Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization.

Bone formation, remodeling and repair are dynamic processes, involving cell migration, ECM assembly, osteocyte embedding, and bone resorption. Using live-cell imaging, we previously showed that osteoblast assembly of the ECM proteins fibronectin and collagen is highly dynamic and is integrated with cell motility. Additionally, osteoblast-to-osteocyte transition involved arrest of cell motility, followed by dendrite extension and retraction that may regulate positioning of embedding osteocytes. To further understand how osteocytes differentiate and embed in collagen, mice were generated that co-expressed GFPtopaz-tagged collagen with a Dmp1-Cre-inducible tdTomato reporter targeted to preosteocytes/osteocytes. Dual live-cell imaging of collagen and osteocyte dynamics in mineralizing primary calvarial cell cultures showed that Dmp1-Cre/tdTomato turned on in early bone nodule forming regions, demarcated by foci of concentrated GFP-collagen bundles that appeared structurally distinct from the surrounding collagen. Dmp1-Cre/tdTomato-positive cells were post-mitotic and were continuously induced throughout the 2 week timecourse, whereas the majority of collagen was assembled by day 7. GFP-collagen fibrils showed global (tissue-level) motions, suggesting coordinated cell layer movement, and local fibril motions mediated by cell-generated forces. Condensation of collagen fibril networks occurred within bone nodules prior to mineralization. Intravital imaging confirmed a similar structural appearance of GFP-collagen in calvarial bone, with analogous global motions of mineralizing areas adjacent to sutures. In early (unmineralized) calvarial cell cultures, Dmp1-Cre/tdTomato-positive cells were motile (mean velocity 4.8 µm/h), moving freely in and around the forming bone nodule, with a small number of these cells embedded in collagen, constraining their motion. In mineralizing cultures, the average velocity of Dmp1-Cre/tdTomato-positive cells was significantly reduced (0.7 µm/h), with many immobilized in the mineralizing nodule. Three apparent mechanisms for embedding of Dmp1-Cre/tdTomato-positive cells were observed. In some cases, a previously motile Dmp1-Cre/tdTomato-positive cell became immobilized in collagen fibril networks that were newly assembled around the cell, thereby entrapping it. In other cases, a motile Dmp1-Cre/tdTomato-positive cell moved into an already formed "collagen lacuna," arrested its motility and became embedded. Alternatively, some cells switched on tdTomato expression in situ within a lacuna. These data provide new insight into the dynamic process of bone collagen assembly and suggest multiple mechanisms for osteocyte entrapment in collagen matrix.

Biochemical composition of urolithiasis from stone dust - a matched-pair analysis.

OBJECTIVE: To determine if the biochemical composition of a renal calculus can be measured from 'dust' obtained during laser fragmentation. PATIENTS AND METHODS: This pilot study was set in a tertiary referral hospital between 2011 and 2013. Stone dust was aspirated through the ureteroscope during lasering and a stone fragment also retrieved. Both samples were analysed by Fourier transform infrared spectroscopy. Pairs of stone (standard) and dust were compared. They were deemed to match if both were of the same pure biochemical composition or if the predominant constituent was the same in mixed compositions, as this would not alter subsequent management. RESULTS: Paired specimens were obtained from 97 ureteroscopies. The dust specimen was sufficient for analysis in 66/97 (68%) cases. Of these, the composition matched that of the stone in 49/66 (74%) cases. In 12/66 (18%) the biochemistry differed only in the relative proportions of each constituent, whilst 5/66 (8%) showed a complete mismatch. The overall sensitivity was 51% and specificity 97%. A limitation of the study is the small number of some stone types analysed (<5 each cystine, atazanavir, mixed uric acid/calcium oxalate). CONCLUSION: We have demonstrated in this pilot study successful proof of principle. Further work is required initially to improve the number of sufficient dust specimens. This technique may offer an option when a stone cannot be retrieved ureteroscopically.

Prospective randomized trial of hexylaminolevulinate photodynamic-assisted transurethral resection of bladder tumour (TURBT) plus single-shot intravesical mitomycin C vs conventional white-light TURBT plus mitomycin C in newly presenting non-muscle-invasive bladder cancer.

OBJECTIVE: To determine if photodynamic 'blue-light'-assisted resection leads to lower recurrence rates in newly presenting non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: We conducted a prospective randomized trial of hexylaminolevulinate (HAL) photodynamic diagnosis (PDD)-assisted transurethral resection of bladder tumour (TURBT) plus single-shot intravesical mitomycin C vs standard white-light-assisted TURBT plus single-shot intravesical mitomycin C. A total of 249 patients with newly presenting suspected NMIBC enrolled at Guy's Hospital between March 2005 and April 2010. Patients with a history of bladder cancer were excluded. The surgery was performed by specialist bladder cancer surgical teams. Of the eligible patients, 90% agreed to be randomized. RESULTS: Of the 249 patients, 209 (84%) had cancer and in 185 patients (89%) the cancer was diagnosed as NMIBC. There were no adverse events related to HAL in any of the patients randomized to the intravesical HAL-PDD arm. Single-shot intravesical mitomycin C was administered to 61/97 patients (63%) in the HAL-PDD arm compared with 68/88 patients (77%) in the white-light arm (P = 0.04) Intravesical HAL was an effective diagnostic tool for occult carcinoma in situ (CIS). Secondary CIS was identified in 25/97 patients (26%) in the HAL-PDD arm compared with 12/88 patients (14%) in the white-light arm ((P = 0.04) There was no significant difference in recurrence between the two arms at 3 or 12 months: in the HAL-PDD and the white-light arms recurrence was found in 17/86 and 14/82 patients (20 vs 17%), respectively ((P = 0.7) at 3 months, and in 10/63 and 15/67 patients (16 vs 22%), respectively ((P = 0.4) at 12 months. CONCLUSIONS: Despite HAL-PDD offering a more accurate diagnostic assessment of a bladder tumour, in this trial we did not show that this led to lower recurrence rates of newly presenting NMIBC compared with the best current standard of care.

Photodynamic diagnosis of non-muscle-invasive bladder cancer with hexaminolevulinate cystoscopy: a meta-analysis of detection and recurrence based on raw data.

BACKGROUND: Studies on hexaminolevulinate (HAL) cystoscopy report improved detection of bladder tumours. However, recent meta-analyses report conflicting effects on recurrence. OBJECTIVE: To assess available clinical data for blue light (BL) HAL cystoscopy on the detection of Ta/T1 and carcinoma in situ (CIS) tumours, and on tumour recurrence. DESIGN, SETTING, AND PARTICIPANTS: This meta-analysis reviewed raw data from prospective studies on 1345 patients with known or suspected non-muscle-invasive bladder cancer (NMIBC). INTERVENTION: A single application of HAL cystoscopy was used as an adjunct to white light (WL) cystoscopy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We studied the detection of NMIBC (intention to treat [ITT]: n=831; six studies) and recurrence (per protocol: n=634; three studies) up to 1 yr. DerSimonian and Laird's random-effects model was used to obtain pooled relative risks (RRs) and associated 95% confidence intervals (CIs) for outcomes for detection. RESULTS AND LIMITATIONS: BL cystoscopy detected significantly more Ta tumours (14.7%; p<0.001; odds ratio [OR]: 4.898; 95% CI, 1.937-12.390) and CIS lesions (40.8%; p<0.001; OR: 12.372; 95% CI, 6.343-24.133) than WL. There were 24.9% patients with at least one additional Ta/T1 tumour seen with BL (p<0.001), significant also in patients with primary (20.7%; p<0.001) and recurrent cancer (27.7%; p<0.001), and in patients at high risk (27.0%; p<0.001) and intermediate risk (35.7%; p=0.004). In 26.7% of patients, CIS was detected only by BL (p<0.001) and was also significant in patients with primary (28.0%; p<0.001) and recurrent cancer (25.0%; p<0.001). Recurrence rates up to 12 mo were significantly lower overall with BL, 34.5% versus 45.4% (p=0.006; RR: 0.761 [0.627-0.924]), and lower in patients with T1 or CIS (p=0.052; RR: 0.696 [0.482-1.003]), Ta (p=0.040; RR: 0.804 [0.653-0.991]), and in high-risk (p=0.050) and low-risk (p=0.029) subgroups. Some subgroups had too few patients to allow statistically meaningful analysis. Heterogeneity was minimised by the statistical analysis method used. CONCLUSIONS: This meta-analysis confirms that HAL BL cystoscopy significantly improves the detection of bladder tumours leading to a reduction of recurrence at 9-12 mo. The benefit is independent of the level of risk and is evident in patients with Ta, T1, CIS, primary, and recurrent cancer.

Prevention of bladder tumours after nephroureterectomy for primary upper urinary tract urothelial carcinoma: a prospective, multicentre, randomised clinical trial of a single postoperative intravesical dose of mitomycin C (the ODMIT-C Trial).

BACKGROUND: Standard treatment for upper urinary tract urothelial carcinoma (UUTUC) is nephroureterectomy. Subsequently, around 40% of patients will develop a bladder tumour potentially because of implantation from the primary tumour. OBJECTIVE: To prevent bladder tumour after nephroureterectomy with a single postoperative dose of intravesical mitomycin C (MMC). DESIGN, SETTING, AND PARTICIPANTS: A prospective, randomised, nonblinded trial (ODMIT-C: One Dose Mitomycin C) was undertaken in 46 British centres between July 2000 and December 2006. The study recruited 284 patients with no previous or concurrent history of bladder cancer undergoing nephroureterectomy for suspected UUTUC. INTERVENTION: A single postoperative intravesical dose of MMC (40 mg in 40 ml saline) or standard management on removal of the urinary catheter. MEASUREMENTS: Bladder tumour formation was judged by visual appearance at cystoscopy at 3, 6, and 12 mo following nephroureterectomy. RESULTS AND LIMITATIONS: One hundred forty-four patients were randomised to receive MMC and 140 patients to receive standard care. In the MMC arm, 105 of 144 patients (73%) and 115 of 140 patients (82%) in the standard care arm received their allocated treatment. Thirteen of 105 patients who received MMC and 20 of 115 patients allocated to standard treatment did not complete follow-up. By modified intention-to-treat analysis, 21 of 120 patients (17%) in the MMC arm developed a bladder recurrence in the first year compared to 32 of 119 patients (27%) in the standard treatment arm (p=0.055). By treatment as per protocol analysis, 17 of 105 patients (16%) in the MMC arm and 31 of 115 patients (27%) in the standard treatment arm developed a recurrence (p=0.03). No serious adverse events were reported. A limitation is that histologic proof of recurrence was not required in this trial. CONCLUSIONS: A single postoperative dose of intravesical MMC appears to reduce the risk of a bladder tumour within the first year following nephroureterectomy for UUTUC. The absolute reduction in risk is 11%, the relative reduction in risk is 40%, and the number needed to treat to prevent one bladder tumour is nine.

Hexylaminolaevulinate fluorescence cystoscopy in patients previously treated with intravesical bacille Calmette-Guérin.

OBJECTIVE To determine if hexylaminolaevulinate fluorescence cystoscopy (HAL-FC) has the potential to improve the diagnosis of bladder cancer in patients who have been treated with bacille Calmette-Guérin (BCG). PATIENTS AND METHODS Patients scheduled for rigid cystoscopy after BCG therapy were recruited prospectively between April 2005 and February 2006. Patients received HAL (Hexvix, PhotoCure ASA, Oslo, Norway) and the D-light system (Storz, Tuttlingen, Germany) was used to detect fluorescence. The bladder was mapped and biopsies taken under white light and then using HAL-FC. The main outcome was the frequency and nature of additional pathology detected by HAL-FC. Twenty-seven patients (21 men and six women; median age 70 years, range 49-82) underwent 32 HAL-FC. RESULTS Recurrent bladder cancer was detected in 11 of the 32 (34%) examinations. HAL-FC detected additional pathology in five of the 27 (19%) patients. In two of these cases the additional pathology was clinically significant (one pT4G3 intraprostatic transitional cell carcinoma and one intravesical pT1G2 + carcinoma in situ), whereas in three cases the pathology was hyperplasia/dysplasia. Overall, the false-positive biopsy rate with HAL-FC was 63%. In the presence of positive voided urine cytology six of eight patients had recurrent bladder tumour and the false-positive biopsy rate was only 34%. Urine cytology was positive in four of five of the patients in whom additional pathology was detected by HAL-FC. CONCLUSIONS Clinically significant occult pathology can be detected using HAL-FC after BCG therapy, but in <10% of cases. The rate of false-positive biopsies is high but in our hands appears to be lower than with white-light guided biopsies after BCG. Our pragmatic approach is to use HAL-FC after BCG when clinical suspicion is high, and when the preoperative voided urine cytology is positive.

Hexylaminolevulinate photodynamic diagnosis for multifocal recurrent nonmuscle invasive bladder cancer.

OBJECTIVE: To determine the potential for hexylaminolevulinate (HAL) photodynamic diagnosis (PDD) to improve the management of multifocal recurrent nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patients with a history of NMIBC and with at least two suspected papillary recurrences were enrolled in this prospective study between April 2005 and October 2006. The photosensitizer was hexylaminolevulinate (HAL) (PhotoCure, Norway), and the Storz D-light system was used to detect fluorescence. The bladder was mapped initially under white light and then using HAL-photodynamic diagnosis (PDD). The number and types of additional lesions detected by HAL-PDD over white light cystoscopy were measured. RESULTS: Eighteen patients (11 men), median age 74 years (range 35-84 yrs), underwent HAL-PDD. The median HAL instillation time was 109 minutes (range 60-250 min). Recurrent bladder cancer was confirmed histologically in 14/18 (78%) patients. Additional pathology was detected in 8/14 (57%) patients with confirmed recurrence and confirmed histologically in 6 of these. Additional pathology was papillary in 5/6 (83%) patients, and flat pathology was found in all six patients with additional foci. Carcinoma in situ (CIS) was detected in 4/6 (67%) patients with additional foci, three of whom were subsequently treated with intravesical bacille Calmette-Guérin (BCG). The sensitivity of HAL-PDD for the detection of tumor was 97.8%, compared with 69.6% for white light cystoscopy. The false-positive fluorescence-guided biopsy rate was 18/63 (29%). CONCLUSION: HAL-PDD allows more complete management of bladder tumor in patients with multifocal recurrence. The high frequency of additional lesions detected and the rate of detection of CIS suggest that HAL-PDD should be the standard of care.

Bladder cancer: new TUR techniques.

Transurethral resection of bladder tumours (TURBT) using a wire loop remains the gold-standard treatment for bladder tumours, but it is associated with unacceptably high early recurrence rates after first resection. Improvements to standard resection techniques and a range of optical and technological advances offer exciting possibilities for improving outcomes. Early second resection has been shown to reduce recurrence rates, and increase response to intravesical chemotherapy and/or immunotherapy. It should be considered in most high-risk non-muscle invasive cancers (T1; G3; multifocal) being managed by bladder conservation. Newer energy sources, such as laser, may facilitate day case management of bladder tumours using local anaesthesia in select groups of patients. The novel technique of photodynamic diagnosis improves tumour detection, and quality of resection, and is likely to become the standard for initial tumour management. The traditional 'incise and scatter' resection technique goes against all oncological surgical principles. En-bloc resection of tumours would be far preferable and demands further development and evaluation. The technique of TURBT needs to evolve to allow first-time clearance of disease and low recurrence rates.

Hexylaminolaevulinate 'blue light' fluorescence cystoscopy in the investigation of clinically unconfirmed positive urine cytology.

OBJECTIVE: To investigate the value of photodynamic diagnosis (PDD) using hexylaminolaevulinate (Hexvix, PhotoCure, Oslo, Norway) in the investigation of patients with positive urine cytology who have no evidence of disease after standard initial investigations. PATIENTS AND METHODS: Twenty-three patients referred with positive urine cytology but no current histological evidence of cancer were investigated between April 2005 and January 2007 with PDD, using Hexvix and the D-light system (Karl Storz, Tuttlingen, Germany) to detect fluorescence. The bladder was mapped initially under white light and then under 'blue-light'. Biopsies were taken from abnormal urothelium detected by white light, fluorescence, or both. All cytological specimens were reviewed by a reference cytopathologist unaware of the result of the PDD. RESULTS: Twenty-five PDD-assisted cystoscopies were carried out on 23 patients (20 men/3 women; median age 64 years, range 24-80 years). Of the 23 patients, 17 (74%) were previously untreated for transitional cell carcinoma (TCC), whilst six were under surveillance for previous TCC. Nineteen of the 23 (83%) cytology specimens were confirmed as suspicious or positive by the reference pathologist. TCC of the bladder or preneoplastic lesions were diagnosed in six patients, i.e. six (26%) of those investigated and six of 19 (32%) with confirmed positive cytology. Four of the six were under surveillance for previous bladder tumour. Additional pathology was detected by fluorescence in five of the six patients, including two carcinoma in situ (CIS), one CIS + G3pT1 tumour, and two dysplasia. Diagnoses in PDD-negative cases included one upper tract TCC and four patients with stones. In addition, one patient had CIS diagnosed on both white light and PDD 6 months later. CONCLUSION: Additional pathology was detected by HAL fluorescence cystoscopy in 32% of patients with confirmed positive urinary cytology. PDD is a key step in the management of patients with positive urinary cytology and no evidence of disease on conventional tests.

Open partial nephrectomy: outcomes from two UK centres.

OBJECTIVE: To define the current achievable outcomes from open partial nephrectomy (OPN) in the UK at a time when other treatments for small kidney tumours are increasingly being advocated. Current knowledge of the effectiveness of OPN is limited by the fact that published data are almost exclusively derived from a very few centres of established world renown. PATIENTS AND METHODS: We retrospectively reviewed 100 consecutive planned OPNs in 90 patients at two UK centres; 93 operations were for suspected cancer. The median (range) tumour size was 3.8 (1.2-9) cm. In all, 42 OPNs were imperative for patients with a single kidney (14), synchronous bilateral tumours (20), or renal impairment alone (eight). In 42 patients with a tumour of < or = 4 cm and a normal contralateral kidney the decision to do OPN was considered elective. There were 10 additional operations in seven patients with Von Hippel-Lindau (VHL) disease. In all, 21 OPNs were in the context of a single kidney. RESULTS: In all, 95 OPNs were successfully completed; one operation was abandoned and there were four nephrectomies, including two for bleeding, one for a positive margin on frozen-section analysis, and one for multifocal tumours. The median warm/cold ischaemia time was 20/33 min. The intraoperative/early complication rate was 36%, including a major complication rate of 11% and re-operation rate for primary bleeding of 3%. Of 36 complications, 30 (83%) were in 23 patients with either an imperative indication or VHL. Complications were more common in the imperative/VHL group (59%) than in the elective/other group (12%). Renal function was preserved in 80 of 100 (80%) OPNs overall. Creatinine levels returned to baseline in 11 of 21 (50%) patients with renal impairment before OPN and in 12 of 20 (60%) with a single kidney, whilst five of 21 (24%) with a single kidney needed dialysis after OPN. The median (range) stay after surgery was 6 (3-50) nights. A malignant diagnosis was confirmed in 76 of 93 (82%) specimens on final histopathology. There were 11 of 100 (11%) positive margins, one managed by immediate conversion to nephrectomy and the remaining 10 managed expectantly. After a median (range) follow-up of 24 (1-69) months there were no deaths from kidney cancer, but three patients had local recurrences and two others had developed metastatic recurrence. CONCLUSION: OPN is complex surgery, especially in the imperative setting, but very good results are achievable outside established centres of world renown. It provides good cancer control in the short term with low renal morbidity. These results may act as a reference point in the UK by which to compare results of new treatments for kidney cancer.