An interobserver reproducibility study on invasiveness of bladder cancer using virtual microscopy and heatmaps.

AIMS: The distinction between non-invasive (pTa) and invasive (pT1) non-muscle invasive bladder cancer (NMIBC) is subject to considerable interobserver variation. We aimed to generate a teaching set of images based on the diagnostic opinions of a panel of expert genitourinary pathologists. METHODS AND RESULTS: Twenty-five transurethral resection specimens initially reported as pT1 NMIBC from two university hospitals were selected on the basis of potential uncertainty of stromal invasion. Digitized slides were reviewed independently by a panel of eight genitourinary pathologists, who annotated any invasive area if present. Annotations were reviewed by the lead panel, and heatmaps of annotated areas were constructed. Reasons for discrepancies were analysed, and kappa scores were calculated to determine agreement among the eight panellists. Full agreement by the eight panellists was obtained in 11 of 25 cases (44%), with a multi-rater (Fleiss) kappa of 0.47 (P < 0.0001). After joint review of the seven discordant (agreement <75% of panellists) cases, consensus was obtained for six cases, and a teaching set of images was generated. CONCLUSIONS: Interobserver agreement among the panellists in the selected cases was moderate, but consensus could be reached in almost all cases. Heatmaps proved to be instrumental in generating a teaching set of images for standardization of histological criteria for NMIBC invasion.

A novel strategy based on histological protein profiling in-silico for identifying potential biomarkers in urinary bladder cancer.

OBJECTIVE: To screen a publicly available immunohistochemistry (IHC) based web-atlas, to identify key proteins in bladder cancer that might serve as potential biomarkers. MATERIALS AND METHODS: The first version of the Human Protein Atlas (HPA 1.0), with 660 proteins, was visually examined to identify proteins with a variable staining pattern among the 12 tissue samples representing bladder cancer. None or limited previous characterization in bladder cancer, as well as a supportive Western blot, were also required. The selected proteins were then evaluated in an independent set of patient samples (106 tumour samples of differing stage and grade) represented in a tissue microarray (TMAi). The IHC expression of the identified proteins in the TMAi was scored and related to tumour stage and grade. RESULTS: The expression profiles of the 13 proteins selected from the web-atlas were confirmed in the TMAi. Expression patterns for seven proteins were significantly altered (P < 0.05) with higher stage and/or grade. Three of those (CN130, DSG3, PHF6) lack characterization in bladder cancer, whereas the remaining four proteins have previously been suggested as key proteins/potential biomarkers in cancer, some of them also in bladder cancer. CONCLUSION: New candidate proteins for urinary bladder cancer were identified through screening of the publicly available HPA 1.0. Although further evaluation is necessary, this strategy is promising in the search for new biomarkers, with potential to improve the management of patients with this disease.

Reliability of the reported size of removed colorectal polyps.

BACKGROUND: The size of colorectal polyps is important in the clinical management of these lesions. AIM: To audit the accuracy in calculating the size of "polyps" by various specialists. MATERIALS AND METHODS: Eighteen pathologists and four surgeons measured, with a conventional millimetre ruler, the largest diameter of 12 polyp phantoms. The results of two independent measurements (two weeks apart) were compared with the gold standard-size assessed at The Royal Institute of Technology, Sweden. RESULTS: Thirty-one percent (83/264-trial 1) and 33% (88/264-trial 2) of the measurements underestimated or overestimated the gold standard size by >1 mm. Of the 22 experienced participants, 95% (21/22-trial 1) and 91% (20/22-trial 2) misjudged by >1 mm the size of one or more polyps. Values given by 13 participants (4.9%) in trial 1 and by 15 participants (5.7%) in trial 2, differed by > or = +/-4 mm from the gold standard size. In addition, a big difference between the highest and the lowest values was recorded in some polyps (up to 11.4 mm). Those disparate values were regarded as a human error in reading the scale on the ruler. CONCLUSION: Using a conventional ruler (the tool of pathologists worldwide) unacceptably high intra-observer and inter-observer variations in assessing the size of polyp-phantoms was found. The volume and the shape of devices, as well as human error in reading the scale of the ruler were confounding factors in size assessment. In praxis, the size is crucial in the management of colorectal polyps. Considering the clinical implications of the results obtained, the possibility of developing a method that will allow assessment of the true size of removed clinical polyps is being explored.

No outgrowth of chondrocytes from non-digested particulated articular cartilage embedded in commercially available fibrin matrix: an in vitro study.

BACKGROUND: Commercially available fibrin is routinely being used as both a matrix in certain cartilage repair techniques and a method for scaffold fixation. Chondrocytes from non-digested particulated cartilage fragments are proposed as a possible source for new cartilage tissue formation in some operative techniques. The goal of this study was to test that chondrocytes from particulated articular cartilage embedded in fibrin have an active role in the process of cartilage repair, as well as if commercially available fibrin should be used as a suitable matrix. METHODS: Articular cartilage was obtained from patients undergoing total knee replacement surgery. The biopsies were particulated in small, 1-2-mm(3) pieces and embedded in fibrin. Two groups were compared in our study, particulated articular cartilage with and without collagenase treatment. The specimens were analyzed by optical microscopy after 2-5 weeks of cultivation in a special construct embedded in a cell culture medium containing particulated cartilage embedded in fibrin in the upper phase and cancellous bone in the lower phase under the perforated nylon membrane. RESULTS: None of the biopsies taken from four different patients showed the outgrowth of chondrocytes or bone marrow-originated cells into the fibrin matrix in our experimental model. CONCLUSIONS: It has been shown in our experimental model in vitro little to support the theory that articular chondrocytes from particulated articular cartilage embedded in fibrin have an active role in cartilage repair in its early stage.

Castration radiosensitizes prostate cancer tissue by impairing DNA double-strand break repair.

Chemical castration improves responses to radiotherapy in prostate cancer, but the mechanism is unknown. We hypothesized that this radiosensitization is caused by castration-mediated down-regulation of nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). To test this, we enrolled 48 patients with localized prostate cancer in two arms of the study: either radiotherapy first or radiotherapy after neoadjuvant castration treatment. We biopsied patients at diagnosis and before and after castration and radiotherapy treatments to monitor androgen receptor, NHEJ, and DSB repair in verified cancer tissue. We show that patients receiving neoadjuvant castration treatment before radiotherapy had reduced amounts of the NHEJ protein Ku70, impaired radiotherapy-induced NHEJ activity, and higher amounts of unrepaired DSBs, measured by γ-H2AX foci in cancer tissues. This study demonstrates that chemical castration impairs NHEJ activity in prostate cancer tissue, explaining the improved response of patients with prostate cancer to radiotherapy after chemical castration.

Interobserver reproducibility of modified Gleason score in radical prostatectomy specimens.

The Gleason score (GS) of prostate cancer is calculated by adding primary and secondary Gleason grades with patterns occupying less than 5% of the tumour often not included despite their probable prognostic significance. A modified Gleason score (mGS) comprising primary and tertiary patterns of higher grade has been proposed, but its interobserver variability has yet to be elucidated. Slides from 69 consecutive prostatectomy specimens were circulated among four genitourinary pathologists. GS and mGS were assessed, and results were compared in pairs. Mean weighted kappa for GS and mGS were 0.56 (range 0.52-0.66) and 0.58 (range 0.49-0.74), respectively. The difference between GS and mGS was 0, 1 and 2 score units in 66%, 26% and 8%, respectively, mean 0.41 score units (range 0.24-0.51). The increment was greater for transition-zone tumours than for peripheral-zone tumours (0.63 and 0.35 score units, respectively, P=0.002). An odd mGS (5, 7 or 9) was more often given than an odd GS (77% and 62%, respectively, P<0.001). Disagreement between observers greater than 1 score unit was more common with mGS than GS (18% and 4%, respectively, P<0.001). In conclusion, overall mean weighted kappa for interobserver reproducibility of mGS is at least as high as that of GS. However, there is a clustering of mGS in odd scores, and severe disagreement is more commonly observed than with GS. Training of mGS assessment would possibly improve agreement. Tertiary Gleason patterns need to be better defined.

Complete androgen insensitivity without Wolffian duct development: the AR-A form of the androgen receptor is not sufficient for male genital development.

BACKGROUND: The androgen receptor (AR) is essential for the differentiation of male external and internal genitalia. It is normally present in two forms, a full-length form B and an N-terminal truncated form A with still unknown function. Mutations in the AR gene cause androgen insensitivity syndrome (AIS), which is divided into subgroups according to the degree of undermasculinization. Patients with completely female external genitalia are classified as complete AIS (CAIS). However, a recent study has shown that some CAIS patients have signs of internal male genital differentiation due to missense mutations that show some degree of residual function. OBJECTIVE: We aimed to study the expression of the different forms of the AR in two CAIS patients in relation to the development of male internal genital structures. One patient had a mutation (L7fsX33) that affects only the full-length AR-B form of the AR, whereas the other had a nonsense mutation (Q733X) affecting both isoforms. MEASUREMENTS AND RESULTS: We thoroughly analysed internal genitalia at surgery and by histological examination. No signs of Wolffian duct (WD) development were present in any of the patients. Western blotting of proteins from gonadal and genital skin fibroblasts was performed with AR antibodies directed against different AR epitopes. The N-terminally truncated A form was expressed in normal amounts in the patient with the L7fsX33 mutation while no AR was detected in the other patient. CONCLUSION: The presence of the AR-A form does not seem to be sufficient for WD maintenance and differentiation.

Interobserver reproducibility of percent Gleason grade 4/5 in total prostatectomy specimens.

PURPOSE: Recently the percent Gleason grade 4/5 was proposed as a predictor of the outcome of prostate cancer and it has been shown that it adds prognostic information to that given by Gleason score. To our knowledge the interobserver variability of percent Gleason grade 4/5 has not yet been investigated. We assessed the percent Gleason grade 4/5, including the identification of high grade patterns and estimation of the percent tumor area, which is potentially more difficult than conventional Gleason grading. MATERIALS AND METHODS: A consecutive series of 69 total prostatectomy specimens was reviewed. A single slide per specimen was circulated among 4 genitourinary pathologists, who assessed Gleason score and the percent Gleason grade 4/5. Results were compared pairwise and a weighted kappa was calculated for Gleason score and the percent Gleason grade 4/5. RESULTS: The 4 observers had a mean weighted kappa for Gleason score and the percent Gleason grade 4/5 of 0.52 to 0.66 (overall mean 0.56) and 0.58 to 0.72 (overall mean 0.66), respectively. The best agreement for percent Gleason grade 4/5 was in 2 pathologists at the same department (weighted kappa 0.86). Transition zone tumors had a lower weighted kappa for Gleason score but a higher weighted kappa for percent Gleason grade 4/5 than peripheral zone tumors. In cases of the greatest disagreement in the percent Gleason grade 4/5 crush artifact, cribriform cancer and high grade PIN within the tumor were significantly more common. An intraobserver reproducibility of weighted kappa 0.91 was achieved for Gleason score and the percent Gleason grade 4/5. CONCLUSIONS: Interobserver reproducibility of the percent Gleason grade 4/5 is substantial and at least as good as that of the Gleason score. Hence, concern about interobserver variability should not deter pathologists from using the percent Gleason grade 4/5 as a prognostic marker for prostate cancer.

Interobserver reproducibility of percent Gleason grade 4/5 in prostate biopsies.

PURPOSE: Percent Gleason grade 4/5 (GG4/5) has been proposed as a predictor of prognosis in prostate cancer and it has been shown to add prognostic information to that given by Gleason score (GS). We recently noted that the interobserver reproducibility of percent GG4/5 in total prostatectomy specimens is at least as good as that of the GS. However, to our knowledge the reproducibility of percent GG4/5 in needle biopsies has not yet been investigated. MATERIALS AND METHODS: A consecutive series of needle biopsies from 69 men with prostate cancer was reviewed. Biopsies were taken according to a standardized octant protocol. All 279 slides containing cancer were circulated among 4 genitourinary pathologists, who assessed GS and percent GG4/5. Results were compared pairwise and weighted kappa was calculated. RESULTS: The 4 observers had a mean weighted kappa for GS and percent GG4/5 of 0.48 to 0.55 (overall mean 0.51) and 0.52 to 0.68 (overall mean 0.60), respectively. There was less disagreement in percent GG4/5 when a single biopsy was positive for cancer than when 6 or more biopsies were positive. The number of positive biopsies showed a stronger correlation with a discrepancy in percent GG4/5 than cancer length. Disagreement was worse when cribriform or fusion patterns were present. CONCLUSIONS: Interobserver reproducibility of percent GG4/5 on prostate biopsies is at least as good as that of GS. Hence, concern about interobserver variability should not deter pathologists from using percent GG4/5. Grading appears to be most difficult when cancer is present in multiple biopsies or it contains cribriform or fusion patterns.