Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cf DNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cf DNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cf DNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cf DNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cf DNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cf DNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cf DNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cf DNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

The purpose of this study was to determine whether primary breast cancer patients showed evidence of circulating tumour cells (CTCs) during follow-up as an alternative to monitoring disseminated bone marrow tumour cells (DTCs) by immunocytochemistry and reverse transcriptase (RT) -PCR for the detection of micrometastases. We planned to compare CTC and DTC frequency in low-risk and high-risk patients. We identified two cohorts of primary breast cancer patients who were at low (group II, T 1 N 0 , n ¼ 18) or high (group III, 43 nodes positive (with one exception, a patient with two positive nodes) n ¼ 33) risk of relapse who were being followed up after primary treatment. We tested each cohort for CTCs using the CellSearch system on 1 -7 occasions and for DTCs by immunocytochemistry and RT -PCR on 1 -2 occasions over a period of 2 years. We also examined patients with confirmed metastatic disease (group IV, n ¼ 12) and 21 control healthy volunteers for CTCs (group I). All group I samples were negative for CTCs. In contrast, 7 out of 18 (39%) group II primary patients and 23 out of 33 (70%) group III patients were positive for CTCs (P ¼ 0.042). If we count only samples with 41 cell as positive: 2 out of 18 (11%) group II patients were positive compared with 10 out of 33 (30%) in group III (P ¼ 0.174). In the case of DTCs, 1 out of 13 (8%) group II patients were positive compared with 19 out of 27 (70%) in group III (Po0.001). Only 10 out of 33 (30%) patients in group III showed no evidence of CTCs in all tests over the period of testing, compared with 11 out of 18 (61%) in group II (P ¼ 0.033). A significant proportion of poor prognosis primary breast cancer patients (group III) have evidence of CTCs on follow-up. Many also have evidence of DTCs, which are more often found in patients who were lymph node positive. As repeat sampling of peripheral blood is more acceptable to patients, the measurement of CTCs warrants further investigation because it enables blood samples to be taken more frequently, thus possibly enabling clinicians to have prior warning of impending overt metastatic disease.

Measurement of EGFR on the surface of CTCs, derived from individuals with metastatic breast cancer patients is possible using the CellSearch system and showed consistent positivity over time. The use of this system will now be validated in a prospective study aiming to identify patients for anti-EGFR therapy based on the expression profile of CTCs.

IntroductionCirculating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.MethodsCellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test.ResultsFor CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90).ConclusionsThe inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Both CTC and plasma DNA analyses together suggested that these patients had little evidence of metastatic disease. Future studies will be designed to assess the utility of these biomarkers in the follow-up of a larger number of women with breast cancer.