Petra Stieber

Comparison of CT colonography, colonoscopy, sigmoidoscopy and faecal occult blood tests for the detection of advanced adenoma in an average risk population

Background and aims: This prospective trial was designed to compare the performance characteristics of five different screening tests in parallel for the detection of advanced colonic neoplasia: CT colonography (CTC), colonoscopy (OC), flexible sigmoidoscopy (FS), faecal immunochemical stool testing (FIT) and faecal occult blood testing (FOBT). Methods: Average risk adults provided stool specimens for FOBT and FIT, and underwent same-day low-dose 64-multidetector row CTC and OC using segmentally unblinded OC as the standard of reference. Sensitivities and specificities were calculated for each single test, and for combinations of FS and stool tests. CTC radiation exposure was measured, and patient comfort levels and preferences were assessed by questionnaire. Results: 221 adenomas were detected in 307 subjects who completed CTC (mean radiation dose, 4.5 mSv) and OC; 269 patients provided stool samples for both FOBT and FIT. Sensitivities of OC, CTC, FS, FIT and FOBT for advanced colonic neoplasia were 100% (95% CI 88.4% to 100%), 96.7% (82.8% to 99.9%), 83.3% (95% CI 65.3% to 94.4%), 32% (95% CI 14.9% to 53.5) and 20% (95% CI 6.8% to 40.7%), respectively. Combination of FS with FOBT or FIT led to no relevant increase in sensitivity. 12 of 45 advanced adenomas were smaller than 10 mm. 46% of patients preferred CTC and 37% preferred OC (p<0.001). Conclusions: High-resolution and low-dose CTC is feasible for colorectal cancer screening and reaches sensitivities comparable with OC for polyps >5 mm. For patients who refuse full bowel preparation and OC or CTC, FS should be preferred over stool tests. However, in cases where stool tests are performed, FIT should be recommended rather than FOBT.

Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients

Purpose: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a) evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b) compare the prognostic significance of VEGF isoforms; and (c) analyze the combined effects of HER-2/neu and VEGF on clinical outcome. Experimental Design: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF121–206 ELISA uses antibodies that bind to VEGF121 and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF165–206 ELISA uses antibodies that bind to VEGF165 and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF121–206 and VEGF165–206 were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Results: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF121–206 and VEGF165–206 expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (χ2 test: P < 0.001 for both VEGF121–206 and VEGF165–206). VEGF121–206 and VEGF165–206 demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF121–206, P = 0.0068; VEGF165–206, P = 0.0046; multivariate analysis: VEGF121–206, P = 0.1475; VEGF165–206, P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF121–206 and VEGF165–206 were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF121–206, P = 0.0003; VEGF165–206, P = 0.0038; multivariate analysis: VEGF121–206, P = 0.0103; VEGF165–206, P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF121–206, P = 0.0280; VEGF165–206, P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF121–206/VEGF165–206 results in additional prognostic information for survival (VEGF121–206, P = 0.0133; VEGF165–206, P = 0.0092). Conclusion: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Tumor Markers in Breast Cancer – European Group on Tumor Markers Recommendations

Recommendations are presented for the routine clinical use of serum and tissue-based markers in the diagnosis and management of patients with breast cancer. Their low sensitivity and specificity preclude the use of serum markers such as the MUC-1 mucin glycoproteins (CA 15.3, BR 27.29) and carcinoembryonic antigen in the diagnosis of early breast cancer. However, serial measurement of these markers can result in the early detection of recurrent disease as well as indicate the efficacy of therapy. Of the tissue-based markers, measurement of estrogen and progesterone receptors is mandatory in the selection of patients for treatment with hormone therapy, while HER-2 is essential in selecting patients with advanced breast cancer for treatment with Herceptin (trastuzumab). Urokinase plasminogen activator and plasminogen activator inhibitor 1 are recently validated prognostic markers for lymph node-negative breast cancer patients and thus may be of value in selecting node-negative patients that do not require adjuvant chemotherapy.

Soluble MICA in malignant diseases.

The immunoreceptor NKG2D activates natural killer cells and costimulates CD8 T cells. The MHC class I–related MICA molecules are ligands of NKG2D and are expressed on malignant, but not on normal, cells. As NKG2D plays an important role in the immunosurveillance of tumors, studies suggest that release of MICA from cancer cells constitutes an immune escape mechanism that systemically impairs antitumor immunity. Here, we investigated the potential of soluble MICA (sMICA) as a marker in cancer. Analysis of sMICA in sera of 512 individuals revealed significantly (p < 0.0001) higher levels in patients with various malignancies (n = 296, median 161 pg/ml) than in healthy individuals (n = 62, median <30 pg/ml). Patients with benign diseases (n = 154, median 84 pg/ml) exhibited intermediate sMICA levels. In cancer patients, elevated sMICA levels correlated significantly with cancer stage and metastasis (p = 0.015 and p = 0.007, respectively). While release of MICA is thought to impair tumor immunity, determination of sMICA levels may provide useful additional information in the diagnosis and staging of cancer.

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 Detectionplus‐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 (≤1 day after the initial peak during therapy) to low minimum levels (≤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.

Clinical use of circulating nucleosomes

Nucleosomes, complexes of DNA and histone proteins, are released from dying and stressed cells into the blood circulation. Concentrations of circulating nucleosomes in plasma and serum are frequently found to be elevated in various cancers, and also in such acute conditions as stroke, trauma, and sepsis as well as in autoimmune diseases. The first part of this review focuses on the structural and functional properties of nucleosomes, the potential sources of nucleosome release into the circulation, the metabolism of circulating nucleosomes, and their pathophysiological role in disease. It goes on to describe the relevance of circulating nucleosomes in the diagnosis and prognosis of non-malignant conditions such as sepsis, stroke, and autoimmune disease. Finally, it describes the clinical value of nucleosomes in the diagnosis, staging, prognosis, and monitoring of therapy in cancer; in particular, their potential as a new diagnostic tool for the early estimation of response to cytotoxic cancer therapy is emphasized.

Methylation of Serum DNA Is an Independent Prognostic Marker in Colorectal Cancer

Purpose: Aberrant CpG island hypermethylation is a feature of a subgroup of colorectal cancers, which can be detected in the serum of affected patients. This study was designed to identify methylation targets with prognostic significance in the serum of patients with colorectal cancer. Experimental Design: In a gene evaluation set consisting of sera from 24 patients with local colorectal cancers, 14 with metastasized disease, and 20 healthy controls, the genes HPP1/TPEF, HLTF, and hMLH1 were identified as potential serum DNA methylation markers. These genes were further analyzed in a test set of sera of 104 patients with colorectal cancer. Results: Methylation of HLTF, HPP1/TPEF, and hMLH1 was found to be significantly correlated with tumor size, and methylation of HLTF and HPP1/TPEF was significantly associated with metastatic disease and tumor stage. Moreover, methylation of HPP1/TPEF was also associated with serum carcinoembryonic antigen. The prognostic relevance of methylation of these genes was tested in pretherapeutic sera of 77 patients with known follow-up. Patients with methylation of HPP1/TPEF or HLTF were found to have unfavorable prognosis (P = 0.001 and 0.008). In contrast, serum methylation of hMLH1 was not associated with a higher risk of death. Multivariate analysis showed methylated HPP1 and/or HLTF serum DNA to be independently associated with poor outcome and a relative risk of death of 3.4 (95% confidence interval, 1.4-8.1; P = 0.007). Conclusions: These data show that the methylation status of specific genes in the serum of patients with colorectal cancer has the potential to become a pretherapeutic predictor of outcome.

Nucleosomes in serum as a marker for cell death.

Abstract 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:3.0–4.1%) 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 °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).

Circulating Nucleosomes Predict the Response to Chemotherapy in Patients with Advanced Non–Small Cell Lung Cancer

Purpose: We investigated the potential of circulating, nucleosomal DNA for the early prediction of the efficacy of chemotherapy in patients with advanced lung cancer. Experimental Design: In serum of 212 patients with newly diagnosed non–small cell lung cancer (stages III and IV) undergoing chemotherapy, nucleosomes (ELISA, Roche) were measured at days 1, 3, 5, and 8 of the first cycle and before each new therapeutic cycle. Additionally, carcinoembryonic antigen and cytokeratin 19 fragments (CYFRA 21–1; Elecsys, Roche) were determined before each cycle. The therapeutic success was classified by computed tomography before start of the third cycle according to the World Health Organization criteria. Results: In univariate analysis, responders (patients with remission) showed significantly (P < 0.05) lower values for the area under the curve of days 1 to 8 (AUC 1–8) of nucleosomes, the pretherapeutic baseline values of cycle 2 (BV2) and cycle 3 (BV3) of nucleosomes, and higher decreases of the baseline values from cycle 1 to 2 (BV1–2) and from cycle 1 to 3 (BV1–3) compared with nonresponders (patients with stable or progressive disease). Additionally, CYFRA 21–1 (BV1, BV2, BV3, BV1–2, BV1–3) and carcinoembryonic antigen (BV1–2) discriminated significantly between both groups. In multivariate analysis including all parameters available until end of the first therapeutic cycle, nucleosomes (AUC 1–8), CYFRA 21–1 (BV1), stage, and age were independent predictors of therapy response with nucleosomes (AUC 1–8) having the strongest impact. Conclusion: Circulating nucleosomes in combination with oncological biomarkers are valuable for the early estimation of the efficacy of chemotherapy in patients with lung cancer.

Prognostic and therapeutic significance of carbohydrate antigen 19-9 as tumor marker in patients with pancreatic cancer

In pancreatic cancer (PC) accurate determination of treatment response by imaging often remains difficult. Various efforts have been undertaken to investigate new factors which may serve as more appropriate surrogate parameters of treatment efficacy. This review focuses on the role of carbohydrate antigen 19-9 (CA 19-9) as a prognostic tumor marker in PC and summarizes its contribution to monitoring treatment efficacy. We undertook a Medline/PubMed literature search to identify relevant trials that had analyzed the prognostic impact of CA 19-9 in patients treated with surgery, chemoradiotherapy and chemotherapy for PC. Additionally, relevant abstract publications from scientific meetings were included. In advanced PC, pretreatment CA 19-9 levels have a prognostic impact regarding overall survival. Also a CA 19-9 decline under chemotherapy can provide prognostic information for median survival. A 20% reduction of CA 19-9 baseline levels within the first 8 weeks of chemotherapy appears to be sufficient to define a prognostic relevant subgroup of patients (‘CA 19-9 responder’). It still remains to be defined whether the CA 19-9 response is a more reliable method for evaluating treatment efficacy compared to conventional imaging.