Egbert R. Boevé

Active Surveillance for Low-Risk Prostate Cancer Worldwide: The PRIAS Study

BACKGROUND Overdiagnosis and subsequent overtreatment are important side effects of screening for, and early detection of, prostate cancer (PCa). Active surveillance (AS) is of growing interest as an alternative to radical treatment of low-risk PCa. OBJECTIVE To update our experience in the largest worldwide prospective AS cohort. DESIGN, SETTING, AND PARTICIPANTS Eligible patients had clinical stage T1/T2 PCa, prostate-specific antigen (PSA) ≤ 10 ng/ml, PSA density <0.2 ng/ml per milliliter, one or two positive biopsy cores, and Gleason score ≤ 6. PSA was measured every 3-6 mo, and volume-based repeat biopsies were scheduled after 1, 4, and 7 yr. Reclassification was defined as more than two positive cores or Gleason >6 at repeat biopsy. Recommendation for treatment was triggered in case of PSA doubling time <3 yr or reclassification. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Multivariate regression analysis was used to evaluate predictors for reclassification at repeat biopsy. Active therapy-free survival (ATFS) was assessed with a Kaplan-Meier analysis, and Cox regression was used to evaluate the association of clinical characteristics with active therapy over time. RESULTS AND LIMITATIONS In total, 2494 patients were included and followed for a median of 1.6 yr. One or more repeat biopsies were performed in 1480 men, of whom 415 men (28%) showed reclassification. Compliance with the first repeat biopsy was estimated to be 81%. During follow-up, 527 patients (21.1%) underwent active therapy. ATFS at 2 yr was 77.3%. The strongest predictors for reclassification and switching to deferred treatment were the number of positive cores (two cores compared with one core) and PSA density. The disease-specific survival rate was 100%. Follow-up was too short to draw definitive conclusions about the safety of AS. CONCLUSIONS Our short-term data support AS as a feasible strategy to reduce overtreatment. Clinical characteristics and PSA kinetics during follow-up can be used for risk stratification. Strict monitoring is even more essential in men with high-risk features to enable timely recognition of potentially aggressive disease and offer curative intervention. Limitations of using surrogate end points and markers in AS should be recognized. TRIAL REGISTRATION The current program is registered at the Dutch Trial Register with ID NTR1718 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1718).

Molecular Grading of Urothelial Cell Carcinoma With Fibroblast Growth Factor Receptor 3 and MIB-1 is Superior to Pathologic Grade for the Prediction of Clinical Outcome

PURPOSE Fibroblast growth factor receptor 3 (FGFR3) mutations were recently found at a high frequency in well-differentiated urothelial cell carcinoma (UCC). We investigated the relationship between FGFR3 status and three molecular markers (MIB-1, P53, and P27kip1) associated with worse prognosis and determined the reproducibility of pathologic grade and molecular variables. PATIENTS AND METHODS In this multicenter study, we included 286 patients with primary (first diagnosis) UCC. The histologic slides were reviewed. FGFR3 status was examined by polymerase chain reaction-single strand conformation polymorphism and sequencing. Expression levels of MIB-1, P53, and P27kip1 were determined by immunohistochemistry. Mean follow-up was 5.5 years (range, 0.4 to 18.4 years). RESULTS FGFR3 mutations were detected in 172 (60%) of 286 UCCs. Grade 1 tumors had an FGFR3 mutation in 88% of patient samples and grade 3 tumors in 16% of patient samples. Conversely, aberrant expression patterns of MIB-1, P53, and P27kip1 were seen in 5%, 2%, and 3% of grade 1 tumors and in 85%, 60%, and 56% of grade 3 tumors, respectively. In multivariate analysis with recurrence rate, progression, and disease-specific survival as end points, the combination of FGFR3 and MIB-1 proved independently significant in all three cases. By using these two molecular markers, three molecular grades (mGs) could be identified: mG1 (mutation; normal expression), favorable prognosis; mG2 (two remaining combinations), intermediate prognosis; and mG3 (no mutation; high expression), poor prognosis. The molecular variables were more reproducible than pathologic grade (85% to 100% v 47% to 61%). CONCLUSION The FGFR3 mutation represents the favorable molecular pathway of UCC. Molecular grading provides a new, simple, and highly reproducible tool for clinical decision making in UCC patients.

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.

Molecular Grade (FGFR3/MIB-1) and EORTC Risk Scores Are Predictive in Primary Non–Muscle-Invasive Bladder Cancer

BACKGROUND The European Organization for Research and Treatment of Cancer (EORTC) risk scores are not validated in an independent patient population. Molecular grade (mG) based on fibroblast growth factor receptor 3 (FGFR3) gene mutation status and MIB-1 expression was proposed as an alternative to pathologic grade in bladder cancer (BCa) [1]. OBJECTIVE To validate the EORTC risk score and to determine its relation to mG in a series with long-term follow-up as well as to determine reproducibility of pathologic grade and mG. DESIGN, SETTING, AND PARTICIPANTS In this multicenter study, we included 230 patients with primary non-muscle-invasive BCa (NMIBC). MEASUREMENTS Four uropathologists reviewed the slides. FGFR3 mutation status was examined by two assays. MIB-1 was assessed by immunohistochemistry. The EORTC risk scores for recurrence and progression were determined. Multivariable analyses were used to find prognostic factors. RESULTS AND LIMITATIONS Median follow-up was 8.62 yr (interquartile range: 6.6-11.8). FGFR3 mutations were significantly related to favorable disease parameters, whereas altered MIB-1 was frequently seen with pT1, high grade, and high EORTC risk scores. EORTC risk scores were significant in multivariable analyses for recurrence and progression. In multivariable analyses for progression and disease-specific survival, the mG had independent significance. The addition of mG to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7% (p<0.001; Mantel-Haenszel test). The mG (89%) was more reproducible than the pathologic grade (41-74%). CONCLUSIONS We validated the EORTC risk scores for primary NMIBC in a clinical and biomarker setting. Next to EORTC risk score, mG proved highly reproducible and predictive. Our long-term results justify an independent prospective analysis of mG and EORTC risk scores.

Calcium oxalate nephrolithiasis: Effect of renal crystal deposition on the cellular composition of the renal interstitium

Urinary calcium oxalate (CaOx) crystals and crystal agglomerates are normally harmlessly excreted, but in nephrolithiasis they are retained by tubular epithelial cells and shifted into the renal interstitium. This crystalline material induces an inflammatory response consisting of an increase in the number of interstitial cells and an expansion of the extracellular matrix. The newly arrived cells either derive from the blood or the connective tissue or they are formed by local proliferation. Identification of the cells that surround the interstitial crystals is a first step in investigating the question of whether the interstitial cells could remove the crystalline material. Therefore, we performed an immunohistochemical study on the kidneys of rats made hyperoxaluric by ethylene glycol (EG) and ammonium chloride (AC). Attention was paid to expression of the leukocyte common antigen (LCA), which identifies all types of leukocytes, the ED1 antigen, which is specific for monocytes and macrophages, and the major histocompatibility class II antigen (MHC II), which is present on dendritic cells, B lymphocytes, and activated macrophages. The results obtained were compared with those seen in two human kidney specimens with acute and chronic oxalosis. In both rat and humans, macrophages and multinucleated giant cells are the major cells that encapsulate the interstitial crystals. This similarity in response underlines the relevance of the rat nephrolithiasis model. The rat experiments showed, furthermore, that the number of interstitial crystals and the amount of biochemically measured kidney-associated oxalate both decrease with time, if the nephrolithiatic agents EG and AC are omitted from the drinking water. Further studies must clarify whether macrophages and multinucleated giant cells are able to remove the interstitial crystals and how these cells are recruited at the inflammatory site.

The pathologist's mean grade is constant and individualizes the prognostic value of bladder cancer grading.

BACKGROUND A new grading system for bladder cancer (BCa) was adopted in 2004 to reduce observer variability and provide better prognostic information. OBJECTIVE We compared the World Health Organization (WHO) 1973 and 2004 systems for observer variability and prognosis. DESIGN, SETTING, AND PARTICIPANTS Slides of 173 primary non-muscle-invasive BCa were reviewed two times by four pathologists. MEASUREMENTS Intra- and interobserver variability were assessed using κ statistics. We determined the mean grade (eg, G1/low malignant potential is 1 grade point, G2/low grade is 2 grade points) of the pathologists per grading cycle. Kaplan-Meier analyses were applied for prediction of recurrence and progression. RESULTS AND LIMITATIONS For WHO 2004 and 1973 grading, the agreement between the pathologists was 39-74% (κ: 0.14-0.58) and 39-64% (κ: 0.15-0.41), respectively. The intraobserver agreement varied from 71% to 88% (κ: 0.55-0.81). The mean grade of a pathologist was constant (difference below 0.1 grade point) irrespective of the grading system. Conversely, mean-grade differences among the pathologists were high, up to 0.7 grade point. The mean grades for the WHO 2004 system were 0.3-0.5 grade point higher than those of WHO 1973. Mean grade distinguished low and high graders among the pathologists and was strongly linked with risk of progression in each grade category. CONCLUSIONS The variation in mean grade among individual pathologists exceeded the grade shift caused by WHO 2004 grading. Knowledge of the pathologists mean grade allows a better assessment of the prognostic value of grading. Mean grade has the potential to become a tool for quality assurance in pathology.

Crystal-Cell Interaction Inhibition by Polysaccharides

PURPOSE We studied the effect of polysaccharides on interactions between calcium oxalate monohydrate (COM) crystals and cultured renal cells. MATERIALS AND METHODS Monolayers of Madin-Darby canine kidney (MDCK) cells were incubated with radiolabeled crystals in the presence of various concentrations of natural glycosaminoglycans (GAGs) and semisynthetic polysaccharides (SSPs). RESULTS While most GAGs were found to have relatively little effect, SSPs (SP54, G871 and G872) were potent inhibitors of crystal-cell association. Pretreatment of crystals, but not of cells, was similarly effective, suggesting polysaccharide-induced modification of crystal surface properties. CONCLUSIONS This result further supports the idea that SSPs, and especially G872, are of potential interest for treatment of recurrent stone disease.

An Ultrastructural Study of Experimentally Induced Microliths in Rat Proximal and Distal Tubules

Calcium oxalate stone formation was induced in rats by oral application of ethylene glycol and ammonium chloride for 4, 8 and 24 days. After each induction period, light-microscopically, birefringent crystals were seen in the tubular lumen and, intracellularly, in proximal and distal tubular cells. After a postfixation which partially removed the crystalline material crystal ghosts were seen by electron microscopy. In the lumen, crystal ghosts were observed ranging from single crystals to crystal agglomerates. The large intraluminal agglomerates were surrounded by epithelial cells and cellular debris. Both crystal types had an organic interior. In the cytoplasm of ultrastructurally changed proximal tubular cells, small (200 to 600 nm. in diameter) single crystal ghosts were present in the terminal web at the basis of the microvilli. Others were present in large vacuolar structures, with a fine granular matrix. After the prolonged microlith induction periods, such vacuolar structures were seen throughout the cell. The organic matrix of the crystal ghosts therein had acquired a more aggregated and complex structure.

Optimization of Nonmuscle Invasive Bladder Cancer Recurrence Detection Using a Urine Based FGFR3 Mutation Assay

PURPOSE FGFR3 mutations occur in 70% of nonmuscle invasive bladder tumors. Although urine based FGFR3 mutation analysis can detect recurrence, its sensitivity may be limited if samples have few or no tumor cells. We determined whether test sensitivity depends on tumor size and the time point of urine collection, and how to increase sensitivity. MATERIALS AND METHODS A total of 440 urine samples from 18 patients with a suspicious bladder lesion at cystoscopy were collected during 6 days before surgery. Eight patients (300 samples) had an FGFR3 mutant tumor, including 4 each with a tumor greater than 3 and less than 1.5 cm. Polymerase chain reaction based FGFR3 analysis was done on all tumors and urine samples. RESULTS FGFR3 mutations were detected in 257 of the 300 urine samples (86%) from patients with an FGFR3 mutant tumor. Assay sensitivity was 100% for tumors greater than 3 cm and 75% for tumors less than 1.5 cm. It increased to 100% in patients with a less than 1.5 cm tumor when samples were pooled during 24 hours. Sensitivity was not influenced by the time of urine collection. All urine samples from patients with an FGFR3 wild-type tumor were negative for FGFR3 mutation. CONCLUSIONS The sensitivity of tumor detection increased with tumor size. FGFR3 assay sensitivity depends on the number of shed tumor cells and improves by increasing urine volume. These findings suggest that there is an upper limit to the sensitivity of the FGFR3 assay when 1 urine sample is analyzed. This may also apply to other DNA or RNA based assays.

Lower Testosterone Levels With Luteinizing Hormone-Releasing Hormone Agonist Therapy Than With Surgical Castration: New Insights Attained by Mass Spectrometry

PURPOSE Androgen deprivation therapy by bilateral orchiectomy (surgical castration) or luteinizing hormone-releasing hormone agonist therapy (medical castration) is recommended for advanced or metastatic prostate cancer. Both methods aim at reducing serum testosterone concentrations to a castrate level which is currently defined as less than 50 ng/dl. The results of previous studies are based on testosterone immunoassays that have insufficient accuracy in the low range. In this study we reevaluated serum testosterone concentrations in men on androgen deprivation therapy using isotope dilution-liquid chromatography-tandem mass spectrometry, an accurate method of measuring testosterone in the castrate range. MATERIALS AND METHODS Subjects underwent surgical castration (34) or received a luteinizing hormone-releasing hormone agonist (32). Serum samples were obtained more than 3 months after surgery or initiation of luteinizing hormone-releasing hormone agonist therapy. Testosterone levels were determined using isotope dilution-liquid chromatography-tandem mass spectrometry. Dihydroepiandrosterone sulfate, androstenedione, sex hormone-binding globulin and inhibin B levels were determined. RESULTS All subjects had serum testosterone values less than 50 ng/dl and 97% had testosterone concentrations less than 20 ng/dl. Medically castrated men had significantly lower testosterone levels (median 4.0 ng/dl, range less than 2.9 to 20.2) than those surgically castrated (median 9.2 ng/dl, range less than 2.9 to 28.8, p <0.001). No difference was found in dehydroepiandrosterone sulfate, androstenedione and sex hormone-binding globulin levels between the groups, whereas inhibin B levels were significantly higher in the luteinizing hormone-releasing hormone agonist treated group. CONCLUSIONS Using an accurate technique for testosterone measurement, subjects on luteinizing hormone-releasing hormone agonist therapy had significantly lower testosterone concentrations than men who underwent surgical castration. The clinical relevance of these findings remains to be determined.