Jacques B. de Kok

Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, β-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, β-2-microglobin, β-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.

Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT‐PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real‐time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle‐invasive tumors. We conclude that standardized real‐time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder. Int. J. Cancer 87:217–220, 2000.

Effects of Blood-Processing Protocols on Cell-free DNA Quantification in Plasma

Recently, qualitative analysis of cell-free DNA in blood plasma has attracted much interest for the diagnosis of cancer, fetal gender, Rhesus D status, and inherited disorders (1)(2). Other studies have shown that quantification of total plasma DNA may indicate fetal chromosomal aneuploidies and pregnancy-associated complications or the presence/recurrence of cancer (1)(3)(4). Irrespective of the type of study (qualitative or quantitative DNA analysis), it is important that cell-free plasma DNA is not contaminated with cellular DNA that interferes with analysis and accurate quantification. To prevent cellular DNA contamination, Chiu et al. (5) emphasized the need of preanalytical standardization of blood-processing protocols. The authors note that after centrifugation of blood at a low speed (800g), the amount of isolated DNA from plasma is affected by the presence of cells that remain in the plasma fraction. An additional centrifugation step (16 000g) or filtering of the plasma is necessary to produce absolutely cell-free plasma DNA. Because most laboratories, including ours, centrifuge blood at relatively low speeds (800–1500g) and store plasma without additional treatment, these results may have a serious impact on the usefulness of previously collected plasma samples for retrospective DNA analysis/quantification. To confirm the results of Chiu et al. (5), we collected EDTA blood in 7-mL Vacutainer Tubes from 18 healthy …

Prediction of recurrence in Ta urothelial cell carcinoma by real-time quantitative PCR analysis: A microarray validation study

Accurate prediction of tumor recurrence in patients with superficial urothelial cell carcinoma (UCC) might result in a significant reduction of invasive follow‐up cystoscopies. A recent study identified a panel of 26 genes from a large cDNA microarray analysis of bladder tumors that discriminated between early‐ and late‐recurring patients with superficial Ta tumors (Dyrskjøt et al., Nat Genet 2003;33:90–6). We aimed to validate this panel of genes in 44 primary Ta UCCs (23 and 21 tumors from patients with short or prolonged recurrence‐free periods, respectively), by real‐time quantitative PCR. Statistical analysis showed marginal significant different mRNA expression levels between the 2 patient groups. To evaluate a supplementary effect of genes for the identification of patients with short or prolonged recurrence‐free intervals, forward logistic regression analysis was applied. This revealed that a combination of the expression profiles of the genes HNRPK, LTB4DH and ANP32B resulted in the best performance, although the combination only marginally increased the predictive value of HNRPK alone. Comparing the receiver‐operating‐characteristic curves for HNRPK expression among patients with short or prolonged recurrence‐free periods, revealed an area under the curve of 0.696 (95% CI, 0.537–0.855). Using the median HNRPK expression level as cut‐off, a sensitivity of 69.6% and a specificity of 71.4% were obtained for the identification of patients with short or prolonged recurrence‐free periods, respectively. In conclusion, we were not able to confirm the microarray gene expression pattern of the 26 genes shown by Dyrskjøt et al. The discovery of accurate recurrence predictive markers, therefore, remains a challenge.

PCR-Restriction Fragment Length Polymorphism Method to Detect the X/Y Polymorphism in the Promoter Site of the Mannose-binding Lectin Gene

Mannose-binding lectin (MBL), a pattern-recognition molecule produced by the liver and present in serum, is an important player in the innate immune system. MBL acts by binding various carbohydrate structures on microbial surfaces, after which it activates the complement system via the lectin pathway. In addition, MBL can promote direct opsonophagocytosis of microorganisms and modulate diverse inflammatory mediators (1). Deficiency of MBL was first identified in association with a common defect of opsonization in children. Additional studies have identified MBL deficiency as a risk factor for diverse infectious diseases (1). In addition, MBL deficiency has been found to be associated with certain autoimmune diseases (1) and, recently, atherosclerosis (2). MBL deficiency is caused by mutations in the coding and promoter regions of the MBL gene, which have a profound effect on plasma concentrations of the MBL protein. Three point mutations have been found in …

CDC91L1 (PIG-U) mRNA expression in urothelial cell carcinomas.

CDC91L1 (PIG‐U) was recently discovered as a new oncogene in human bladder cancer and showed mRNA overexpression in 36% of primary bladder tumor tissues compared to normal urothelium. We further investigated CDC91L1 mRNA expression in 8 bladder cancer cell lines, 14 normal bladder tissues and 42 urothelial cell carcinomas by real‐time quantitative PCR. The prognostic value of CDC91L1 mRNA expression was also investigated. Surprisingly, only one (2.4%) tumor tissue showed overexpression compared to normal urothelium. No significant relationship of CDC91L1 mRNA expression with increasing pathologic stage (p = 0.962) or grade (p = 0.557) was observed. Median normalized CDC91L1 mRNA expression values were 0.19 for superficial tumors (n = 21) and 0.18 for invasive tumors (n = 21). Grade I, grade II and grade III tumors had median normalized expression values of 0.26, 0.18 and 0.33, respectively. CDC91L1 mRNA expression level was not indicative of early tumor recurrence (log rank p = 0.1629), tumor progression (log rank p = 0.9307) or overall and disease‐specific survival (log rank p = 0.9193 and 0.4710, respectively). Our results suggest, in contrast to those of Guo et al. (Nat Med 2004;10:374–81), that the oncogene CDC91L1 is not overexpressed at the mRNA level in urothelial cell carcinomas and cannot be used to predict the course of the disease.