Evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy

Abstract

Serological cell death biomarkers and circulating tumor cells (CTCs) have potential uses as tools for pharmacodynamic blood-based assays and their subsequent application to early clinical trials. In this study, we evaluated both the expression and clinical significance of CTCs and serological cell death biomarkers in patients with small cell lung cancer. Blood samples from 88 patients were assayed using enzyme-linked immunosorbent assays for various cytokeratin 18 products (eg, M65, cell death, M30, and apoptosis) as well as nucleosomal DNA. CTCs (per 7.5 ml of blood) were quantified using Veridex CellSearch technology. Before therapeutic treatment, cell death biomarkers were elevated in patients compared with controls. CTCs were detected in 86% of patients; additionally, CD56 was detectable in CTCs, confirming their neoplastic origin. M30 levels correlated with the percentage of apoptotic CTCs. M30, M65, lactate dehydrogenase, and CTC number were prognostic for patient survival as determined by univariate analysis. Using multivariate analysis, both lactate dehydrogenase and M65 levels remained significant. CTC number fell following chemotherapy, whereas levels of serological cell death biomarkers peaked at 48 hours and fell by day 22, mirroring the tumor response. A 48-hour rise in nucleosomal DNA and M30 levels was associated with early response and severe toxicity, respectively. Our results provide a rationale to include the use of serological biomarkers and CTCs in early clinical trials of new agents for small cell lung cancer.

Figures

Figure 1

SCLC CTC morphology and effect of chemotherapy on CTC number. Based on CK and DAPI staining profile, CTCs represent as a heterogeneous population among which there are mitotic, apoptotic, and aggregated CTCs (A). A majority of patients have decreased CTC number on day 22 after the first cycle of chemotherapy (B).

Figure 2

CD56 in SCLC CTC and tumor biopsies. The CD56 staining profile is consistent in the matched tumor biopsies and CTC samples. A and B: Tumor biopsies from primary lung lesions and isolated CTCs from the same patients. C: Tumor biopsy from liver metastasis and paired CTCs. The asterisk shows a SCLC CTC with dual staining for CK and CD56 that can be seen next to a white blood cell (DAPI positive only).

Figure 3

Kaplan-Meier curve demonstrating overall survival according to M65 determined before therapy.

Figure 4

Serial assays of serological cell death biomarkers during chemotherapy. A: Determination of optimal time points for sampling M30/M65 (n = 12). B: Determination of optimal time points for sampling nDNA (n = 12). C: Comparison of patient values (n = 39) with controls (n = 85).