Circulating nucleosomes and immunogenic cell death markers HMGB1, sRAGE and DNAse in patients with advanced pancreatic cancer undergoing chemotherapy.

Serum biomarkers are urgently needed for patient stratification and efficient treatment monitoring in pancreatic cancer (PC). Within a prospective diagnostic observation study, blood samples were obtained from 78 patients with advanced PC before and weekly during the course of palliative chemotherapy. Circulating nucleosomes and immunogenic cell death markers, high-mobility group box 1 (HMGB1), soluble receptors of advanced glycation end products (sRAGE) and DNAse activity, were measured by enzyme-linked immunosorbent assay and correlated with results of radiological staging after 2 months of treatment, with time to progression (TTP) and overall survival (OS). Median TTP and OS of PC patients were 3.9 and 7.7 months, respectively. Pretherapeutic baseline biomarker levels did not correlate with objective response; however, nucleosome levels on day (d) 28 were higher (p = 0.048) and sRAGE levels at time of staging (d56) were lower in progressive patients (p = 0.046). Concerning estimation of prognosis, high nucleosome levels (d7, d14, d21 and d56), low sRAGE levels (d56) and DNAse activity courses (d0-d7) correlated with TTP, whereas high nucleosomes (d7, d14 and d56), high HMGB1 (d21 and d56) and DNAse (d0-d7) were associated with OS. After adjustment to Karnofsky performance score, nucleosomes and HMGB1 (both d56) and DNAse (d0-d7) remained independent prognostic factors. Thus, courses of circulating nucleosomes and immunogenic cell death markers HMGB1 and sRAGE show prognostic relevance in PC patients undergoing chemotherapy.

Circulating nucleosomes and biomarkers of immunogenic cell death as predictive and prognostic markers in cancer patients undergoing cytotoxic therapy.

INTRODUCTION: Immunogenic cell death markers are released from apoptotic and necrotic cells upon pathologic or therapeutic causes and stimulate the innate and adaptive immune system. Cell death products such as nucleosomes, damage-associated molecular pattern (DAMP) molecules such as the high-mobility group box 1 protein (HMGB1) and its receptor of advanced glycation end products (sRAGE) are supposed to play an essential role in driving this process. However, this immunogenic activation may have dual effects, either by sensitizing the immune system for more efficient tumor cell removal or by creating a favorable tumor microenvironment that facilitates tumor growth, proliferation and invasiveness. AREAS COVERED: Here, we review recent findings on the relevance of serum nucleosomes, DNAse activity, HMGB1 and sRAGE as biomarkers for the diagnosis, prognosis and therapy prediction in cancer disease. EXPERT OPINION: In comparison with healthy controls, cancer patients demonstrated elevated serum levels of nucleosomes and HMGB1 while sRAGE levels were decreased. During locoregional and systemic cytotoxic therapies, a high release of nucleosomes and HMGB1 as well as low release of sRAGE before and during the initial phase of the treatment was found to be associated with poor response to the therapy and patient survival. Therefore, immunogenic cell death markers are promising tools for the prognosis, therapy prediction and monitoring in cancer patients.

Methodological and preanalytical evaluation of an HMGB1 immunoassay.

BACKGROUND: Soluble high-mobility group box 1 (sHMGB1) is a promising biomarker for the prognosis and the monitoring of cancer and of acute diseases such as trauma and sepsis. MATERIALS AND METHODS: We investigated the methodological characteristics of an ELISA for sHMGB1 (Shino-Test, Tokyo, Japan and IBL, Hamburg, Germany) including intra- and inter-assay imprecision, dilution linearity and differences in serum and plasma materials. Furthermore, the influence of various preanalytical factors such as time and storage temperature before and after centrifugation prior to definite deep freezing, as well as multiple freeze-thaw cycles were tested. By the use of sera from 28 healthy individuals, a reference range and the dependency on patient characteristics were established. RESULTS: Intra-assay imprecision (coefficients of variation (CV)=1.2-4.8%) and inter-assay imprecision (10.3-14.0%) were in an acceptable range of manual assays. HMGB1 levels were found to be considerably lower in EDTA plasma as compared to serum samples. Linearity testing yielded satisfying results with dilution recoveries of 100-121% (mean=112.3%). sHMGB1 results were the same when samples were kept at 4°C and 25°C after centrifugation, for up to 7 days (recoveries 87-128%). Delay before centrifugation led to a considerable increase in some samples. The median values for healthy individuals was 1.3 ng/ml, and the 95th percentile was 4.1 ng/ml. HMGB1 levels correlated inversely with age (R=0.33). CONCLUSION: The sHMGB1 ELISA is a robust and safe assay producing reliable quantitative results in sera.

Methodological and preanalytical evaluation of a RAGE immunoassay.

BACKGROUND: Soluble receptor of advanced glycation end products (sRAGE) is a promising biomarker for the prognosis and the monitoring of cancer and of acute diseases such as trauma and sepsis. MATERIALS AND METHODS: We investigated the methodological characteristics of an ELISA for sRAGE (R&D Diagnostics) including intra- and inter-assay imprecision, dilution linearity and differences in various serum and plasma materials. Furthermore, the influence of various preanalytical factors such as time and storage temperature before and after centrifugation prior to definite deep freezing, as well as multiple freeze-thaw cycles, were tested. By the use of sera from 30 healthy individuals, a reference range and the dependency on patient characteristics was established. RESULTS: Intra-assay imprecision (coefficients of variation (CV): 6.0-11.5%) and inter-assay imprecision (5.9-7.8%) were in an acceptable range of manual assays. Linearity testing yielded satisfying results with dilution recoveries of 99-131%. Results of serum, EDTA-plasma (recovery of 85.9-114.7%), and heparin-plasma samples (88-102%) were quite comparable, while results from citrate-plasma were slightly lower (78-96%). There was no influence of the time to centrifugation after 6 and 24 hours (recoveries 87-102%) at storage temperatures of 4°C and 25°C. Similarly, results were the same when samples were kept at 4°C and 25°C after centrifugation for up to 7 days (recoveries 88-109%). Repeated freeze-thawing of samples did not affect the results obtained for the RAGE protein (recoveries 92-104%). The median value of healthy individuals was 1.10 ng/ml, with 90% limits of 0.52 to 1.49 ng/ml. CONCLUSION: sRAGE ELISA is a very robust and safe assay which produces reliable quantitative results for sera and plasma measurements.