, Bartholomeusz C. PEA15-AA, an unphosphorylatable mutant of PEA15, as a novel therapeutic gene for triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-22.

Background: Patients achieving a pathologic complete response (pCR) following neoadjuvant therapy have significantly improved event-free survival relative to those who do not; and pCR is an FDA-accepted endpoint to support accelerated approval of novel agents/combinations in the neoadjuvant treatment of high risk early stage breast cancer. Previous studies have shown that recurrence risk increased with increasing burden of residual disease (as assessed by the RCB index). As well, these studies suggest that patients with minimum residual disease (RCB-I class) also have favorable outcomes (comparable to those achieving a pCR) within high risk tumor subtypes. In this study, we assess whether integrating RCB with MRI functional tumor volume (FTV), which in itself is prognostic, can improve prediction of distant recurrence free survival (DRFS); and identify a subset of patients with minimal residual disease with comparable DRFS as those who achieved a pCR. Imaging tools can then be used to identify the subset that will do well early and guide the timing of surgical therapy. Method: We performed a pooled analysis of 596 patients from the I-SPY2 TRIAL with RCB, pre-surgical MRI FTV data and known follow-up (median 2.5 years). We first assessed whether FTV predicts residual disease (pCR or pCR/RCB-I) using ROC analysis. We applied a power transformation to normalize the pre-surgical FTV distribution; and assessed its association with DRFS using a bi-variate Cox proportional hazard model adjusting for HR/HER2 subtype. We also fitted a bivariate Cox model of RCB index adjusting for subtype; and assessed whether adding pre-surgical FTV to this model further improves association with DRFS using a likelihood ratio (LR) test. For the Cox modeling, penalized splines approximation of the transformed FTV and RCB index with 2 degrees of freedom was used to allow for non-linear effects of FTV and RCB on DRFS. Result: Pre-surgical MRI FTV is significantly associated with DRFS (Wald p Conclusion: Pre-surgical MRI FTV is effective at predicting minimal residual disease (RCB0/I) in the I-SPY 2 TRIAL. Despite the association between FTV and RCB, FTV appears to provide independent added prognostic value (to RCB and subtype), suggesting that integrating MRI volume measures and RCB into a composite predictor may improve DRFS prediction. Citation Format: Hylton NM, Symmans WF, Yau C, Li W, Hatzis C, Isaacs C, Albain KS, Chen Y-Y, Krings G, Wei S, Harada S, Datnow B, Fadare O, Klein M, Pambuccian S, Chen B, Adamson K, Sams S, Mhawech-Fauceglia P, Magliocco A, Feldman M, Rendi M, Sattar H, Zeck J, Ocal I, Tawfik O, Grasso LeBeau L, Sahoo S, Vinh T, Yang S, Adams A, Chien AJ, Ferero-Torres A, Stringer-Reasor E, Wallace A, Boughey JC, Ellis ED, Elias AD, Lang JE, Lu J, Han HS, Clark AS, Korde L, Nanda R, Northfelt DW, Khan QJ, Viscusi RK, Euhus DM, Edmiston KK, Chui SY, Kemmer K, Wood WC, Park JW, Liu MC, Olopade O, Tripathy D, Moulder SL, Rugo HS, Schwab R, Lo S, Helsten T, Beckwith H, Haugen PK, van't Veer LJ, Perlmutter J, Melisko ME, Wilson A, Peterson G, Asare AL, Buxton MB, Paoloni M, Clennell JL, Hirst GL, Singhrao R, Steeg K, Matthews JB, Sanil A, Berry SM, Abe H, Wolverton D, Crane EP, Ward KA, Nelson M, Niell BL, Oh K, Brandt KR, Bang DH, Ojeda-Fournier H, Eghtedari M, Sheth PA, Bernreuter WK, Umphrey H, Rosen MA, Dogan B, Yang W, Joe B, I-SPY 2 TRIAL Consortium, Yee D, Pusztai L, DeMichele A, Asare SM, Berry DA, Esserman LJ. Refining neoadjuvant predictors of three year distant metastasis free survival: Integrating volume change as measured by MRI with residual cancer burden [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-07-03.

Background: In women with breast cancer receiving neoadjuvant chemotherapy, residual cancer burden (RCB) predicts distant recurrence and survival. In those with high risk tumors, locoregional recurrence (LRR) remains a concern, and has been associated with type of local therapy received. We evaluated the impact of local therapy on LRR in the ISPY-2 TRIAL. Methods: Data were analyzed in Stata 14.2, using Chi2 test, log rank test, and a Cox proportional hazards model. RCB was considered a categorical variable (0/1 versus 2/3), as described in prior publications. Breast surgery categories were lumpectomy +/- radiotherapy, or mastectomy +/- radiotherapy. Axillary surgery was defined as sentinel lymph node (SLN) surgery (≤6 nodes removed) or axillary dissection (>6 nodes). Results: Follow up data from the I-SPY2 TRIAL were available for 630 patients (median follow up 2.76 yrs, range 0.4-7.2). Type of local therapy was significantly associated with clinical stage at presentation, with stage III patients most frequently undergoing mastectomy + radiation (p In a Cox model adjusting for clinical stage, tumor subtype, surgical therapy, RCB status, nodal radiation, and age, significant predictors for LRR were tumor subtype and RCB status. Hazard ratio (HR) for LRR in those with RCB 0/1 was 0.39 compared to those with RCB 2/3 (95% CI 0.17-0.87, p=0.021). There was no difference in LRR between breast conservation and mastectomy; within the breast conservation group, those who had lumpectomy alone had higher hazard of LRR compared to those having lumpectomy + radiation (HR 3.1, 95% CI 1.1-9.2, p=0.043). Conclusions: Extent of surgical therapy was not associated with local tumor control, regardless of advanced tumor stage at presentation. Rather, tumor biology and response to therapy were the best predictors of LRR. These data highlight the opportunity to minimize the morbidity of extensive surgical therapy for patients with excellent response to systemic therapy. LRR rates by clinical features and treatment status FrequencyLRR RateP valueClinical Stage 0.5I240 (47.5%)5.8% II185 (36.6%)8.7% III80 (15.8%)6.3% Tumor Subtype 0.014ER+PR+Her2-161 (26.4%)3.1% ER+PR-Her2-56 (9.2%)3.6% Her2+176 (28.9%)6.3% Triple negative216 (35.5%)11.1% Local therapy 0.169Lumpectomy85 (13.5%)11.8% Lumpectomy with radiation198 (31.4%)5.6% Mastectomy173 (27.5%)5.2% Mastectomy with radiation174 (27.6%)8.6% Axillary surgery 0.23None5 (0.8%)20% SLN329 (52.2%)5.8% ALND296 (47%)8.5% Axillary radiation 0.535Yes42 (6.7%)9.5% No588 (93.3%)7.0% Axillary management 0.2No surgery or radiation5 (0.8%)20.0% SLN312 (50%)5.3% SLN+Axillary radiation17 (2.7%)8.3% ALND271 (43%)10.3% ALND+Axillary radiation25 (4%)5.4% RCB 0.0020/1293 (50.1%)3.8% 2/3292 (49.9%)10.3% Citation Format: Silverstein J, Suleiman L, Yau C, Price ER, Singhrao R, Yee D, DeMichele A, Isaacs C, Albain KS, Chien AJ, Forero-Torres A, Wallace AM, Pusztai L, Ellis ED, Elias AD, Lang JE, Lu J, Han HS, Clark AS, Korde L, Nanda R, Northfelt DW, Khan QJ, Viscusi RK, Euhus DM, Edmiston KK, Chui SY, Kemmer K, Wood WC, Park JW, Liu MC, Olopade O, Leyland-Jones B, Tripathy D, Moulder SL, Rugo HS, Schwab R, Lo S, Helsten T, Beckwith H, I-SPY 2 TRIAL Consortium, Berry DA, Asare SM, Esserman LJ, Boughey JC, Mukhtar RA. The impact of local therapy on locoregional recurrence in women with high risk breast cancer in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-14-01.

BackgroundWe examined the prognostic significance of circulating tumor cell (CTC) dynamics during treatment in metastatic breast cancer (MBC) patients receiving first-line chemotherapy.MethodsSerial CTC data from 469 patients (2202 samples) were used to build a novel latent mixture model to identify groups with similar CTC trajectory (tCTC) patterns during the course of treatment. Cox regression was used to estimate hazard ratios for progression-free survival (PFS) and overall survival (OS) in groups based on baseline CTCs, combined CTC status at baseline to the end of cycle 1, and tCTC. Akaike information criterion was used to select the model that best predicted PFS and OS.ResultsLatent mixture modeling revealed 4 distinct tCTC patterns: undetectable CTCs (56.9% ), low (23.7%), intermediate (14.5%), or high (4.9%). Patients with low, intermediate, and high tCTC patterns had statistically significant inferior PFS and OS compared with those with undetectable CTCs (P < .001). Akaike Information Criterion indicated that the tCTC model best predicted PFS and OS compared with baseline CTCs and combined CTC status at baseline to the end of cycle 1 models. Validation studies in an independent cohort of 1856 MBC patients confirmed these findings. Further validation using only a single pretreatment CTC measurement confirmed prognostic performance of the tCTC model.ConclusionsWe identified 4 novel prognostic groups in MBC based on similarities in tCTC patterns during chemotherapy. Prognostic groups included patients with very poor outcome (intermediate + high CTCs, 19.4%) who could benefit from more effective treatment. Our novel prognostic classification approach may be used for fine-tuning of CTC-based risk stratification strategies to guide future prospective clinical trials in MBC.

Liquid chromatography-mass spectrometry based metabolomics studies are increasingly applied to large population cohorts, running for several weeks to months, even extending to years of data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalization approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we designed an experiment with an arrangement to embed biological sample replicates to measure the variance within and between batches for over 1,000 human plasma samples run over 44 days. We integrate these replicates in a novel workflow to remove unwanted variation in a hierarchical structure (hRUV) by progressively merging the adjustments in neighbouring batches. We demonstrate significant improvement of hRUV over existing methods in maintaining biological signals whilst removing unwanted variation for large scale metabolomics studies.

Purpose: We evaluated the prognostic and predictive value of circulating tumor cells (CTCs) hormone receptor–positive (HR+) metastatic breast cancer (MBC) patients randomized to letrozole alone or letrozole plus bevacizumab in the first-line setting (CALGB 40503). Experimental Design: Blood samples were collected at pretreatment and three additional time points during therapy. The presence of ≥5 CTCs per 7.5 mL of blood was considered CTC positive. Association of CTCs with progression-free survival (PFS) and overall survival (OS) was assessed using Cox regression models. Results: Of 343 patients treated, 294 had CTC data and were included in this analysis. Median follow-up was 39 months. In multivariable analysis, CTC-positive patients at baseline (31%) had significantly reduced PFS [HR, 1.49; 95% confidence interval (CI), 1.12–1.97] and OS (HR, 2.08; 95% CI, 1.49–2.93) compared with CTC negative. Failure to clear CTCs during treatment was associated with significantly increased risk of progression (HR, 2.2; 95% CI, 1.58–3.07) and death (HR, 3.4; 95% CI, 2.36–4.88). CTC-positive patients who received only letrozole had the worse PFS (HR, 2.3; 95% CI, 1.54–3.47) and OS (HR, 2.6; 95% CI, 1.59–4.40). Median PFS in CTC-positive patients was significantly longer (18.0 vs. 7.0 months) in letrozole plus bevacizumab versus letrozole arm (P = 0.0009). Restricted mean survival time analysis further revealed that addition of bevacizumab was associated with PFS benefit in both CTC-positive and CTC-negative patients, but OS benefit was only observed in CTC-positive patients. Conclusions: CTCs were highly prognostic for the addition of bevacizumab to first-line letrozole in patients with HR+ MBC in CALGB 40503. Further research to determine the potential predictive value of CTCs in this setting is warranted.

Detection of disseminated tumor cells (DTCs) in bone marrow is an established negative prognostic factor. We isolated small pools of (~20) EPCAM-positive DTCs from early breast cancer patients for genomic profiling. Genome-wide copy number profiles of DTC pools (n = 45) appeared less aberrant than the corresponding primary tumors (PT, n = 16). PIK3CA mutations were detected in 26% of DTC pools (n = 53), none of them were shared with matched PTs. Expression profiling of DTC pools (n = 30) confirmed the upregulation of EPCAM expression and certain oncogenes (e.g., MYC and CCNE1), as well as the absence of hematopoietic features. Two expression subtypes were observed: (1) luminal with dual epithelial–mesenchymal properties (high ESR1 and VIM/CAV1 expression), and (2) basal-like with proliferative/stem cell-like phenotype (low ESR1 and high MKI67/ALDH1A1 expression). We observed high discordance between ESR1 (40%) and ERRB2 (43%) expression in DTC pools vs. the clinical ER and HER2 status of the corresponding primary tumors, suggesting plasticity of biomarker status during dissemination to the bone marrow. Comparison of expression profiles of DTC pools with available data from circulating tumor cells (CTCs) of metastatic breast cancer patients revealed gene expression signatures in DTCs that were unique from those of CTCs. For example, ALDH1A1, CAV1, and VIM were upregulated in DTC pools relative to CTCs. Taken together, analysis of pooled DTCs revealed molecular heterogeneity, possible genetic divergence from corresponding primary tumor, and two distinct subpopulations. Validation in larger cohorts is needed to confirm the presence of these molecular subtypes and to evaluate their biological and clinical significance., Genetics: two subtypes of disseminated tumor cells in bone marrow Tumor cells found in the bone marrow of women with early-stage breast cancer constitute molecularly distinct populations that could impact patient outcomes and treatment responses. Mark Jesus Magbanua and coworkers from the University of California, San Francisco, USA, performed genome-wide copy number variation analysis, expression profiling on 64 cancer-related genes and screened for PIK3CA mutations in pools of tumor cells isolated from bone marrow aspirates of patients undergoing surgery. These disseminated tumor cells (DTCs) contained fewer genomic abnormalities on average than their corresponding primary tumors, although the DTCs often acquired oncogenic mutations in PIK3CA. Expression profiling revealed two distinct subtypes of DTCs, both of which were different from circulating tumor cells found in the bloodstream. Further studies are needed to evaluate the biological and clinical significance of this molecular and genomic heterogeneity in DTCs.

Purpose: We profiled circulating tumor cells (CTCs) to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).Methods: CTCs were isolated from 105 patients with MBC using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACS), 28 of whom had serial CTC analysis (74 samples, 2–5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (n = 151) and genome-wide copy-number analysis by array comparative genomic hybridization (aCGH; n = 49).Results: Combined transcriptional and genomic profiling showed that CTCs were 26% ESR1−ERBB2−, 48% ESR1+ERBB2−, and 27% ERBB2+. Serial testing showed that ERBB2 status was more stable over time compared with ESR1 and proliferation (MKI67) status. While cell-to-cell heterogeneity was observed at the single-cell level, with increasingly stable expression in larger pools, patient-specific CTC expression “fingerprints” were also observed. CTC copy-number profiles clustered into three groups based on the extent of genomic aberrations and the presence of large chromosomal imbalances. Comparative analysis showed discordance in ESR1/ER (27%) and ERBB2/HER2 (23%) status between CTCs and matched primary tumors. CTCs in 65% of the patients were considered to have low proliferation potential. Patients who harbored CTCs with high proliferation (MKI67) status had significantly reduced progression-free survival (P = 0.0011) and overall survival (P = 0.0095) compared with patients with low proliferative CTCs.Conclusions: We demonstrate an approach for complete isolation of EPCAM-positive CTCs and downstream comprehensive transcriptional/genomic characterization to examine the biology and assess breast cancer biomarkers in these cells over time. Clin Cancer Res; 24(6); 1486–99. ©2018 AACR.

165 Background: Young women diagnosed with breast cancer face unique challenges. The desire to have a biologic child (bchild) is often a factor in treatment decisions at diagnosis and follow-up. The aim of this study is to characterize the intentions, attitudes, and decision factors considered by young women who desire to have a bchild after a diagnosis of EBC. Methods: This prospective study included young women diagnosed with stage 1-3 EBC under age 45 who saw an oncologist at UCSF’s Breast Care Center (BCC). Young women < 6 months from diagnosis were invited to complete a baseline REDCap survey on fertility, child-bearing, and family-building. Descriptive statistics were used to summarize responses. Chi square and independent samples t tests were used to compare demographics. Covariates were analyzed using odds ratios. Results: From Feb 2018 to Dec 2020, 166 eligible pts seen at the UCSF BCC were contacted, 143 pts consented, and 108 (75.5%) completed the baseline survey. Of the 108 pts, 57 (53%) were interested in having a bchild in the future. Age was the biggest driver of whether participants were interested in future child-bearing vs. not (Mean = 35.4 vs 40.9, p < 0.001) and was not influenced by stage, receptor status, or treatment. 73.6% (N = 42) of those interested in future child-bearing underwent or planned to undergo fertility preservation (FP). FP was associated with full time employment (p = 0.03) and higher education (p = 0.02). Of the 57 pts interested in future child-bearing (42 HR+, 11 TN, 12 HER2+), 30% wished to start trying to conceive within 2 years from diagnosis, and only 20% would wait 5 years or more. 43% (N = 18) of those with HR+ disease were willing to complete 5 years of hormone therapy (HT) before trying to conceive. Given hypothetical risk scenarios of an incurable cancer recurrence, 16.3% of participants were interested in a future bchild despite a 75-100% hypothetical risk of recurrence (ROR). Young women self-identified as Asian were less inclined to pursue child-bearing with increasing risk (OR = 0.3 vs. Caucasian, p = 0.04), while BRCA-carriers were more likely to remain interested in future child-bearing despite increasing risk (OR = 6.43 vs. non-carriers, p = 0.03). 59% would stop adjuvant HT early if the hypothetical absolute increased ROR from early discontinuation was < 10%. Conclusions: In this single-institution study, over half of young women with EBC expressed a desire to have a future bchild. This was independent of stage, receptor status, and treatment; the majority of women wished to conceive < 5 years from diagnosis. The desire for a future bchild decreased with increasing hypothetical risk of incurable recurrence; however, a subset wished to attempt child-bearing even when an incurable recurrence was certain. Having a bchild after EBC is a priority for many young women and should be addressed at diagnosis and throughout the continuum of care.

Breast cancer patients with progressing central nervous system (CNS) disease have limited treatment options. Few chemotherapy drugs with activity in breast cancer have well-documented CNS penetration. This phase 2 trial evaluated efficacy and safety of irinotecan 125 mg/mBreast cancer patients of any biological subtype and progressing brain metastases and/or leptomeningeal disease (LMD) were eligible. The primary endpoint was CNS response rate. Secondary endpoints were clinical benefit rate (CBR), time to progression (TTP), and overall survival (OS). Imaging studies evaluating intracranial and extracranial response were performed every 8 weeks.Thirty patients were evaluable for safety and efficacy. The most common hematologic and non-hematologic adverse events were neutropenia, and nausea and fatigue, respectively. There were two confirmed CNS partial responses (PR) and five patients with stable disease in the CNS ≥ 16 weeks, resulting in a 7% PR and 23% CBR. Median TTP was 2.3 months (range 13-444 days), and median OS from treatment initiation until death was 4.9 months (range 20-1023 days). Excluding patients with LMD, median TTP and OS were 3.1 and 5.6 months, respectively. Only one patient progressed systemically before CNS progression.The combination of irinotecan and temozolomide was well tolerated, demonstrated some clinical activity across multiple breast cancer subtypes with progressing CNS disease, and offers a reasonable option for patients who are not candidates for further radiation or clinical trials.

Background: The goal of I-SPY 2 is to rapidly test novel therapies in addition to standard of care in high-risk breast cancer patients. It has resulted in increasing response rates, where pCR rates in TNBC and HR-HER2+ subsets have reached ~60% and ~75%, respectively. Yet, there remains a sizeable subset of non-responders, especially among HR+ patients. Identification of ‘universal’ resistance mechanisms may guide rational selection of agents to improve these patient's outcomes. Thus, we analyzed reverse phase protein array (RPPA) based quantitative protein/phosphoprotein data across arms to assess whether there are common mechanisms rendering these cancers resistant to all agent classes tested to date. Methods: 736 patients (260 HR+HER2-, 252 TN, 142 HR+HER2-, and 82 HR-HER2+; over 8 arms: 194 Ctr, 105 neratinib (N), 63 veliparib/carboplatin (VC), 128 AMG386 (anti-ANG1/2), 87 MK2206 (anti-AKT), 43 TH/pertuzumab (P), 49 TDM1/P, and 67 pembrolizumab (Pembro)) with pCR and RPPA data at the pre-treatment time point were considered for this analysis. 141 RPPA endpoints representing key cancer pathways were assessed for association with pCR using logistic regression modeling, with HR, HER2 and treatment arm as covariates (likelihood ratio test; p Citation Format: Julia Wulfkuhle, Denise Wolf, Christina Yau, Lamorna Brown-Swigart, Rosa I Gallagher, Gillian Hirst, Laura Sit, Smita Asare, I-SPY 2 TRIAL Investigators, Nola Hylton, Angela DeMichele, Douglas Yee, Jo Chien, Hope Rugo, John Park, Kathy Albain, Rita Nanda, Debu Tripathy, Richard Schwab, Don Berry, Laura Esserman, Laura van t' Veer, Emanual Petricoin, III. Identification of biomarkers associated with therapeutic resistance: Quantitative protein/phosphoprotein analysis of ~750 patients across 8 arms of the neoadjuvant I-SPY 2 TRIAL for high-risk early stage breast cancer [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 PD9-04.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1. Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Background: We have previously described that TNBC patients whose tumors have both HER2 Y1248 phosphorylation (pHER2) “high” and phospho-EGFR Y1173 (pEGFR) “high” have increased response (pCR) to neratinib in the I-SPY2 TRIAL. We hypothesize that the paradoxical finding of a response prediction signature comprised of HER2 activation in a HER2 IHC/FISH-negative population means there must be a ligand-driven biochemical event responsible for the HER2 phosphorylation because HER2 mutations were also not found to be significant. Exploratory analysis of additional cellular signaling events and protein expression levels in pre-treatment, LCM-purified tumor epithelium by reverse phase protein microarray (RPPA) included semi-quantitative measurement of total levels of estrogen receptor alpha (ERα), which has been previously shown to be able to act as a membrane non-genomic signaling molecule through direct interaction with various tyrosine kinases including EGFR and HER2. Since ERα has been previously shown to act as a ligand and co-stimulate (activate) HER2 and EGFR when present at low levels, we investigated whether or not RPPA-measured ERα levels in the TNBC cohort analyzed to date were higher in tumors with both pHER2 “high” and pEGFR “high” levels and thus provide evidence explaining how HER2-EGFR activation is occurring in TNBC. Methods: Using RPPA analysis, we measured 118 analytes in lysates of LCM tumor epithelium obtained from the pre-treatment biopsy samples of 86 TNBC (Allred=0) patients in the I-SPY2 TRIAL analyzed to date. Cutpoints for pEGFR and pHER2 were determined previously by ROC analysis for pCR correlation in the neratinib treated TNBC population, and used here to dichotomize the pHER2 and pEGFR data in the larger TNBC population. Wilcoxon Rank Sum testing was performed using the continuous variable total ERα data and compared the TNBC that were both pHER2 and pEGFR “high” (N=39) to the rest of the TNBC population (N=47). Total ERα values were then divided into “high” and “low” groups based on the TNBC population median value in order to determine frequency/percentages within each class. Our study is exploratory with no claims for generalizability of the data, and calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Results: Total ERα values were obtained in 84/86 TNBC tumors analyzed. Total levels of ERα were higher (p< 0.006) in TNBC tumors with pHER2 and pEGFR “high” levels. 68% (26/38) of tumors in the pHER2 and pEGFR “high” group had ERα levels above the population median compared to 35% (16/46) in the rest of the TNBC population. Conclusion: Our exploratory analysis reveals that ERα levels are significantly higher in TNBC with pHER2 and pEGFR activation and may be behaving as a direct signaling ligand in TNBC and driving HER2-EGFR signaling. This ERα-pHER2/pEGFR association was missed by current ER and HER2 clinical laboratory testing techniques, and if validated in larger independent study sets could suggest that utilization of new protein-based techniques defining ER more quantitatively could be helpful to understand tumor biology and therapeutic response prediction, especially in the context of TNBC that are ostensibly ER negative. Citation Format: Gallagher RI, Yau C, Wolf DM, Dong T, Hirst G, Brown-Swigart L, ISPY-2 TRIAL Investigators, Buxton M, DeMichele A, van't Veer L, Yee D, Paoloni M, Esserman L, Berry D, Park J, Petricoin EF, Wulfkuhle JD. Quantitative ERα measurements in TNBC from the I-SPY 2 TRIAL correlate with HER2-EGFR co-activation and heterodimerization [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 P3-05-02.

Importance Pathologic complete response (pCR) is a known prognostic biomarker for long-term outcomes. The I-SPY2 trial evaluated if the strength of this clinical association persists in the context of a phase 2 neoadjuvant platform trial. Objective To evaluate the association of pCR with event-free survival (EFS) and pCR with distant recurrence–free survival (DRFS) in subpopulations of women with high-risk operable breast cancer treated with standard therapy or one of several novel agents. Design, Setting, and Participants Multicenter platform trial of women with operable clinical stage 2 or 3 breast cancer with no prior surgery or systemic therapy for breast cancer; primary tumors were 2.5 cm or larger. Women with tumors that were ERBB2 negative/hormone receptor (HR) positive with low 70-gene assay score were excluded. Participants were adaptively randomized to one of several different investigational regimens or control therapy within molecular subtypes from March 2010 through 2016. The analysis included participants with follow-up data available as of February 26, 2019. Interventions Standard-of-care neoadjuvant therapy consisting of taxane treatment with or without (as control) one of several investigational agents or combinations followed by doxorubicin and cyclophosphamide. Main Outcomes and Measures Pathologic complete response and 3-year EFS and DRFS. Results Of the 950 participants (median [range] age, 49 [23-77] years), 330 (34.7%) achieved pCR. Three-year EFS and DRFS for patients who achieved pCR were both 95%. Hazard ratios for pCR vs non-pCR were 0.19 for EFS (95% CI, 0.12-0.31) and 0.21 for DRFS (95% CI, 0.13-0.34) and were similar across molecular subtypes, varying from 0.14 to 0.18 for EFS and 0.10 to 0.20 for DRFS. Conclusions and Relevance The 3-year outcomes from the I-SPY2 trial show that, regardless of subtype and/or treatment regimen, including 9 novel therapeutic combinations, achieving pCR after neoadjuvant therapy implies approximately an 80% reduction in recurrence rate. The goal of the I-SPY2 trial is to rapidly identify investigational therapies that may improve pCR when validated in a phase 3 confirmatory trial. Whether pCR is a validated surrogate in the sense that a therapy that improves pCR rate can be assumed to also improve long-term outcome requires further study. Trial Registration ClinicalTrials.gov Identifier:NCT01042379

e13079 Background: Eribulin is an effective microtubule inhibitor for the treatment of ABC. Based on encouraging efficacy with docetaxel/cyclophosphamide, we hypothesized that eribulin combined with cyclophosphamide (EC) would be effective with tolerable toxicity. The aim of the study was to determine the maximum tolerated dose (MTD) of EC, followed by a dose expansion study to estimate the clinical benefit rate (CBR) of EC in pts with ABC. Methods: Study eligibility included pts with histologically confirmed ABC with any number of prior lines of therapy. Pts were treated using a dose escalation strategy with cohort expansion once MTD was determined. Dose level 0 (DL0): E 1.1 mg/m2 on days 1 and 8, C 600 mg/m2 on day 1 of a 21-day cycle. DL1: E 1.4 mg/m2 on days 1 and 8, C 600 mg/m2 on day 1 of a 21-day cycle. Phase II expanded enrollment at DL1. The primary objective of the phase II study was CBR [(complete response (CR), partial response (PR), and stable disease (SD)]. Secondary objectives were response rate (RR), duration of response (DOR), time to progression (TTP), and safety. Using a 2-stage design, responses in 3 of the first 22 pts allowed continued enrollment to a planned 40. Results: No dose limiting toxicities (DLT) were identified in phase Ib (n = 6). 3 pts were treated at DL0 and 3 at DL1, the MTD. 44 pts with ABC were enrolled at the MTD and are included in the analysis. 31 pts had HR+/HER2- disease, 12 pts had triple negative disease (TNBC), 1 pt had HR+/HER2+ disease. Median age was 56 yrs, prior treatment (rx) for ABC included a median of 1 line of hormone rx (range 0-6) and 2 lines of prior chemorx (range 0-7). CBR was 79.5% (35/44; 7 PR, 28 SD) and median PFS 16.4 wks (95%CI:13.8-21.1 wks). Longer PFS was observed in those with HR+ disease vs TNBC (18.1 vs 10.8 wks; P = 0.067). Adverse events (AE) of any grade included fatigue (68.2%, n = 30), neutropenia (ntp) (59.1%, n = 26), nausea (56.8%, n = 25), constipation (50%, n = 22), peripheral neuropathy (47.7%, n = 21), dyspnea (40.9%, n = 18), headache (36.4%, n = 16), and anorexia (36.4%, n = 16). The most common grade 3/4 AE ntp (47.7%, n = 21); 3 pts had febrile ntp. Dose reductions due to ntp were required to 500 mg/m2 C (n = 17) and to 1.1 mg/m2 in eribulin (n = 12). Conclusions: EC in heavily pretreated ABC resulted in an encouraging CBR of 79.5% and PFS of 16.4 wks, comparing favorably to single agent E (PFS 14.8 wks). Dose reduction and delays were due primarily to ntp. EC is an effective combination therapy with manageable toxicity in ABC. Clinical trial information: NCT01554371 .

1068 Background: A major limitation of endocrine therapy in hormone receptor positive (HR+) ABC is the development of resistance. Preclinical data suggests that higher levels of vascular endothelial growth factor (VEGF) are associated with endocrine therapy resistance. We conducted a phase II trial to evaluate the clinical benefit (CB) of PZ, a VEGF receptor tyrosine kinase inhibitor (TKI) combined with nonsteroidal aromatase inhibitors (NSAIs) in pts with ABC resistant to NSAIs. Methods: Eligibility included postmenopausal women with HR+ ABC and progressive disease (PD) after at least one month of NSAIs. Treatment was PZ 800 mg/day plus either letrozole or anastrozole. The primary endpoint was clinical benefit rate at 12 weeks (CBR12, wks). Secondary endpoints were PFS and safety. A CBR of 20% was considered a clinically meaningful comparison to the expected CBR of < 5% with continued NSAIs after PD. Using a 2-stage design, stable disease in at least 1 of the first 13 pts allowed continued enrollment to a planned 28. Results: 32 pts were enrolled; 28 are evaluable for study endpoints and all patients completed the study. The median age was 58 years (range: 41-77). Pts were heavily pre-treated, with a median of 2 prior hormone therapies (range 1-6) and 1 prior chemotherapy (range 0-8). 8 pts (28.6%) stopped treatment due to adverse events (AE) including hypertension (HTN), fever, transaminitis, nausea, vomiting, rash, hand foot syndrome and pulmonary embolism (PE); 6 pts progressed before wk 12. CBR12 was 46.4% (12 SD, 1 PR); CBR24 was 25% (5 SD, 2 PR). Median PFS was 20 wks (95% CI 11-48, and median PFS for pts with CBR12 was 24 wks. 7 pts had PFS > 6 months (24, 32, 36, 36, 48, 184 and 274 wks); 2 pts had PFS > 3 years (184 and 274 weeks). The most common grade 1/2 AE were nausea (48.2%), fatigue (33.3%), diarrhea (29.6%), back pain (22.2%), and arthralgias (22.2%). Grade 3/4 AEs included HTN (3/28; 11.1), transaminitis (3/28; 11.1%), headache (2/28; 7.4), heart failure, vertigo, nausea, oral pain, vomiting, fever, fatigue, and hypokalemia (one patient each: 3.7%). Conclusions: The addition of PZ to NSAIs resulted in a CBR12 of 46.4%, and a CBR24 of 25% in pts with heavily pre-treated ABC resistant to NSAIs. These results support clinical efficacy of antiangiogenic TKI in HR+ ABC, and suggest benefit in hormone resistant disease. Expected toxicities resulted in early discontinuation in 28.6%, which limited drug exposure. Clinical trial information: NCT 01466972 .

1076 Background: Increased expression of the MYC transcriptional oncogene is found in about 70% of TNBC and is associated with poor prognosis. In MYC-overexpressing TN xenografts, CDK1 inhibition with dina results in synthetic lethality, and attenuates distant metastasis. In syngeneic models, the combination of dina with anti-PD1 therapy is synergistic and increases immune cell tumor infiltration and activation. Methods: Patients (pts) with advanced TNBC received dina IV day 1 and 8 in combination with fixed dose P 200 mg once every 21 days. Dina was dose escalated using a toxicity probability interval design targeting a dose limiting toxicity (DLT) rate of 25%. Evaluable disease and pre-treatment metastatic biopsies were required. After 17 pts were enrolled, eligibility was amended to require ≤2 lines of prior chemotherapy, and LDH ≤1.5x normal. MYC IHC was performed on baseline tumor biopsies and correlated with clinical response using Welch’s unequal variances t-test. Results: 32 pts were enrolled (median age 53, median 2 prior lines of therapy, 13 pts (41%) had disease which was previously ER+). 18 pts were enrolled in the dose escalation phase with no observed DLTs; 33 mg/m2 was determined to be the recommended phase 2 dose (RP2D). 14 additional patients were enrolled in dose expansion at 33 mg/m2, completing trial accrual. Grade ≥3 adverse events (AEs) in all pts included neutropenia (ntp) (37.5%), febrile ntp (12.5%), fatigue (12.5%), transaminitis (3.2%), and neuromuscular weakness (3.2%). Most common all grade AEs included fatigue (63%), diarrhea (63%), nausea (63%), and ntp (53%). Possible immune-related AEs included sinusitis (4 pts), hemolytic anemia (1 pt), pneumonitis (1 pt), rash (2 pts), neuromuscular weakness (1 pt), and transaminitis (1 pt). Of the 29 patients evaluable for response, 1 pt (3.4%) had a CR, 4 pts (13.8%) had a PR, and 6 pts (20.6%) had SD. MYC IHC staining on baseline metastatic tumor biopsies in 19 pts correlated significantly with clinical response. Additional biomarker testing will be reported. Conclusions: The RP2D of dina given in combination with standard dose P is 33 mg/m2 on D1, 8 of a 21-day cycle. Toxicities were generally manageable and non-overlapping. In exploratory analysis, greater MYC IHC staining correlated significantly with response to study therapy. Clinical trial information: NCT01676753 .

Purpose: We examined the prognostic impact of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) detected at the time of surgery in 742 untreated patients with early breast cancer. Experimental Design: DTCs in bone marrow were enumerated using the EPCAM-based immunomagnetic enrichment and flow cytometry (IE/FC) assay. CTCs in blood were enumerated either by IE/FC or CellSearch. Median follow-up was 7.1 years for distant recurrence-free survival (DRFS) and 9.1 years for breast cancer–specific survival (BCSS) and overall survival (OS). Cox regressions were used to estimate hazard ratios for DRFS, BCSS, and OS in all patients, as well as in hormone receptor–positive (HR-positive, 87%) and HR-negative (13%) subsets. Results: In multivariate models, CTC positivity by IE/FC was significantly associated with reduced BCSS in both all (n = 288; P = 0.0138) and HR-positive patients (n = 249; P = 0.0454). CTC positivity by CellSearch was significantly associated with reduced DRFS in both all (n = 380; P = 0.0067) and HR-positive patients (n = 328; P = 0.0002). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n = 273), double positivity (CTC+/DTC+, 8%) was significantly associated with reduced DRFS (P = 0.0270), BCSS (P = 0.0205), and OS (P = 0.0168). In HR-positive patients, double positivity (9% of 235) was significantly associated with reduced DRFS (P = 0.0285), BCSS (P = 0.0357), and OS (P = 0.0092). Conclusions: Detection of CTCs in patients with HR-positive early breast cancer was an independent prognostic factor for DRFS (using CellSearch) and BCSS (using IE/FC). Simultaneous detection of DTCs provided additional prognostic power for outcome, including OS.