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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.

Purpose: HSP90 is a chaperone protein required for the stability of a variety of client proteins. 17-Demethoxygeldanamycin (17-AAG) is a natural product that binds to HSP90 and inhibits its activity, thereby inducing the degradation of these clients. In preclinical studies, HER2 is one of the most sensitive known client proteins of 17-AAG. On the basis of these data and activity in a phase I study, we conducted a phase II study of 17-AAG (tanespimycin) with trastuzumab in advanced trastuzumab-refractory HER2-positive breast cancer.Experimental Design: We enrolled patients with metastatic HER2+ breast cancer whose disease had previously progressed on trastuzumab. All patients received weekly treatment with tanespimycin at 450 mg/m2 intravenously and trastuzumab at a conventional dose. Therapy was continued until disease progression. The primary endpoint was response rate by Response Evaluation Criteria in Solid Tumors (RECIST) criteria.Results: Thirty-one patients were enrolled with a median age of 53 years and a median Karnofsky performance status (KPS) of 90%. The most common toxicities, largely grade 1, were diarrhea, fatigue, nausea, and headache. The overall response rate was 22%, the clinical benefit rate [complete response + partial response + stable disease] was 59%, the median progression-free survival was 6 months (95% CI: 4–9), and the median overall survival was 17 months (95% CI: 16–28).Conclusions: This is the first phase II study to definitively show RECIST-defined responses for 17-AAG in solid tumors. Tanespimycin plus trastuzumab has significant anticancer activity in patients with HER2-positive, metastatic breast cancer previously progressing on trastuzumab. Further research exploring this therapeutic interaction and the activity of HSP90 inhibitors is clearly warranted. Clin Cancer Res; 17(15); 5132–9. ©2011 AACR.

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.

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.

CCR Translation for This Article from HSP90 Inhibition Is Effective in Breast Cancer: A Phase II Trial of Tanespimycin (17-AAG) Plus Trastuzumab in Patients with HER2-Positive Metastatic Breast Cancer Progressing on Trastuzumab

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(18)F-labeled 1-amino-3-fluorocyclobutane-1-carboxylic acid ((18)F-fluciclovine) is a leucine analog radiotracer that depicts amino acid transport into cells. (18)F-fluciclovine PET/CT visualizes malignancy, including prostate cancer, invasive ductal breast cancer, and invasive lobular breast cancer. Whether changes in (18)F-fluciclovine avidity reflect changes in tumor burden resulting from treatment has not been shown. In this prospective clinical trial (clinical trials.gov: NCT01864083), changes in (18)F-fluciclovine avidity after neoadjuvant therapy were compared to breast cancer therapy response, as determined by residual tumor burden on pathology, were evaluated. Methods: Twenty-four women with a new diagnosis of locally advanced invasive ductal breast cancer (n = 18) or invasive lobular breast cancer (n = 6) underwent (18)F-fluciclovine PET/CT before and after the completion of neoadjuvant systemic therapy. SUV(max), SUV(mean), metabolic tumor volume, and total lesion avidity were obtained for the primary breast tumor, axillary lymph nodes, and extraaxillary lymph nodes on each examination and corrected for background (18)F-fluciclovine avidity. The relationship between changes in (18)F-fluciclovine avidity and the percentage of reduction of tumor on pathology was assessed with the Spearman rank correlation. Results: The median decrease in the corrected SUV(max) of the primary breast lesions was 99% (range, 33%–100%). The median reduction of tumor on pathology was 92% (range, 10%–100%). Changes in (18)F-fluciclovine avidity were strongly correlated with the percentage of reduction of tumor on pathology (Spearman ρ, 0.79; 95% CI, 0.56–0.90; P < 0.001). Conclusion: Changes in (18)F-fluciclovine avidity strongly correlated with the tumor response on pathology in this pilot study.