Using the VALGENT-3 framework to assess the clinical and analytical performance of the RIATOL qPCR HPV genotyping assay

Background and objective: The VALGENT framework is developed to assess the clinical performance of HPV tests that offer genotyping capability. Samples from the VALGENT-3 panel are used to identify an optimal viral concentration threshold for the RIATOL qPCR HPV genotyping assay (RIATOL qPCR) to assure non-inferior accuracy to detect high-grade cervical intraepithelial neoplasia (CIN), compared to Qiagen Hybrid Capture 2 (HC2), a standard comparator test validated for cervical cancer screening. Study design: The VALGENT-3 panel comprised 1300 samples from women participating in the Slovenian cervical cancer screening programme, enriched with 300 samples from women with abnormal cytology. In follow-up, 126 women were diagnosed with CIN2+ (defined as diseased) and 1167 women had two consecutive negative Pap smears (defined as non-diseased). All 1600 samples were analyzed with the RIATOL qPCR. Viral concentration was expressed as viral log10 of the number of copies/ml. A zone of viral concentration cut-offs was defined by relative ROC analysis where the sensitivity and specificity were not inferior to HC2. Results: The RIATOL qPCR had a sensitivity and specificity for CIN2+ of 97.6% (CI: 93.2-99.5%) and 85.1% (CI: 82.9-87.1%), respectively, when the analytical cut off was used. At a cut off of 6.5, RIATOL qPCR had a sensitivity of 96.0% (CI: 91.0-98.7%) and a specificity of 89.5% (87.6-91.2%). At optimized cut off, accuracy of the qPCR was non-inferior to the HC2 with a relative sensitivity of 1.00 [CI: 0.95-1.05 (p= 0.006)] and relative specificity of 1.00 [CI: 0.98-1.01 (p= 0.0069)]. Conclusions: The RIATOL qPCR has a high sensitivity and specificity for the detection of CIN2+. By using a fixed cut-off based on viral concentration, the test is non-inferior to HC2. HPV tests that provide viral concentration measurements or other quantifiable signals allow flexibility to optimize accuracy required for cervical cancer screening

Elevated levels of the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in sera of cancer patients.

The concentration of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml(-1) for bFGF and 500 pg ml(-1) for VEGF, were identified as 'elevated'. One measurement during follow-up was included into the analysis per patient. For 19 treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic factors. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with 15% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic factors compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas

International Delegations and the Values of Federalism

Inland water sediments receive large quantities of terrestrial organic matter(1-5) and are globally important sites for organic carbon preservation(5,6). Sediment organic matter mineralization is positively related to temperature across a wide range of high-latitude ecosystems(6-10), but the situation in the tropics remains unclear. Here we assessed temperature effects on the biological production of CO2 and CH4 in anaerobic sediments of tropical lakes in the Amazon and boreal lakes in Sweden. On the basis of conservative regional warming projections until 2100 (ref. 11), we estimate that sediment CO2 and CH4 production will increase 9-61% above present rates. Combining the CO2 and CH4 as CO2 equivalents (CO(2)eq; ref. 11), the predicted increase is 2.4-4.5 times higher in tropical than boreal sediments. Although the estimated lake area in low latitudes is 3.2 times smaller than that of the boreal zone, we estimate that the increase in gas production from tropical lake sediments would be on average 2.4 times higher for CO2 and 2.8 times higher for CH4. The exponential temperature response of organic matter mineralization, coupled with higher increases in the proportion of CH4 relative to CO2 on warming, suggests that the production of greenhouse gases in tropical sediments will increase substantially. This represents a potential large-scale positive feedback to climate change

Arterio-venous gradients of IL-6, plasma and serum VEGF and D-dimers in human cancer

The circulating angiogenic factors vascular endothelial growth factor-A, interleukin-6 and the fibrin D-dimer fragment were measured in the mesenteric vein, the uterine vein, as well as in peripheral venous and arterial samples in 21 randomly selected patients with operable colorectal, ovarian and cervical carcinoma. In addition, immunohistochemistry for vascular endothelial growth factor-A and interleukin-6 was performed on colorectal tumours of such patients. Serum and plasma vascular endothelial growth factor-A were not significantly elevated in the vein draining the tumours, despite tumour cell expression of vascular endothelial growth factor-A. Serum vascular endothelial growth factor-A is therefore not all tumour-derived. In contrast, serum interleukin-6 was highly elevated in the draining veins in agreement with expression of interleukin-6 in the cytoplasm of tumour cells. In the megakaryoblastic cell line MEG-01, the expression of vascular endothelial growth factor-A was found to be regulated by interleukin-6. Thus, the higher platelet vascular endothelial growth factor-A load resulting in higher serum vascular endothelial growth factor levels in cancer patients may partly result from an interleukin-6 mediated up-regulation of the expression of vascular endothelial growth factor-A in the precursor of the platelet, i.e. the megakaryocyte. We also confirmed by immunohistochemistry that platelets adhere and aggregate on tumour endothelium. We propose that interleukin-6 indirectly promotes tumour angiogenesis through its up-regulation of the vascular endothelial growth factor-A load in platelets. In addition, the correlations found between peripheral venous interleukin-6 and peripheral venous fibrinogen and D-dimers levels, and the high D-dimer levels found in the draining vein of the tumour, in agreement with fibrin deposits found in the tumour stroma, suggest an important role for interleukin-6 in extra-vascular fibrinogen metabolism. Our results suggest a pivotal role for interleukin-6 in the intrinsic link between haemostasis and angiogenesis. This might be of importance in the development of anti-angiogenic agents based on interference with haemostasis

Real-time RT–PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results

Vascular endothelial growth factor A (VEGF-A) mRNA expression levels decrease after menopause in normal breast tissue but not in breast cancer lesions

We hypothesized that the regulation of microvascular functions and angiogenesis in breast tissue, a well known target of ovarian steroid action, is dependent on the hormonal exposure of the breast. Relative expression levels of VEGF-A (vascular endothelial growth factor A), a putative key regulator of angiogenesis in breast cancer, were analysed in the tumour and the adjacent non-neoplastic breast tissue of 19 breast cancer patients by quantitative reverse transcriptase polymerase chain reaction. In non-neoplastic breast specimens the expression levels of all detected VEGF-A-isoforms (189, 165, 121) were significantly higher in premenopausal compared to post-menopausal women (P = 0.02) and were inversely correlated with the patient's age (P = 0.006). In contrast, in cancerous tissues menopausal status had no influence on VEGF-A-expression levels. Benign and malignant tissues exhibited a similar expression pattern of VEGF-A-isoforms relative to each other. Thus, the regulation of the vasculature in normal breast tissue, as opposed to breast cancer tissue, appears to be hormonally dependent. Endogenous and therapeutically used hormonal steroids might, therefore, cause clinically relevant changes of the angiogenic phenotype of the human breast. © 1999 Cancer Research Campaig