Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression

<p>Abstract</p> <p>Background</p> <p>As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases.</p> <p>Methods</p> <p>The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer (M0, n = 31), metastatic breast cancer (M1, n = 32), and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo^®3/7 Assay, respectively.</p> <p>Results</p> <p>Patients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001), whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008).</p> <p>Conclusion</p> <p>Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression.</p

Prognostic impact of circulating tumor cells assessed with the CellSearch System (TM) and AdnaTest Breast (TM) in metastatic breast cancer patients: the DETECT study

Introduction: There is a multitude of assays for the detection of circulating tumor cells (CTCs) but a very limited number of studies comparing the clinical relevance of results obtained with different test methods. The DETECT trial for metastatic breast cancer patients was designed to directly compare the prognostic impact of two commercially available CTC assays that are prominent representatives of immunocytochemical and RT-PCR based technologies. Methods: In total, 254 metastatic breast cancer patients were enrolled in this prospective multicenter trial. CTCs were assessed using both the AdnaTest Breast Cancer and the CellSearch system according to the manufacturers' instructions. Results: With the CellSearch system, 116 of 221 (50%) evaluable patients were CTC-positive based on a cut-off level at 5 or more CTCs. The median overall survival (OS) was 18.1 months in CTC-positive patients. (95%-CI: 15.1-22.1 months) compared to 27 months in CTC-negative patients (23.5-30.7 months; p&lt;0.001). This prognostic impact for OS was also significant in the subgroups of patients with triple negative, HER2-positive and hormone receptor-positive/HER2-negative primary tumors. The progression free survival (PFS) was not correlated with CTC status in our cohort receiving different types and lines of systemic treatment (p = 0.197). In multivariate analysis, the presence of CTCs was an independent predictor for OS (HR: 2.7, 95%-CI: 1.6-4.2). When the AdnaTest Breast was performed, 88 of 221 (40%) patients were CTC-positive. CTC-positivity assessed by the AdnaTest Breast had no association with PFS or OS. Conclusions: The prognostic relevance of CTC detection in metastatic breast cancer patients depends on the test method. The present results indicate that the CellSearch system is superior to the AdnaTest Breast Cancer in predicting clinical outcome in advanced breast cancer

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients

IntroductionTriple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets.Materials and methodsTo follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3’ UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival.ResultssHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3’ UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy responseConclusionThe results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets

Pooled Analysis of the Prognostic Relevance of Disseminated Tumor Cells in the Bone Marrow of Patients With Ovarian Cancer

Objective: Detection of disseminated tumor cells (DTCs) in the bone marrow (BM) of patients with breast cancer is associated with poor outcomes. Recent studies demonstrated that DTCs may serve as a prognostic factor in ovarian cancer. The aim of this 3-center study was to evaluate the impact of BM status on survival in a large cohort of patients with ovarian cancer. Materials and Methods: Four hundred ninety-five patients with primary ovarian cancer were included in this 3-center prospective study. Bone marrow aspirates were collected intraoperatively from the iliac crest. Disseminated tumor cells were identified by antibody staining and by cytomorphology. Clinical outcome was correlated with the presence of DTCs. Results: Disseminated tumor cells were detected in 27% of all BM aspirates. The number of cytokeratin-positive cells ranged from 1 to 42 per 2 x 10(6) mononuclear cells. Disseminated tumor cell status did correlate with histologic subtype but not with any of the other established clinicopathologic factors. The overall survival was significantly shorter among DTC-positive patients compared to DTC-negative patients (51 months; 95% confidence interval, 37-65 months vs 33 months; 95% confidence interval, 23-43 months; P = 0.023). In the multivariate analysis, BM status, International Federation of Gynecology and Obstetrics stage, nodal status, resection status, and age were independent predictors of reduced overall survival, whereas only BM status, International Federation of Gynecology and Obstetrics stage, and resection status independently predicted progression-free survival. Conclusions: Tumor cell dissemination into the BM is a common phenomenon in ovarian cancer. Disseminated tumor cell detection has the potential to become an important biomarker for prognostication and disease monitoring in patients with ovarian cancer

Definition and Independent Validation of a Proteomic-Classifier in Ovarian Cancer

Simple Summary: The heterogeneity of epithelial ovarian cancer and its associated molecular biological characteristics are continuously integrated in the development of therapy guidelines. In a next step, future therapy recommendations might also be able to focus on the patient's systemic status, not only the tumor's molecular pattern. Therefore, new methods to identify and validate host-related biomarkers need to be established. Using mass spectrometry, we developed and independently validated a blood-based proteomic classifier, stratifying epithelial ovarian cancer patients into good and poor survival groups. We also determined an age dependence of the prognostic performance of this classifier and its association with important biological processes. This work highlights that, just like molecular markers of the tumor itself, the systemic condition of a patient (partly reflected in proteomic patterns) also influences survival and therapy response and could therefore be integrated into future processes of therapy planning. Abstract: Mass-spectrometry-based analyses have identified a variety of candidate protein biomarkers that might be crucial for epithelial ovarian cancer (EOC) development and therapy response. Comprehensive validation studies of the biological and clinical implications of proteomics are needed to advance them toward clinical use. Using the Deep MALDI method of mass spectrometry, we developed and independently validated (development cohort: n = 199, validation cohort: n = 135) a blood-based proteomic classifier, stratifying EOC patients into good and poor survival groups. We also determined an age dependency of the prognostic performance of this classifier, and our protein set enrichment analysis showed that the good and poor proteomic phenotypes were associated with, respectively, lower and higher levels of complement activation, inflammatory response, and acute phase reactants. This work highlights that, just like molecular markers of the tumor itself, the systemic condition of a patient (partly reflected in proteomic patterns) also influences survival and therapy response in a subset of ovarian cancer patients and could therefore be integrated into future processes of therapy planning

Pooled Analysis of the Prognostic Relevance of Disseminated Tumor Cells in the Bone Marrow of Patients With Ovarian Cancer

Objective: Detection of disseminated tumor cells (DTCs) in the bone marrow (BM) of patients with breast cancer is associated with poor outcomes. Recent studies demonstrated that DTCs may serve as a prognostic factor in ovarian cancer. The aim of this 3-center study was to evaluate the impact of BM status on survival in a large cohort of patients with ovarian cancer. Materials and Methods: Four hundred ninety-five patients with primary ovarian cancer were included in this 3-center prospective study. Bone marrow aspirates were collected intraoperatively from the iliac crest. Disseminated tumor cells were identified by antibody staining and by cytomorphology. Clinical outcome was correlated with the presence of DTCs. Results: Disseminated tumor cells were detected in 27% of all BM aspirates. The number of cytokeratin-positive cells ranged from 1 to 42 per 2 x 10(6) mononuclear cells. Disseminated tumor cell status did correlate with histologic subtype but not with any of the other established clinicopathologic factors. The overall survival was significantly shorter among DTC-positive patients compared to DTC-negative patients (51 months; 95% confidence interval, 37-65 months vs 33 months; 95% confidence interval, 23-43 months; P = 0.023). In the multivariate analysis, BM status, International Federation of Gynecology and Obstetrics stage, nodal status, resection status, and age were independent predictors of reduced overall survival, whereas only BM status, International Federation of Gynecology and Obstetrics stage, and resection status independently predicted progression-free survival. Conclusions: Tumor cell dissemination into the BM is a common phenomenon in ovarian cancer. Disseminated tumor cell detection has the potential to become an important biomarker for prognostication and disease monitoring in patients with ovarian cancer