DNA: stabiel en veranderlijk

DNA en kanker zijn onlosmakelijk met elkaar verbonden. Kanker is een ziekte van het DNA. Het zijn veranderingen in het DNA die van een gewone cel een kankercel maken. In de Verenigde Staten van Amerika is kanker inmiddels de belangrijkste doodsoorzaak|1. De sterfte aan hart en vaatziekten is de laatste decennia hard omlaag gegaan. Bij kanker gaat dat helaas beduidend minder snel. Waarom is dat het geval en wat maakt kanker zo’n lastig probleem? Ik zal proberen u in grote lijnen een beeld te schetsen van de huidige stand van de wetenschap met betrekking tot de moleculaire achtergronden van tumorontwikkeling en de problemen die er zijn in het onderzoek aan kanker. Rede, in verkorte vorm uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht Moleculaire Pathologie van Tumorontwikkeling aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam op 10 oktober 2008

CDNA cloning and mRNA expression of the six mouse insulin-like growth factor binding proteins

The insulin-like growth factor binding proteins (IGFBPs) comprise a family of six distinct proteins which modulate insulin-like growth factor action. We have isolated cDNAs encoding the six mouse IGFBPs (mIGFBPs). In addition, we studied the mRNA expression of the six mIGFBPs during development and in various adult tissues. Our results show that each of the six mIGFBPs is highly homologous to their human and rat counterparts, whereas only the N and C terminal ends are conserved between the six mIGFBPs. Northern blotting revealed that mIGFBP-2, -3, -4 and -5 genes are already expressed at gestational day 11.5, suggesting a role for these mIGFBPs in embryonal development. In liver, a peak of mIGFBP-1 mRNA expression was found around birth, suggesting a function for mIGFBP-1 in the newborn mouse. Finally, tissue-specific expression of the six mouse IGFBP genes was observed in adult tissues suggesting different roles or modes of actions in adult life

Insulin-like growth factor binding protein-2, 28 kDa an 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles, obtained from normal and polycystic ovaries

In order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with regular menstrual cycles and of subjects with cycle abnormalities and polycystic ovaries (diagnosed as polycystic ovary syndrome (PCOS)) and analyzed by Western ligand blotting. IGFBPs of 43 kDa, 37 kDa, 31 kDa, a doublet around 28 kDa and a minor band of 24 kDa were detected in follicle fluid of normal non-dominant (size 4) follicles of both regularly menstruating women and PCOS patients. The 43 and 37 kDa IGFBPs could be identified as IGFBP-3 and the 31 kDa IGFBP as IGFBP-2, whereas the 28 kDa IGFBP could not be identified as IGFBP-1, all by immunoblotting techniques. A dramatic decrease in IGFBP-2, the 28 kDa and 24 kDa IGFBPs was observed in follicular fluid of dominant follicles (size > 10 mm) of both regular menstruating individuals and one PCOS patient as compared with follicular fluid of normal non-dominant or atretic follicles. These observations indicate that the PCOS follicle may not be different from normal with respect to IGFBP profiles. Furthermore, these results suggest that at least one of these IGFBPs might be involved in human folliculogenesis

Modulation of intra-epithelial expansion of human T24 bladder-carcinoma cells in murine urothelium by growth factors and extracellular-matrix components

The high recurrence rate of bladder cancer is probably due to an efficient repopulation of the bladder by residual transformed cells after resection of the tumour. However, the regenerating capacity of the normal urothelial cells is very high. To study the balance between regenerating normal urothelium and outgrowth of transformed urothelial cells, we recently developed an in vitro co-cultivation model. With this model system we studied the effects of growth factors and extracellular-matrix components on the intra-epithelial expansion of human T24 bladder-carcinoma cells in primary mouse-bladder explants. Exposure of the cultures to acidic fibroblast growth factor (aFGF) and laminin led to a dramatic increase in the number of invasive T24 cells into the primary urothelium. Epidermal growth factor (EGF) and collagen types I and IV counteracted the infiltration of individual T24 cells. EGF, aFGF, laminin and collagen types I and IV did not directly affect the migration and proliferation of T24 cells. Apparently, the efficacy of invasion of transformed urothelial cells into primary urothelium is not only dependent on the intrinsic characteristics of the transformed cells, but can be influenced to a considerable extent by exogenous components exerting their influence on the normal urothelium. The clinical relevance of this observation needs to be studied further

FGFR3 and P53 characterize alternative genetic pathways in the pathogenesis of urothelial cell carcinoma

Fibroblast growth factor receptor 3 (FGFR3) and P53 mutations are frequently observed in bladder cancer. We here describe the distribution of FGFR3 mutations and P53 overexpression in 260 primary urothelial cell carcinomas. FGFR3 mutations were observed in 59% and P53 overexpression in 25%. Interestingly, FGFR3 and P53 alterations were mutually exclusive, because they coincided in only 5.7% of tumors. Consequently, we propose that they characterize two alternative genetic pathways in urothelial cell carcinoma pathogenesis. The genetic alterations were reflected in the pathology and the clinical outcome, i.e., FGFR3 mutations were found in low-stage/-grade tumors and were associated with a favorable disease course, whereas P53 alterations were tied to adverse disease parameters

Targeted Therapy in Metastatic Bladder Cancer: Present Status and Future Directions

The recommended treatment for metastatic urothelial carcinoma (mUC) patients is platinum-based chemotherapy. Although initial response rates are moderate, the vast majority of patients experience a relapse due to chemoresistance and eventually succumb to their disease. Furthermore, platinum-based chemotherapy is toxic and approximately 30% of mUC patients are unfit for chemotherapy. Thus, there is a clear unmet need for novel, more efficacious treatment options in mUC with a safer toxicity profile. To propel the advancement of novel treatment options, we present a summary of key signaling pathways and molecular mechanisms that are known to be involved in bladder cancer tumorigenesis with a focus on promising candidate druggable molecular targets and innovative targeted therapies currently under clinical investigation. Targetable alterations were mainly described in fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (ErbB) tyrosine kinase receptor (RTK) families, downstream pathways, and chromatin remodelers, which are major bladder cancer driver genes. Drugs targeting the FGFR family members are emerging as personalized treatment options for selected mUC patients with tumor-specific FGFR alterations. The pan-FGFR inhibitor, erdafitinib, was first-in-class to receive U.S. Food and Drug Administration (FDA) approval in 2019, while inhibitors of ErbB family members have shown less potential. Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that deliver cytotoxic drugs in close proximity to cancer cells by targeting RTKs or other transmembrane proteins. Enfortumab vedotin is the first-in-class ADC that was FDA approved for the treatment of locally advanced or mUC in 2019

An in vitro model of urothelial regeneration: Effects of growth factors and extracellular matrix proteins

Although the cellular turnover of resting urothelium is very low, its regenerative capacity is known to be outstanding. In organotypic mouse urothelial cultures closely mimicking the differentiation and multilayering of normal urothelium, we examined the cell biological mechanisms underlying urothelial regeneration and the specific role of growth factors and several extracellular matrix (ECM) components. Exposure to epidermal growth factor (EGF) and acidic fibroblast growth factor (aFGF) and culture on laminin resulted in enhanced expansion of the urothelium. Microscopy and assessment of proliferative activity revealed that enhanced urothelial expansion due to EGF could be attributed to increased proliferative activity and an increase in cell numbers, whereas aFGF-stimulated expansion must be considered the consequence of increased cellularity and migration. Laminin-enhanced urothelial expansion was shown to be the result of spreading of the entire urothelial organotypic culture. This was associated with a considerable decrease in the number of cell layers. A synergistic effect of growth factors and laminin was not found. This organotypic urothelial cell culture model seems to be very useful in studying strategies to improve urothelial regeneration

A noninvasive multi-analyte diagnostic assay: Combining protein and DNA markers to stratify bladder cancer patients

Purpose: The authors recently reported the development of a noninvasive diagnostic assay using urinary matrix metalloproteinases (MMPs) as monitors of disease-free status and bladder cancer in high-risk populations. Using an approach called clinical intervention determining diagnostic (CIDD), they identified with high confidence those patients who could be excluded from additional intervention. To maximize performance, MMPs were combined with DNAbased markers and CIDD was applied to a population of patients undergoing monitoring for recurrence. Patients and methods: Urine samples were obtained from 323 patients, 48 of whom had a recurrence and 275 of whom did not have cancer upon cytoscopic evaluation. Twist1 and Nid2 methylation status was determined using methylation-specific polymerase chain reaction, FGFR3 mutational status by quantitative PCR, and MMP levels by enzyme-linked immunosorbent assay. Results: Using a combination of these DNA and protein markers, the authors identified with high confidence (97% negative predicted value) those patients who do not have cancer. Cutoffs were adjusted such that at 92% sensitivity, 51% of disease-free patients might be triaged from receiving further tests. Conclusion: The multi-analyte diagnostic readout assay described here is the first to combine protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of patients undergoing bladder cancer recurrence screening increase the sensitivity and negative predictive value at an established MMP protein cutoff. This noninvasive urinary diagnostic assay could lead to the more efficient triage of patients undergoing recurrence monitoring

A reported 20-gene expression signature to predict lymph node-positive disease at radical cystectomy for muscle-invasive bladder cancer is clinically not applicable

Background Neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) provides a small but significant survival benefit. Nevertheless, controversies on applying NAC remain because the limited benefit must be weight against chemotherapy-related toxicity and the delay of definitive local treatment. Therefore, there is a clear clinical need for tools to guide treatment decisions on NAC in MIBC. Here, we aimed to validate a previously reported 20-gene expression signature that predicted lymph node-positive disease at radical cystectomy in clinically node-negative MIBC patients, which would be a justification for upfront chemotherapy. Methods We studied diagnostic transurethral resection of bladder tumors (dTURBT) of 150 MIBC patients (urothelial carcinoma) who were subsequently treated by radical cystectomy and pelvic lymph node dissection. RNA was isolated and the expression level of the 20 genes was determined on a qRT-PCR platform. Normalized Ct values were used to calculate a risk score to predict the presence of node-positive disease. The Cancer Genome Atlas (TCGA) RNA expression data was analyzed to subsequently validate the results. Results In a univariate regression analysis, none of the 20 genes significantly correlated with nodepositive disease. The area under the curve of the risk score calculated by the 20-gene expression signature was 0.54 (95% Confidence Interval: 0.44-0.65) versus 0.67 for the model published by Smith et al. Node-negative patients had a significantly lower tumor grade at TURBT (p = 0.03), a lower pT stage (p<0.01) and less frequent lymphovascular invasion (13% versus 38%, p<0.01) at radical cystectomy than node-positive patients. In addition, in the TCGA data, none of the 20 genes was differentially expressed in node-negative versus node-positive patients. Conclusions We conclude that a 20-gene expression signature developed for nodal staging of MIBC at radical cystectomy could not be validated on a qRT-PCR platform in a large cohort of dTURBT specimens

Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine

PURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of UCC in voided urine. EXPERIMENTAL DESIGN: In a prospective setting, 59 UCC tissues and matched urine samples were obtained, and subjected to MA (23 markers) and FGFR3 mutation analysis (exons 7, 10, and 15). In each case, a clinical record with tumor and urine features was provided. Fifteen patients with a negative cystoscopy during follow-up served as controls. RESULTS: A mutation in the FGFR3 gene was found in 26 (44%) UCCs of which 22 concerned solitary pTaG1/2 lesions. These mutations were absent in the 15 G3 tumors. For the 6 cases with leukocyturia, 46 microsatellite alterations were found in the tumor. Only 1 of these was also detected in the urine. This was 125 of 357 for the 53 cases without leukocyte contamination. The sensitivity of MA on voided urine was lower for FGFR3-positive UCC (15 of 21; 71%) as compared with FGFR3 wild-type UCC (29 of 32; 91%). By including the FGFR3 mutation, the sensitivity of molecular cytology increased to 89% and was superior to the sensitivity of morphological cytology (25%) for every clinical subdivision. The specificity was 14 of 15 (93%) for the two (molecular and morphological) cytological approaches. CONCLUSIONS: Molecular urine cytology by MA and FGFR3 mutation analysis enables a highly sensitive and specific detection of UCC. The similarity of molecular profiles in tumor and urine corroborate their clonal relation