Circulating immunogenic cell death biomarkers HMGB1 and RAGE in breast cancer patients during neoadjuvant chemotherapy.

Neoadjuvant chemotherapy in breast cancer patients aims at preoperative reduction of tumor volume for better resection results and prognosis. As not all patients respond to neoadjuvant therapy, predictive biomarkers are needed for more efficient individual management. In prospectively collected sera of 51 consecutive locally confined breast cancer (LBC) patients receiving preoperative, neoadjuvant chemotherapy, value level kinetics of soluble high mobility group box 1 (HMGB1), soluble receptor for advanced glycation end products (sRAGE) as well as the established breast cancer biomarkers CA 15-3 and carcinoembryonic antigen (CEA) were investigated and correlated with therapy response objectified by pathological staging at surgery. In addition, biomarkers were measured in sera of 30 healthy controls (HC), 13 patients with benign breast diseases, and 28 metastatic breast cancer (MBC) patients. Pretherapeutic levels of soluble HMGB1 were decreased in MBC, while sRAGE was already decreased in LBC. In contrast, CA 15-3 and CEA were strongly elevated in MBC, but not in LBC. Combination of sRAGE and CA 15-3 enabled best discrimination of LBC from HC (AUC 78.2 %; sens 58 % at 95 % spec), while CA15-3 and CEA discriminated best between MBC and all controls (AUC 90.9 %; sens 70 % at 95 % spec). In LBC patients undergoing neoadjuvant chemotherapy, nine patients achieved complete remission (CR), 29 achieved partial remission (PR), while 13 had no change of disease (NC). NC patients tended to have higher HMGB1 and lower sRAGE levels before therapy onset (p = 0.056 and p = 0.054), while CA 15-3 and CEA did not predict therapeutic outcome. Furthermore, kinetics of HMGB1 during therapy correlated with efficacy of the treatment (p = 0.053). Markers of immunogenic cell death are valuable for the diagnosis of MBC and early estimation of response to neoadjuvant therapy in LBC patients.

Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating apoptotic biomarkers nucleosomes, DNAse, cytokeratin-18 fragments and survivin.

Biomarkers predicting response to neoadjuvant chemotherapy in locally confined breast cancer (LBC) are highly needed. We prospectively assessed serial blood levels of apoptotic biomarkers nucleosomes, DNAse activity, cytokeratin-18 fragments (M30) and survivin in 51 LBC patients and correlated them with response to neoadjuvant treatment and established tumor markers. As controls, we used 31 healthy subjects, 13 patients with benign diseases and 28 with metastatic breast cancer (MBC). Levels of nucleosomes and survivin were elevated in LBC and MBC while M30, CEA and CA 15-3 levels were only elevated in MBC. During neoadjuvant chemotherapy, LBC patients with no change of disease (N=13) had significantly higher pretherapeutic levels of nucleosomes than patients with remission (N=38). We conclude that apoptotic biomarkers bear valuable information for diagnosis and therapy response prediction in LBC patients.

Relevance of histone marks H3K9me3 and H4K20me3 in cancer.

BACKGROUND: Circulating nucleosomes are valuable biomarkers for therapy monitoring and estimation of prognosis in cancer disease. While epigenetic and genetic modifications of DNA have been reported in blood of cancer patients, little is known about modifications of histones on circulating nucleosomes. PATIENTS AND METHODS: Sera of 45 cancer patients (21 colorectal, 4 pancreatic, 15 breast, 5 lung cancer), 12 patients with benign gastrointestinal and inflammatory diseases, and 28 healthy individuals were investigated. Histone modifications were detected by chromatin-immunoprecipitation (ChIP) using antibodies for triple histone methylations at sites H3K9me3 and H4K20me3 and subsequent real-time polymerase chain reaction using primers for the centromeric satellites SAT2. Additionally, the amount of circulating nucleosomes, as well as of carcino-embryonic antigen (CEA) and cancer antigen (CA) 19-9 were measured. RESULTS: Levels of SAT2 on H3K9me3 (median 0.507 ng/ml) and on H4K20me3 (0.292 ng/ml) were elevated in sera of patients with breast cancer when compared with healthy controls (0.049 and 0.035 ng/ml), but were lower in patients with colorectal cancer (0.039 and 0.027 ng/ml). Both histone marks were correlated with each other but did not correlate with CEA or CA 19-9 in cancer patients. When H3K9me3 and H4K20me3 were normalized to nucleosome content in sera, ratios were significantly higher in all types of cancer as well as in colorectal and breast subtypes when compared with healthy controls. Best discrimination was achieved by normalized H4K20me3 reaching areas under the curves (AUC) of 79.1%, 90.4% and 81.2% in receiver operating characteristic (ROC) curves of these three comparisons. CONCLUSION: SAT2 levels on H3K9me3 and H4K20me3 are up-regulated in breast cancer and down-regulated in colorectal cancer. Normalization to total nucleosome content enables better discrimination between cancer and control groups.

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.

Apoptosis-related biomarkers sFAS, MIF, ICAM-1 and PAI-1 in serum of breast cancer patients undergoing neoadjuvant chemotherapy.

BACKGROUND: Neoadjuvant chemotherapy improves surgical options and prognosis in patients with operable breast cancer. Predictive biomarkers are needed to choose the most effective therapy and to avoid unnecessary toxicity. PATIENTS AND METHODS: We analyzed the courses of apoptosis-related serum biomarkers macrophage migration-inhibitory factor (MIF), soluble cell death receptor sFAS, soluble intercellular adhesion molecule (sICAM), plasminogen activator inhibitor 1 (PAI-1) as well as the oncological biomarkers carcino-embryonic antigen (CEA) and carbohydrate antigen 15-3 (CA15-3) in prospectively collected sera of 51 patients with locally confined breast cancer undergoing preoperative chemotherapy. As controls 31 healthy women, 13 patients with benign breast disease and 28 patients with metastasized breast cancer were included. RESULTS: sFAS, MIF, CEA and CA15-3 showed significantly higher serum concentrations in patients with metastasized breast cancer than in healthy and benign controls. Additionally, sFAS and MIF discriminated between locally confined breast cancer and healthy controls with an area under the curve (AUC) in receiver operating characteristic (ROC) curves of 73.4% and 70.7%. After neoadjuvant chemotherapy, 38 patients achieved complete (N=9) or partial (N=29) remission, while 13 patients had no change of disease. Pretherapeutic levels of MIF were considerably higher in non-responsive patients (p=0.082). In addition, post-therapeutic sICAM and CA15-3 levels were higher in patients without complete remission. CONCLUSION: Apoptosis-related biomarkers are valuable markers in breast cancer patients and show potential for early estimation of the efficiency of neoadjuvant chemotherapy.

Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.