Serial measurements of circulating tumor cells by quantitative RT-PCR in metastatic breast cancer patients receiving systemic therapy.

Abstract Abstract #5027 Background: Tumor marker assays with reproducible correlation with clinical outcome and rapid turnaround for clinical use are desperately needed. Quantitative RtPCR (qPCR) is a high-throughput method that can quantify circulating tumor cells (CTC). We tested a qPCR assay using cytokeratin-19 (K19) mRNA as a CTC marker on a series of blood collections from a cohort of 20 metastatic breast cancer (MBC) patients.
 Methods: We designed a Taqman qPCR assay targeted to K19 mRNA, with a dynamic range of 0.01 to 1000 fg. Primer/probe sequences were designed to avoid known K19 pseudogenes, and appropriate controls were used to assess RNA integrity and exclude genomic DNA amplification. A prospective Seattle Cancer Care Alliance (SCCA) biospecimen registry of female breast cancer patients was used to test the K19 assay. Serial blood collections were obtained every 6-8 weeks from MBC registry participants. MBC subjects who had ≥2 specimens collected ≥2 months apart, with one drawn prior to initiating a new line of therapy, were included in the analytical cohort. The initial sample was labeled baseline (BL), and the first sample after initiation of treatment was labeled first follow-up (FFU). K19 levels were determined from BL, FFU, and subsequent blood samples. All results were compared to treatment outcomes using Kaplan-Meier plots, log-rank statistics, and Cox regression models.
 Results: K19 levels at FFU (FFU-K19) were more predictive of outcome than either BL or subsequent follow-up levels. FFU-K19 was detectable in 8 of 19 evaluable patients (42%). Of those, all had progressive disease within 6 months (100% specificity). FFU-K19 was below assay detection limit (BDL) for all 6 subjects who were progression-free for >6 months; however, 5 subjects with BDL FFU-K19 were misclassified (62% sensitivity). Nevertheless, survival analyses demonstrated that detectable FFU-K19 was associated with a median time to progression (TTP) of 2.4 months, compared to 7.9 months when K19 was not detected at FFU (p<0.01). Detection of K19 at FFU was associated with a median overall survival (OS) of 12.8 months, compared to a median OS >47 months when FFU-K19 was not detectable, but this difference did not achieve statistical significance (p=0.09). Multivariable models demonstrated that FFU-K19 remained an independent predictor of TTP after controlling for age, ER, and Her2-neu status (p=0.01).
 Conclusions: Preliminary results suggest that detectable K19 levels by qPCR after initiation of therapy is associated with poor outcome in MBC patients. While the assay had superb specificity in this small sample, it had limited sensitivity. Additional CTC markers are being considered to try to further improve identification of MBC patients with poor prognosis early in their treatment course. A separate MBC cohort has been recruited for results validation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5027.