Repeated mutKRAS ctDNA measurements represent a novel and promising tool for early response prediction and therapy monitoring in advanced pancreatic cancer.

Background: The presence of mutated KRAS (mutKRAS ctDNA) in plasma samples has been consistently shown to be a negative prognostic indicator in pancreatic cancer (PC). Only small pilot studies have evaluated the value of serial mutKRAS ctDNA-measurements in PC. Patients and methods: The aim of the present study was to explore the potential of repeated mutKRAS ctDNA measurements for response prediction and therapy monitoring in advanced PC patients. We used the BEAMing technology to determine levels of mutKRAS ctDNA, CA 19-9, CEA and CYFRA 21-1 in 284 plasma samples of 54 patients with advanced PC receiving gemcitabine-based chemotherapy. Absolute levels and kinetics of mutKRAS ctDNA, CA 19-9, CEA and CYFRA 21-1 were correlated to radiological response, progression-free and overall survival. Results: mutKRAS ctDNA was present in a majority of advanced PC patients (n = 36/54, 67%) and indicated tissue KRAS mutation status with a high sensitivity (75%) and specificity (100%). The presence of mutKRAS ctDNA, as well as higher levels of CA 19-9, CEA and CYFRA 21-1 before initiation of the first-line chemotherapy, was significantly correlated to an adverse overall survival. During therapy, changes in mutKRAS ctDNA levels were more rapid and pronounced than changes in protein-based tumor markers. A decrease in mutKRAS ctDNA levels during therapy was an early indicator of response to therapy, while there was no significant correlation between kinetics of CA 19-9, CEA or CYFRA 21-1 and response to chemotherapy during the first four weeks of treatment. Repeated mutKRAS ctDNA measurements during follow-up appeared to be superior to protein-based tumor markers in detecting progressive disease (sensitivity: 83%, specificity: 100%). Conclusion: mutKRAS ctDNA kinetics appear to be a powerful and highly specific tool in early response prediction and therapy monitoring of advanced PC patients receiving chemotherapy.

A comparison of gamma-glutamyl transferase and alkaline phosphatase as prognostic markers in patients with coronary heart disease.

BACKGROUND AND AIMS: Whether gamma-glutamyl transferase (GGT) or alkaline phosphatase (ALP) is a better prognostic marker in patients with coronary heart disease (CHD) remains unknown. The aim of this study was to compare the prognostic value of GGT and ALP in patients with CHD. METHODS AND RESULTS: This study included 3768 patients with CHD. The main study outcome was 3-year all-cause mortality. The median values of GGT and ALP were 36.2 U/L and 69.3 U/L. Patients were divided into subgroups according to GGT or ALP activity > or ≤median. Overall, there were 304 deaths: 195 deaths occurred in patients with GGT >median (n = 1882) and 109 deaths occurred in patients with GGT ≤median (n = 1886); Kaplan-Meier [KM] estimates of all-cause mortality were 11.9% and 6.4% (unadjusted hazard ratio [HR] = 1.85, 95% confidence interval [CI], 1.46 to 2.34]; P < 0.001). According to ALP activity, 186 deaths occurred in patients with ALP >median (n = 1883) and 118 deaths occurred in patients with ALP ≤median (n = 1885); KM estimates of all-cause mortality were 11.4% and 7.1% (unadjusted HR = 1.64 [1.30-2.06]; P < 0.001). After adjustment, GGT (adjusted HR = 1.32 [1.11-1.58]; P = 0.002) but not ALP (adjusted HR = 1.20 [1.00-1.43]; P = 0.051, with both HR calculated per 1 unit increment in logarithmic GGT or ALP scale) remained significantly associated with the risk for mortality. The C statistic of the mortality model with GGT was greater than the C statistic of the model with ALP (0.831 [0.802-0.859] vs. 0.826 [0.793-0.855]; P < 0.001). CONCLUSIONS: In patients with CHD, GGT was a stronger correlate of all-cause mortality than ALP.

[Detection of cell-free lncRNA in serum of cancer patients]. / Nachweis von zellfreier lncRNA im Serum von Tumorpatienten.

BACKGROUND: The analysis of circulating RNA molecules is of increasing interest since tumor-specific RNA expression patterns could be a useful cancer biomarker. A new entity of RNA molecules, the so-called long non-coding RNAs (lncRNA), are of particular interest because of its high tissue- and tumor-specificity. The importance of analytical factors in the quantification of lncRNAs is largely unclear and should therefore be investigated in the present study. PATIENTS AND METHODS: Serum RNA was isolated from patients with bladder, prostate and kidney cancer as well as patients with non-malignant disease. Analytical variables like different RNA isolation procedures, cDNA synthesis and preamplification were studied with respect to quantification of MALAT1 and ACTB via real-time PCR. RESULTS: The quantification of cell-free serum RNA is feasible although the levels of ACTB and MALAT1 were often only slightly above the detection limit. RNA isolation with a combined phenol-based column purification (Ambion mirVana PARIS miRNA Isolation Kit; Qiagen miRNeasy Serum/Plasma Kit) was most effective. The elimination of DNA contamination was most successful during cDNA synthesis with (Takara-Bio PrimeScript RT Reagent Kit with gDNA Eraser). Preamplification with the Applied Biosystems TaqMan PreAmp Master Mix Kit improved sensitivity. Serum ACTB and MALAT1 levels were not significantly increased in patients with urological tumors compared to patients with non-malignant diseases. CONCLUSION: An optimized protocol for the analysis of circulating lncRNAs is described in the present study.

Cytokeratin 19-fragments (CYFRA 21-1) as a novel serum biomarker for response and survival in patients with advanced pancreatic cancer.

BACKGROUND: CYFRA 21-1 serves as biomarker in several epithelial malignancies. However, its role in pancreatic cancer (PC) has not yet been investigated. METHODS: Within a prospective single-centre study serial blood samples were collected from patients with confirmed advanced PC. Pre-treatment values and weekly measurements of CYFRA 21-1, carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (assessed by Elecsys 2010, Roche Diagnostics) during palliative first-line chemotherapy were obtained. Biomarker data were correlated with objective response (determined by RECIST) as well as time to progression (TTP) and overall survival (OS) using uni- and multivariate analyses. RESULTS: Seventy-eight patients were included, 45% of these received treatment in prospective clinical trials. Median TTP was 3.9 months, median OS 7.7 months. Pre-treatment CYFRA 21-1 levels were significantly associated with performance status (P=0.0399) and stage of disease (P=0.0001). Marker values before chemotherapy and at the 2-month staging of all three markers were considered significant predictors for objective treatment response. Pre-treatment CYFRA 21-1 levels, as well as CA 19-9 values, could be applied to define subgroups (categorised by tertiles) with a different OS outcome (CYFRA: 14.8 vs 7.1 vs 4.8 months, CA 19-9: 14.2 vs 7.1 vs 5.2 months; P<0.0001). CYFRA 21-1 and CA 19-9 (both as categorised and as continuous variables) showed a highly significant correlation with TTP and OS at nearly all-time points assessed in univariate analysis. In multivariate analysis, only CYFRA 21-1 and performance status were independent predictors for OS. CONCLUSIONS: CYFRA 21-1 may serve as a valuable tool for monitoring treatment response and assessing prognosis in advanced PC.

Cytokeratin serum biomarkers in patients with colorectal cancer.

BACKGROUND: Circulating cytokeratins have shown to be important for management of patients with lung cancer. Here we investigated their role for differential diagnosis, therapy monitoring and prognosis in colorectal cancer (CRC). PATIENTS AND METHODS: Pretherapeutic levels of cytokeratin-19 fragments (CYFRA 21-1), carcino-embryonic antigen (CEA) and cancer antigen (CA) 19-9 were measured in 42 patients with CRC, 45 with benign colorectal diseases and 51 healthy controls. Furthermore, courses of CYFRA 21-1, tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), M30-antigen, CEA and CA 19-9 were analyzed in prospectively collected sera of 15 patients with CRC during primary chemotherapy and were correlated with therapy response and overall survival (OS). RESULTS: Similar to CEA and CA 19-9, CYFRA 21-1 was significantly elevated in serum from patients with CRC (median 2.1 ng/ml) as compared with healthy (1.2 ng/ml; p<0.0001) and benign gastrointestinal controls (1.7 ng/ml; p=0.0178) and showed stage dependency in CRC (p=0.0118). CYFRA 21-1 correlated with CEA in benign diseases and CRC but not with CA 19-9. The best discrimination between healthy controls and patients with CRC was achieved by combination of CYFRA 21-1 and CA 19-9 (area under the curve; AUC=86.7%), while the combination of CEA and CA 19-9 discriminated best between benign diseases and CRC (AUC=73.9%). In CRC patients during primary chemotherapy, levels of cytokeratins CYFRA 21-1, TPA, TPS, CEA and CA 19-9 tended to be higher in patients with poor response to therapy and with poor prognosis. CONCLUSION: Cytokeratins are elevated in patients with CRC and show some association with response to primary therapy and prognosis.

Clinical validation of an autoantibody test for lung cancer.

BACKGROUND: Autoantibodies may be present in a variety of underlying cancers several years before tumours can be detected and testing for their presence may allow earlier diagnosis. We report the clinical validation of an autoantibody panel in newly diagnosed patients with lung cancer (LC). PATIENTS AND METHODS: Three cohorts of patients with newly diagnosed LC were identified: group 1 (n = 145), group 2 (n = 241) and group 3 (n = 269). Patients were individually matched by gender, age and smoking history to a control individual with no history of malignant disease. Serum samples were obtained after diagnosis but before any anticancer treatment. Autoantibody levels were measured against a panel of six tumour-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2). Assay sensitivity was tested in relation to demographic variables and cancer type/stage. RESULTS: The autoantibody panel demonstrated a sensitivity/specificity of 36%/91%, 39%/89% and 37%/90% in groups 1, 2 and 3, respectively, with good reproducibility. There was no significant difference between different LC stages, indicating that the antigens included covered the different types of LC well. CONCLUSION: This assay confirms the value of an autoantibody panel as a diagnostic tool and offers a potential system for monitoring patients at high risk of LC.

Clinical relevance of tumor markers for the control of chemotherapy.

This presentation is based on our experience with tumor marker monitoring of surgery therapy and chemotherapy effects. The control of chemotherapy is one of the most important problems in oncological practice. The correlation between the clinical status of the patient and tumor size changes, based on the results of different imaging methods, has been the most important and most frequently used method. However, the therapy effect has been recently assessed by markers of the biological activity of the tumor. Using tumor markers for the assessment of the effect of surgery therapy is already part of routine practice in many different types of cancer. Pre-operative and post-operative values of tumor markers are essential for a proper evaluation. However, tumor marker monitoring of the effect of radiotherapy and chemotherapy has been used very rarely, mostly only for research purposes. Besides monitoring by classical tumor markers, monitoring by markers of angiogenesis and apoptosis seem to be promising for the assessment of chemotherapy effect. Measurement of circulating cancer cells by means of mRNA also seems to be intriguing for therapy effect control and monitoring of the course of disease. Unfortunately, the routine use of these methods has been applied in only a few institutes worldwide. A completely different situation has been observed in palliative treatment. In most cases, changes of serum levels of tumor markers correlate with therapy effect. Thus, the effect of treatment on tumor proliferation can be successfully estimated by decreasing tumor marker levels.

Changes of thymidine kinase (TK) during adjuvant and palliative chemotherapy.

UNLABELLED: Thymidine kinase is involved in nucleic acid synthesis and is, therefore, considered to be an important proliferation tumor marker. For this reason, we monitored this marker in the course of colorectal cancer chemotherapy. MATERIALS AND METHODS: We examined thymidine kinase (TK) levels in 30 patients with colorectal cancer who underwent adjuvant or palliative chemotherapy (CHT schemes). The condition for being included in the study was a minimum of 3 cycles of chemotherapy. TK was always assessed with radio-receptor analysis, before and after every chemotherapy cycle, together with other tumor markers. RESULTS: From the monitored tumor markers, only TK changed typically in the course of chemotherapy. In adjuvant chemotherapy, it was mostly low at the beginning of the cycle and its values usually increased considerably at the end. On the other hand, in palliative chemotherapy the dynamics of TK varied mainly depending on the effect of the therapy. Other tumor markers showed nonstandard behavior and minimal correlation with TK changes. CONCLUSION: Thymidine kinase seems to be a suitable parameter for monitoring the effect of adjuvant and palliative chemotherapy in colorectal cancer.

Markers of cellular adhesion in diagnosis and therapy control of colorectal carcinoma.

AIM: Early diagnosis of the progressive tumor disease and control of the effect of therapy in colorectal carcinoma are most frequently performed by monitoring CEA or CA 19-9 tumor markers. Their clinical application is, however, limited. The aim of our study was to demonstrate the contribution of adhesive molecule assessment to the early diagnosis of progression. We also wanted to find out if changes in the levels of cellular adhesion parameters correlate with the effect of antitumor therapy. MATERIALS AND METHODS: Intercellular cell adhesive molecule-1 (ICAM-1) and Vascular cell adhesive molecule-1 (VCAM-1) were assessed using the ELISA method, and the results were correlated with CEA and CA 19-9 tumor markers. Three hundred and sixty-four patients with colorectal carcinoma in Dukes' stages B-D were monitored. The results were processed with the SAS 6.2. statistical program and Statistica. RESULTS: In 92 patients with first clinical progression (occurrence of distant metastases irrespective of localization), significantly increased ICAM-1 and VCAM-1 values were demonstrated. In ROC evaluation of curves, we also demonstrated high sensitivity of adhesive molecules against both the control healthy group (n =89) and the no evidence of disease group (NED) (n=183). Adhesive molecule levels were closely connected with the type and course of therapy and are presented in the form of case reports. CONCLUSION: Soluble adhesive molecules are a prospective parameter both for the early diagnosis of progression and for control of the effect of therapy. There is a need for a large-scale study, preferably multicentric, which would verify the suitability of introducing cellular adhesion parameter assessment into routine practice.

Therapy control in oncology by circulating nucleosomes.

The courses of circulating nucleosomes in the serum of patients with various solid tumors correlate with the clinical outcome after chemo- and radiotherapy. Already during the initial phase of treatment they showed considerable alterations consisting of a rapid increase followed by a decrease during the first therapeutic week. Among patients with advanced lung cancer undergoing chemotherapy, those patients who responded to therapy exhibited less pronounced increases and more complete decreases compared to those patients with insufficient response. In addition, response to therapy was correlated with stronger decreases of the precyclic baseline values from cycle 1 to 2 and from cycle 1 to 3. Thus, circulating nucleosomes are a valuable tool for the early prediction of therapeutic efficacy and can help to modulate therapy strategies early and on an individual basis.

Apoptotic markers in cancer.

In cancer, apoptotic processes occur both spontaneously and induced by antitumor therapies. Qualitative and quantitative changes in cancer cell death along with proliferative alterations are essential determinants in the pathogenesis and progression of malignant disease and its responsiveness to therapy. Besides detecting apoptosis by invasive means in tumor tissue, apoptotic products can be quantified in the circulation. Although circulating apoptotic products usually lack organ and tumor specificity, they contribute in the assessment of disease extent or aggressiveness. The ease of drawing blood facilitates the serial measurement of circulating apoptotic markers to monitor antitumor treatment and predict early response to therapy. This review describes the features of apoptotic and necrotic cell death along with the role the balance between the rates of cell death and cell proliferation plays in the progression of malignancy. The intracellular pathways mediating apoptosis are next summarized. The focus then shifts to the apoptotic markers found in the circulation and their diagnostic, prognostic, predictive, and management utility in cancer.

Effects of single-dose irradiation on bronchial epithelium: a comparison of BEAS 2B cell monolayers, human organ cultures, and Goettinger minipigs.

To assess the effects of radiation on bronchial epithelium, BEAS 2B cells cultured as monolayers and human bronchial epithelium cultured as organ cultures were exposed to single doses of 0, 10 and 30 Gy. The lactate dehydrogenase in the supernatant of the BEAS 2B cells increased markedly 24 h after irradiation, whereas in the organ cultures only a minor increase was found after 48 h. The nucleosomes in the supernatant of the BEAS 2B cells showed a massive increase in response to irradiation, whereas in the organ cultures no change could be seen. The number of BEAS 2B cells was dramatically diminished after 96 h, whereas in the organ cultures a smaller decrease was observed no earlier than 21 days after irradiation. To assess the effects of brachytherapy in bronchial epithelium in vivo, brachytherapy with 30 Gy was performed in Goettinger minipigs, and histological sections of the bronchi were analyzed for morphological alterations and cell numbers. After 2 weeks, only slight cell damage was observable, and after 3 weeks, moderate morphological changes and decreased cell numbers were found. However, after 8 weeks, the epithelium had nearly regained its normal structure. We conclude that the bronchial epithelium has a remarkably high radioresistance and that organ cultures, but not monolayers of BEAS 2B cells, reflect the effects of radiation in vivo.

Circulating nucleosomes in serum.

In the nucleus of eukaryotic cells, DNA is associated with several protein components and forms complexes known as nucleosomes. During cell death, particularly during apoptosis, endonucleases are activated that cleave the chromatin into multiple oligo- and mononucleosomes. Subsequently, these nucleosomes are packed into apoptotic bodies and are engulfed by macrophages or neighboring cells. In cases of high rates of cellular turnover and cell death, they also are released into the circulation and can be detected in serum or plasma. As enhanced cell death occurs under various pathologic conditions, elevated amounts of circulating nucleosomes are not specific for any benign or malignant disorder. However, the course of change in the nucleosomal levels in circulation of patients with malignant tumors during chemotherapy or radiotherapy is associated with the clinical outcome and can be useful for the therapeutic monitoring and the prediction of the therapeutic efficacy.

Nucleosomes in serum as a marker for cell death.

The concentration of nucleosomes is elevated in blood of patients with diseases which are associated with enhanced cell death. In order to detect these circulating nucleosomes, we used the Cell Death Detection-ELISAplus (CDDE) from Roche Diagnostics (Mannheim, Germany) (details at http:\\biochem.roche.com). For its application in liquid materials we performed various modifications: we introduced a standard curve with nucleosome-rich material, which enabled direct quantification and improved comparability of the values within (CVintraassay:3.0-4.11%) and between several runs (CVinterassay:8.6-13.5%), and tested the analytical specificity of the ELISA. Because of the fast elimination of nucleosomes from circulation and their limited stability, we compared plasma and serum matrix and investigated in detail the pre-analytical handling of serum samples which can considerably influence the test results. Careless venipuncture producing hemolysis, delayed centrifugation and bacterial contamination of the blood samples led to false-positive results; delayed stabilization with EDTA and insufficient storage conditions resulted in false-negative values. At temperatures of -20 degrees C, serum samples which were treated with 10 mM EDTA were stable for at least 6 months. In order to avoid possible interfering factors, we recommend a schedule for the pre-analytical handling of the samples. As the first stage, the possible clinical application was investigated in the sera of 310 persons. Patients with solid tumors (n=220; mean=361 Arbitrary Units (AU)) had considerably higher values than healthy persons (n=50; mean=30 AU; p=0.0001) and patients with inflammatory diseases (n=40; mean= 296 AU; p=0.096). Within the group of patients with tumors, those in advanced stages (UICC 4) showed significantly higher values than those in early stages (UICC 1-3) (p=0.0004).

Nucleosomes in serum of patients with benign and malignant diseases.

High quantities of mono- and oligonucleosomes circulate in the blood of patients with malignant tumors. For their direct quantification in serum, we modified the Cell Death Detection(plus)-ELISA for its application in liquid materials. We examined sera samples from 590 persons, including 418 patients with malignant tumors, 109 patients with benign diseases and 63 healthy persons. We also observed the kinetics of the concentration of nucleosomes in serum samples from 20 patients undergoing chemotherapy and from 16 patients undergoing radiotherapy. Sera of patients with malignant tumors contained considerably higher concentrations of nucleosomes (mean = 350 arbitrary units [AU], median = 190 AU) compared with those of healthy persons (mean = 36 AU, median = 24 AU; p = 0.0001) and patients with benign diseases (mean = 264 AU, median = 146 AU; p = 0.072). Concerning the follow-up investigations, the concentration of nucleosomes in serum increased 24-72 hr after the first application of chemotherapy and 6-24 hr after the start of radiotherapy. A subsequent decrease was often correlated with regression of the tumor. In patients undergoing chemotherapy, an increase in the baseline values of circulating nucleosomes >50%, which were determined before each new therapeutic cycle, was correlated with progression of disease; all patients with disease regression showed a decrease >50% of the baseline values. In patients undergoing radiotherapy, an early decrease of the nucleosomal concentration (< or = 1 day after the initial peak during therapy) to low minimum levels (< or = 100 AU) correlated with good clinical outcome; a late decrease (>1 day) to higher minimum levels (>100 AU) was associated with a worse clinical outcome. Thus, the concentration of nucleosomes in serum might be a useful tool for monitoring the biochemical response during antitumor therapy, especially for the early estimation of therapeutic efficacy.

The significance of CEA, CA19-9 and CA72-4 in the detection of colorectal carcinoma recurrence.

UNLABELLED: The significance of CEA, CA19-9 and CA72-4 was evaluated the for early detection of disease recurrence, on the basis of retrospective evaluation of routine data in patients with colorectal carcinoma. They also considered the dependence of the results of these data analyses on the definition of groups of patients, both with no evidence of disease (NED) and with recurrence of disease (RD). PATIENTS AND METHODS: From January 1994 to March 1999 serum levels of CEA, CA19-9 and CA72-4 were determined in the follow-up of 517 patients with colorectal cancer and compared with the retrospectively confirmed clinical status of the patients. RESULTS: CEA and CA19-9 showed comparable sensitivities in the detection of locoregional recurrence of colorectal carcinoma, whilst the sensitivity of CA72-4 was considerably lower. CEA is an optimal marker for detecting distant metastases, in particular liver metastases, since its sensitivity considerably exceeds the sensitivities of the other two monitored markers. CONCLUSION: Using routine data required detailed analysis and clear definitions of groups of patients with NED and RD. The following conclusions for the evaluation of data were drawn from this analysis: a) Tumor marker cut-off values and sensitivities related to 95% specificity of remission values depended strongly on the given definition of the groups of patients with NED and RD. b) The patient group with NED is best characterized as the group of patients who never developed progression and where all the values which were assessed within a period shorter than six months from the end of therapy and follow-up, or less than six months before progression, death, or before the last marker assessment in the patient, were excluded. c) For the optimal characterisation of the group of patients with RD it is recommended only to consider values obtained during the first progression, after the period of complete post-operative or post-therapeutic remission. d) These conclusions refer not only to routine data, where this correction represents a condition for reliable evaluation, but also to any research done, since they ensure complete homogeneity of the group and mutual comparability of the results.

Apoptosis in serum of patients with solid tumours.

Apoptosis, which occurs in highly proliferating tumours spontaneously or during anticancer therapy, many lead to an elevated concentration of circulating nucleosomes in blood. In order to quantify the concentration of nucleosomes, we used the Cell Death Detectionplus-ELISA (CDDE) (Boehringer Mannheim, Germany) based on antibodies against histone and DNA, adapted it to the demands of liquid materials and standardized test performance and handling of serum. Furthermore, we investigated serum samples of 185 patients with solid tumours (additionally 24, treated with radio- or chemotherapy), 30 with acute inflammations and 50 healthy persons. Many patients with tumours (78%) and inflammations (77%) showed higher concentrations of serum-nucleosomes (> 100 AU), whereas 96% of all healthy persons had low values (< 100 AU). Follow up-studies revealed an early peak after initiation of therapy and correlated to the clinical outcome. The concentration of nucleosomes is a sensitive marker of cell death and could be used for monitoring the efficacy of antitumour therapy.