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 biomark- 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 sub-

Circulating Tumor Cells as a Window on Metastasis Biology in Lung Cancer

“…Using CellSearch, which defines tumor cells as both EpCam and cytokeratin positive, SCLC CTM were composed of clusters, rings, and elongated strands ( Figure 1D) consistent with some of the morphologies described for collective migration. 15 Blood samples from SCLC patients I and II had high CTC counts of Ͼ1000 cells in 7.5 ml blood consistent with our previous study 24 and contained numerous CTM allowing a comparative appraisal of the prevalence of nuclei that exhibited the classical condensed and fragmented nuclear morphologies of apoptosis ( Figure 1E). 22 While numerous apoptotic CTCs were detected in both of these patients, no cells with nuclear apoptotic morphology (DAPI stained) were observed in any of the cells forming CTM (summarized in Figure 1F).…”

Considerations for the use of plasma cytokeratin 18 as a biomarker in pancreatic cancer

“…The results from the M30 and M65 ELISA assays showed significant variability between patients with regard to the baseline levels of both full-length CK18 (M65) and the proportion of caspase-cleaved CK18 (M30 : M65). Circulating total CK18 concentrations in this study were relatively high compared with prostate (Kramer et al, 2006) and breast (Olofsson et al, 2007) cancer and comparable with those of other gastrointestinal malignancies (Scott et al, 2009) and non-small-cell lung cancer (Hou et al, 2009). In keeping with the other malignant tumour types (Ulukaya et al, 2007;Hou et al, 2009;Koelink et al, 2009), elevated CK18 levels were associated with poorer survival in the overall patient group on univariate analysis, but in this series failed to reach significance on multivariate analysis.…”

“…Early studies suggest that these assays may have important clinical biomarker utility, as increased levels of circulating CK18 may be prognostic and/or predict tumour response to chemotherapy in a number of different solid tumours (Steele et al, 2008;Scott et al, 2009). Studies on specific tumour types include lung (Ulukaya et al, 2007;Hou et al, 2009), breast (Olofsson et al, 2007), prostate (Kramer et al, 2006), head and neck (Ozturk et al, 2009), colorectal (Ausch et al, 2009;Koelink et al, 2009) and testicular (de Haas et al, 2008) tumours. Although a recent study concluded that plasma levels of CK18 are a potential marker of tumour response in patients with advanced gastrointestinal malignancy (Scott et al, 2009) no previous studies have been reported in patients with pancreatic cancer.…”

Variant Ciz1 is a circulating biomarker for early-stage lung cancer

“…For sample set 1, blood samples were collected with written, informed consent, according to National Health Service-approved protocols at the Paterson Institute for Cancer Research, University of Manchester, and were processed as described previously (22). Additional plasma samples listed in Dataset S1 were obtained from biobanks and were collected according to Institutional Review Board-approved protocols.…”