Evaluation of Circulating Tumor Cells and Serological Cell Death Biomarkers in Small Cell Lung Cancer Patients Undergoing Chemotherapy

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.Small cell lung cancer (SCLC) is initially chemosensitive but invariably relapses with a chemoresistant phenotype.¹ A number of molecularly targeted therapies have been evaluated attempting to improve outcome, but none have succeeded to date.² Ideally, early clinical trials should incorporate validated pharmacodynamic biomarkers, conducted to good clinical laboratory practice, that demonstrate both proof of mechanism (drug hits target) and proof of concept (tumor responds to drug).³ Although possible, serial biopsies are rare in SCLC, and the tissue obtained often insufficient for extensive molecular profiling. Thus, there is a pressing need for blood-based biomarkers that report therapeutic response.Assays of drug-induced cell death are potential proof of concept biomarkers for multiple therapeutics.⁴ The M30 Apoptosense and M65 assays (Peviva, Bromma, Sweden) detect cytokeratin (CK) 18, expressed in epithelial but not hematopoietic cells, and released into the blood following cytoskeletal disassembly and degradation during apoptotic and/or necrotic cell death.⁵ The M30 antibody recognizes a caspase-cleaved neoepitope of CK18 that is only revealed during apoptosis, whereas the M65 assay detects full length and cleaved forms of CK18 reporting apoptosis and necrosis.⁶ Nucleosomal DNA (nDNA) results from cleavage of chromatin by apoptotic endonucleases into membrane bound DNA fragments that are phagocytosed by macrophages and subsequently released into the blood.⁷ nDNA release is also detected when levels of apoptosis overwhelm macrophage capacity for phagocytosis and a more necrotic cell fate ensues.⁷ We have previously validated these cell death biomarkers^8,9 and optimized them for application to a busy, clinical setting.⁶ Here we report on the behavior and clinical utility of these assays in patients with SCLC.Importantly, cell death assays may report host toxicity in addition to tumor response. However, circulating tumor cell (CTC) numbers can, in theory, be used to directly evaluate drug effect on malignant cells.¹⁰ The cytometric approach using CellSearch technology (Veridex Inc., Huntingdon Valley, PA) is now approved by the Food and Drug Administration for clinical decision-making in patients with metastatic breast, colorectal and prostate cancers.^11,12,13 This is the first report on the use of this technology platform for CTC enumeration in patients with SCLC and the first direct comparison of serological biomarkers of cell death and cell death in CTCs.This study was conducted to evaluate cell death assays (M30, M65, and nDNA) and CTC profiles in patients with SCLC undergoing standard chemotherapy, as a prelude to their incorporation as biomarkers in early clinical trials. The hypothesis tested was that increases in cell death biomarkers immediately following therapy would predict outcome and that given the metastatic potential of SCLC high numbers of CTCs would be detectable. The results from this study are most encouraging for the development of CTCs as pharmacodynamic biomarkers in SCLC, provide novel insights into the clinical significance of serological cell death assays, and demonstrate agreement between measures of cell death at the molecular and cellular level in this disease.