Renal tumours in a Tsc2(+/-) mouse model do not show feedback inhibition of Akt and are effectively prevented by rapamycin.

Tuberous sclerosis (TSC) is an inherited syndrome in which tumours in multiple organs are characterised by activation of mammalian target of rapamycin complex 1 (mTORC1). Previous work suggests that mTORC1 activation is associated with feedback inhibition of Akt, a substrate of mTORC2. This could limit TSC-associated tumour growth but lead to paradoxical promotion of tumour cell survival upon treatment with mTOR inhibitors. However, Akt/mTOR signalling has not been fully investigated in TSC-associated tumours and it has been uncertain whether mTOR inhibition can prevent TSC-associated renal tumourigenesis. In this study, we investigated Akt/mTOR signalling in renal tumours using a Tsc2(+/-) mouse model and tested whether mTOR inhibition could prevent renal tumourigenesis. We found that all renal lesions including cysts, adenomas and carcinomas exhibited activation of both Akt and mTORC1 as evidenced by increased protein expression and phosphorylation of Akt and mTOR and their downstream targets. Protein kinase Cα was also highly expressed and phosphorylated in these lesions, consistent with activation of mTORC2. Surprisingly, IRS proteins were highly expressed, in contrast to a striking decrease seen in cultured Tsc2(-/-) mouse embryonic fibroblasts, suggesting one mechanism through which loss of feedback inhibition of Akt may occur in mTORC1 hyperactivated Tsc-associated tumours. Long-term treatment with rapamycin reduced both Akt and mTORC1 activity in normal kidney tissues and blocked the development of all types of renal lesions. In conclusion, in contrast to previous studies, we found that Akt signalling is not inhibited in Tsc-associated renal lesions and that by partially inhibiting the Akt/mTOR pathway, rapamycin is highly effective in preventing Tsc-associated tumours.

Suitability of PSA-detected localised prostate cancers for focal therapy: experience from the ProtecT study.

BACKGROUND: Contemporary screening for prostate cancer frequently identifies small volume, low-grade lesions. Some clinicians have advocated focal prostatic ablation as an alternative to more aggressive interventions to manage these lesions. To identify which patients might benefit from focal ablative techniques, we analysed the surgical specimens of a large sample of population-detected men undergoing radical prostatectomy as part of a randomised clinical trial. METHODS: Surgical specimens from 525 men who underwent prostatectomy within the ProtecT study were analysed to determine tumour volume, location and grade. These findings were compared with information available in the biopsy specimen to examine whether focal therapy could be provided appropriately. RESULTS: Solitary cancers were found in prostatectomy specimens from 19% (100 out of 525) of men. In addition, 73 out of 425 (17%) men had multiple cancers with a solitary significant tumour focus. Thus, 173 out of 525 (33%) men had tumours potentially suitable for focal therapy. The majority of these were small, well-differentiated lesions that appeared to be pathologically insignificant (38-66%). Criteria used to select patients for focal prostatic ablation underestimated the cancer's significance in 26% (34 out of 130) of men and resulted in overtreatment in more than half. Only 18% (24 out of 130) of men presumed eligible for focal therapy, actually had significant solitary lesions. CONCLUSION: Focal therapy appears inappropriate for the majority of men presenting with prostate-specific antigen-detected localised prostate cancer. Unifocal prostate cancers suitable for focal ablation are difficult to identify pre-operatively using biopsy alone. Most lesions meeting criteria for focal ablation were either more aggressive than expected or posed little threat of progression.

Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP).

AIMS: To survey current European practices in handling and reporting of radical prostatectomy (RP) specimens. METHODS AND RESULTS: A European Network of Uropathology (ENUP) was organized for the dissemination of information, survey studies and research collaborations. Contact data of uropathologists were collected from 321 pathology laboratories in 15 West European countries. In the first ENUP survey, 67.6% (217/321) of the members replied to a web-based questionnaire. Some practices were adopted by a large majority, e.g. inking of the specimen (96.6%), Gleason grading (99.5%), stratifying extraprostatic extension (EPE) according to extent (88.2%), reporting TNM stage (88.6%) and reporting location of positive margins (98%). As many as 71.6% of respondents always embedded the entire prostate and only 10.8% always practised partial embedding. Whole mounts were routinely used by 37.5% and standard blocks by 55.5%. Among areas with variable routines were methods to define focal versus extensive EPE and methods to quantify margin positivity, probably reflecting that the optimal method has yet to be determined. CONCLUSIONS: Some practices are almost universally adopted in Europe, whereas others still need to be standardized. The results of the study may be helpful when judging what recommendations are reasonable to issue.

Combined expression of caveolin-1 and an activated AKT/mTOR pathway predicts reduced disease-free survival in clinically confined renal cell carcinoma.

We previously reported that tumour-associated caveolin-1 is a potential biomarker in renal cell carcinoma (RCC), whose overexpression predicts metastasis following surgical resection for clinically confined disease. Much attention has recently focused on the AKT/mTOR pathway in a number of malignancies, including RCC. Since caveolin-1 and the AKT/mTOR signalling cascade are independently shown to be important regulators of tumour angiogenesis, we hypothesised that caveolin-1 interacts with the AKT/mTOR pathway to drive disease progression and metastasis in RCC. The aims of this study were to determine (i) the expression status of the activated AKT/mTOR pathway components (phosphorylated forms) in RCC and (ii) their prognostic value when combined with caveolin-1. Immunohistochemistry for caveolin-1, pAKT, pmTOR, pS6 and p4E-BP1 was performed on tissue microarrays from 174 clinically confined RCCs. Significantly decreased mean disease-free survival was observed when caveolin-1 was coexpressed with either pAKT (2.95 vs 6.14 years), pmTOR (3.17 vs 6.28 years), pS6 (1.45 vs 6.62 years) or p4E-BP1 (2.07 vs 6.09 years) than when neither or any one single biomarker was expressed alone. On multivariate analysis, the covariate of 'caveolin-1/AKT' (neither alone were influential covariates) was a significant influential indicator of poor disease-free survival with a hazard ratio of 2.13 (95% CI: 1.15-3.92), higher than that for vascular invasion. Tumours that coexpressed caveolin-1 and activated mTOR components were more likely to be larger, higher grade and to show vascular invasion. Our results provide the first clinical evidence that caveolin-1 cooperates with an activated AKT/mTOR pathway in cancer and may play an important role in disease progression. We conclude that evaluation of the 'caveolin-1/AKT/mTOR axis' in primary kidney tumours will identify subsets of RCC patients who require greater postoperative surveillance and more intensive treatment.

A UK-based investigation of inter- and intra-observer reproducibility of Gleason grading of prostatic biopsies.

AIMS: The frequency of prostatic core biopsies to detect cancer has been increasing with more widespread prostate specific antigen (PSA) testing. Gleason score has important implications for patient management but morphological reproducibility data for British practice are limited. Using literature-based criteria nine uropathologists took part in a reproducibility study. METHODS: Each of the nine participants submitted slides from consecutive cases of biopsy-diagnosed cancer assigned to the Gleason score groups 2-4, 5-6, 7 and 8-10 in the original report. A random selection of slides was taken within each group and examined by all pathologists, who were blind to the original score. Over six circulations, new slides were mixed with previously read slides, resulting in a total of 47 of 81 slides being read more than once. RESULTS: For the first readings of the 81 slides, the agreement with the consensus score was 78% and overall interobserver agreement was kappa 0.54 for Gleason score groups 2-4, 5-6, 7, 8-10. Kappa values for each category were 0.33, 0.56, 0.44 and 0.68, respectively. For the 47 slides read more than once, intra-observer agreement was 77%, kappa 0.66. The study identified problems in core biopsy interpretation of Gleason score at levels 2-4 and 7. Patterns illustrated by Gleason as 2 tended to be categorized as 3 because of the variable acinar size and unassessable lesional margin. In slides with consensus Gleason score 7, 13% of readings were scored 6 and in slides with consensus 6, 18% of readings were scored 7. CONCLUSIONS: Recommendations include the need to increase objectivity of the Gleason criteria but limits of descriptive morphology may have to be accepted.

A study of Gleason score interpretation in different groups of UK pathologists; techniques for improving reproducibility.

AIMS: To test the effectiveness of a teaching resource (a decision tree with diagnostic criteria based on published literature) in improving the proficiency of Gleason grading of prostatic cancer by general pathologists. METHODS: A decision tree with diagnostic criteria was developed by a panel of urological pathologists during a reproducibility study. Twenty-four general histopathologists tested this teaching resource. Twenty slides were selected to include a range of Gleason score groups 2-4, 5-6, 7 and 8-10. Interobserver agreement was studied before and after a presentation of the decision tree and criteria. The results were compared with those of the panel of urological pathologists. RESULTS: Before the teaching session, 83% of readings agreed within +/- 1 of the panel's consensus scores. Interobserver agreement was low (kappa = 0.33) compared with that for the panel (kappa = 0.62). After the presentation, 90% of readings agreed within +/- 1 of the panel's consensus scores and interobserver agreement amongst the pathologists increased to kappa = 0.41. Most improvement in agreement was seen for the Gleason score group 5-6. CONCLUSIONS: The lower level of agreement among general pathologists highlights the need to improve observer reproducibility. Improvement associated with a single training session is likely to be limited. Additional strategies include external quality assurance and second opinion within cancer networks.

Best Practice No 180. Nephrectomy for renal tumour; dissection guide and dataset.

Renal tumours constitute 2.5% of all malignancies and are among the 10 most common malignancies in the UK. Most of these are renal cell carcinomas (RCC) of various subtypes. Although historically RCC has been shown to be resistant to radiotherapy and chemotherapy, recent data suggest that the use of biological treatments, such as adjuvants, may be beneficial in patients with disease that has progressed at the time of presentation. The accurate diagnosis, staging, and grading of RCC is now a crucial element in optimal patient management. There are data to support the importance of histological type, tumour size, stage (especially patterns of extrarenal spread), and grade in determining outcome, and these data have been used to develop the published classification (Heidelberg/Rochester), staging (TNM), and grading (Fuhrman) systems. This article describes a dissection and histological sampling protocol that has been shown to increase the yield of staging information, a guide to histological classification and grading, and finally a minimum dataset for the completion of a satisfactory pathology report.

Caveolin-1 overexpression predicts poor disease-free survival of patients with clinically confined renal cell carcinoma.

Renal cell carcinomas, although usually apparently fully resected at surgery, commonly recur as distant metastasis. New markers are needed to predict which patients may relapse especially as novel methods of treatment (e.g. laproscopic resection) may make it impossible to assess conventional pathological prognostic markers. The caveolins are a family of proteins that represent the major structural components of caveolae; recent work suggests that these may have influence on several signalling pathways and they are thus potential prognostic markers. Immunohistochemistry for caveolin-1 was performed on sections of peripheral tumour from 114 consecutative nonmetastatic RCCs. Cytoplasmic caveolin-1 immunohistochemical (ICC) reaction was scored on a semiquantative scale of 1-3. Immunohistochemical score was tested for impact on disease-free survival by Kaplan-Meier and Cox regression methods. A total of 50 tumours had ICC score 1; 43 had score 2 and 21 score 3. Larger, higher grade and tumours with vascular invasion had significantly higher scores. On univariate survival analysis (Kaplan-Meier), patients with tumours scoring 1 had a mean disease-free survival of 6.61 years (95% CI 5.76-7.46) compared with 5.4 years (4.53-6.30) and 3.15 years (1.87-4.44) for scores 2 and 3, respectively. This is a significant difference (P=0.0017 log rank test). On multivariate analysis with size, grade and caveolin ICC score as independent covariates, caveolin ICC score 3 was an influential predictor of poor disease-free survival with a hazard ratio of 2.6 (P=0.03). We conclude that cytoplasmic overexpression of caveolin-1 predicts a poor prognosis in RCC; that this is likely to be a useful prognostic marker and that it may have importance in tumour progression.

Analysis of the prognostic implications of different tumour margin types in renal cell carcinoma.

AIMS: Invasion of perinephric tissues is part of the Union Internationale Contre le Cancer (UICC) (1997) T staging criteria for renal cell carcinoma (RCC) and appears to confer a worse prognosis. However, there are no established histological criteria to determine if this has occurred and histopathologists differ in their interpretation of the tumour margin. The purpose of this study was to determine histological criteria for invasion of perinephric tissues that may be used in staging. METHODS AND RESULTS: We assessed the prognostic implications of different margin types in 176 cases of RCC with good follow-up data. The tumour margin type in each cases was classified as follows: fibrous tumour capsule; rim of kidney; fibrous capsule with 'collar stud' invasion; pushing margin, no capsule; and tumour cell invasion of fat. The margin types were used in univariate and multivariate survival analysis to determine which had most impact on disease-free survival. In Cox regression analysis with all other influential covariates cellular invasion of fat was the only margin type that had any prognostic impact, conferring a 2.9 relative hazard compared with tumours with a fibrous capsule (P = 0.007). CONCLUSIONS: For staging purposes the designation of a tumour as invading perinephric tissues should be limited to those cases that have tumour cells invading the perinephric fat.

Evaluation of a protocol for examining nephrectomy specimens with renal cell carcinoma.

AIMS: To evaluate the practicality of use and the effectiveness of a standard protocol for examining nephrectomy specimens for renal cell carcinoma (RCC), with emphasis on the identification of vascular invasion. METHODS: A standard protocol, devised to identify the major prognostic determinants, was used to examine 79 consecutive tumours submitted to four histopathology departments. The incidence of vascular invasion found was compared with the incidence in a historical series of tumours. RESULTS: The protocol proved easy to follow, and appeared to increase the incidence of observed vascular invasion (40 of 69 cases compared with 69 of 176 cases in the historical series; p = 0.059, Fishers exact test, one sided) CONCLUSIONS: If pathological prognostic determinants are to be used for clinical management, then it is important that they are identified and recorded consistently. The protocol described provides a method of examining nephrectomy specimens that can be used in routine practice and would probably reliably identify recognised prognostic variables.

Elastography in the detection of prostatic cancer.

We describe a simple, practical technique for producing elastography images of the prostate. Our standard technique for the detection of prostatic cancer is ultrasound guided systematic biopsy of the prostate, with extra targeted biopsies of any abnormal areas detected by grey-scale ultrasound. The aim of this study to determine whether adding elastography imaging with targeted biopsies of abnormal areas detects more cancers. The results of elastography imaging were evaluated against grey-scale images and biopsy data in 100 patients. Elastography detected an additional 5 cancers in the 100 patients and detected an extra 3 patients with cancer at the expense of 8 extra biopsies. This represents a significant improvement in the detection rate of prostatic cancer.

Contribution of grade, vascular invasion and age to outcome in clinically localized renal cell carcinoma.

OBJECTIVE: To determine the relative prognostic importance of microvascular invasion in apparently localized renal cell carcinoma (RCC). PATIENTS AND METHODS: A retrospective clinical and pathological review was conducted of 176 consecutive patients identified from pathology records who had a nephrectomy for RCC with a median follow-up of 44 months. Vascular invasion was recorded and categorized by the level of microvascular invasion (MVI), renal vein invasion (RVI) and inferior vena cava invasion (IVCI). Tumour type, grade and size were also assessed. These variables were assessed by univariate and multivariate analysis to determine their effect on disease-free survival. RESULTS: In the univariate analysis tumour size, grade, vascular invasion and young age each predicted reduced disease-free survival. On multivariate analysis for all 176 patients, grade, vascular invasion and young age were the significant independent predictors of reduced disease-free survival. In a subgroup of 149 patients from whom those with very high risk determinants were excluded (those with grade 4 tumours and/or IVCI) most of the risk of metastasis could be accounted for by vascular invasion and young age alone (MVI vs no vascular invasion, hazard ratio 3.18, 95% confidence interval 1.29-7.84; RVI vs no vascular invasion 2.41, 0.989-5.89; and age per year 0.963, 0.94-0.992). CONCLUSIONS: Grade, vascular invasion and young age are the main independent predictors of relapse in clinically localized RCC after nephrectomy. For most patients, who do not have very high risk indicators, the main adverse predictors are vascular invasion and young age. These findings are important when selecting patients for trials of adjuvant therapy and have implications for pathological staging.

Effects of a selective adenosine A1 receptor antagonist on the development of cyclosporin nephrotoxicity.

1. The clinical application of cyclosporin as an immunosuppressive agent is limited by its nephrotoxicity. 2. The effect of FK453, a selective A1-receptor antagonist, administered twice daily to rats at a dose of 100 mg kg-1 was assessed on the development of nephrotoxicity induced by cyclosporin (10 mg kg-1 i.p. daily) administered for 14 days. The effects of nifedipine administered twice daily (0.3 mg kg-1 s.c.) for 14 days, on cyclosporin nephrotoxicity were also studied. 3. Cyclosporin induced a 46.58% and 35.78% decline in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) respectively and a reduction of 16.69% in filtration fraction (FF). Co-administration of FK453 resulted in falls of 30.5%, 18.59% and 14.7% in GFR, ERPF and FF respectively, the former two significantly less than the falls seen with cyclosporin (CyA) alone (P < 0.05 vs CyA, ANOVA). 4. Nifedipine appeared to have a more pronounced protective effect resulting in a decline of only 20.91% in GFR, with no significant change in ERPF (increase of 0.93%) when co-administered with CyA. 5. These observations indicate adenosine plays a minor role in the pathophysiology of CyA nephrotoxicity.

Permselectivity in thin membrane nephropathy.

The glomerular permselectivity to polydisperse neutral dextrans was compared in 6 patients with thin membrane nephropathy (TMN) and 10 healthy controls. Despite having normal renal hemodynamics and minimal proteinuria, the patients with TMN had significantly increased fractional clearance of neutral molecules with Stokes radius > 42 A. Conventional theories of glomerular barrier size selectivity cannot fully explain these data since they would predict that our patients would have had nephrotic range proteinuria.

Quantitative audit of the content of histopathology reports.

An audit of histopathology reports presents the problem that the output is textual and difficult to quantify. This makes the definition of an adequate report subjective and susceptible to observer variation. A procedure has been developed which allows the quantitative analysis of reports and facilitates the development of local reporting guidelines. A topic is selected; the auditor then lists the possible details that may be included in the report and notes how many reports from a sample include each detail. The results are discussed at a departmental meeting with the aim of agreeing on reporting guidelines. At a later date another sample of reports can be analysed for compliance with the guidelines and compared with the previous reports. Problems with compliance can be discussed further and at the audit meeting the guidelines may be amended appropriately, thus completing the audit cycle. This method of audit has the advantage that the results are quantitative and that the group discussion and re-examination of the guidelines has educational value.