Development and evaluation of the MiCheck® Prostate test for clinically significant prostate cancer.

BACKGROUND: There is a clinical need to identify patients with an elevated PSA who would benefit from prostate biopsy due to the presence of clinically significant prostate cancer (CSCaP). We have previously reported the development of the MiCheck® Test for clinically significant prostate cancer. Here, we report MiCheck's further development and incorporation of the Roche Cobas standard clinical chemistry analyzer. OBJECTIVES: To further develop and adapt the MiCheck® Prostate test so it can be performed using a standard clinical chemistry analyzer and characterize its performance using the MiCheck-01 clinical trial sample set. DESIGN, SETTINGS, AND PARTICIPANTS: About 358 patient samples from the MiCheck-01 US clinical trial were used for the development of the MiCheck® Prostate test. These consisted of 46 controls, 137 non-CaP, 62 non-CSCaP, and 113 CSCaP. METHODS: Serum analyte concentrations for cellular growth factors were determined using custom-made Luminex-based R&D Systems multi-analyte kits. Analytes that can also be measured using standard chemistry analyzers were examined for their ability to contribute to an algorithm with high sensitivity for the detection of clinically significant prostate cancer. Samples were then re-measured using a Roche Cobas analyzer for development of the final algorithm. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Logistic regression modeling with Monte Carlo cross-validation was used to identify Human Epidydimal Protein 4 (HE4) as an analyte able to significantly improve the algorithm specificity at 95% sensitivity. A final model was developed using analyte measurements from the Cobas analzyer. RESULTS: The MiCheck® logistic regression model was developed and consisted of PSA, %free PSA, DRE, and HE4. The model differentiated clinically significant cancer from no cancer or not-clinically significant cancer with AUC of 0.85, sensitivity of 95%, and specificity of 50%. Applying the MiCheck® test to all evaluable 358 patients from the MiCheck-01 study demonstrated that up to 50% of unnecessary biopsies could be avoided while delaying diagnosis of only 5.3% of Gleason Score (GS) ≥3+4 cancers, 1.8% of GS≥4+3 cancers and no cancers of GS 8 to 10. CONCLUSIONS: The MiCheck® Prostate test identifies clinically significant prostate cancer with high sensitivity and negative predictive value (NPV). It can be performed in a clinical laboratory using a Roche Cobas clinical chemistry analyzer. The MiCheck® Prostate test could assist in reducing unnecessary prostate biopsies with a marginal number of patients experiencing a delayed diagnosis.

Glypican-1 as a target for fluorescence molecular imaging of bladder cancer.

OBJECTIVES: To investigate whether anti-glypican-1 antibody Miltuximab conjugated with near-infrared dye IRDye800CW can be used for in vivo fluorescence imaging of urothelial carcinoma. METHODS: The conjugate, Miltuximab-IRDye800CW, was produced and characterized by size exclusion chromatography and flow cytometry with glypican-1-expressing cells. Balb/c nude mice bearing subcutaneous urothelial carcinoma xenografts were intravenously injected with Miltuximab-IRDye800CW or control IgG-IRDye800CW and imaged daily by fluorescence imaging. After 10 days, tumors and major organs were collected for ex vivo study of the conjugate biodistribution, including its accumulation in the tumor. RESULTS: The intravenous injection of Miltuximab-IRDye800CW to tumor-bearing mice showed its specific accumulation in the tumors with the tumor-to-background ratio of 12.7 ± 2.4, which was significantly higher than that in the control group (4.6 ± 0.9, P < 0.005). The ex vivo imaging was consistent with the in vivo findings, with tumors from the mice injected with Miltuximab-IRDye800CW being significantly brighter than the organs or the control tumors. CONCLUSIONS: The highly specific accumulation and retention of Miltuximab-IRDye800CW in glypican-1-expressing tumors in vivo shows its high potential for fluorescence imaging of urothelial carcinoma and warrants its further investigation toward clinical translation.

The feasibility of Miltuximab®-IRDye700DX-mediated photoimmunotherapy of solid tumors.

BACKGROUND: Photoimmunotherapy (PIT) is an emerging method of cancer treatment based on the use of a photosensitizer near-infrared dye IRDye700DX (IR700) conjugated to a monoclonal antibody. The antibody selectively delivers IR700 to cancer cells, which can then be killed after photoexcitation. Glypican-1 (GPC-1) is a novel target expressed specifically in malignant tumors. We aimed to investigate whether anti-GPC-1 antibody Miltuximab® (Glytherix Ltd., Sydney, Australia) can be conjugated with IR700 for PIT of solid tumors. METHODS: The dye IR700 was conjugated with Miltuximab® and characterized by spectrophotometry and flow cytometry. Miltuximab®-IR700-mediated PIT was tested in prostate (DU-145), bladder (C3 and T-24), brain (U-87 and U-251) and ovarian (SKOV-3) cancer cell lines. After 1 h incubation with Miltuximab®-IR700, the cells were washed by PBS and illuminated using a 690-nm light-emitting diode. The viability of the cells was assessed by a CCK-8 viability kit 24 h later. RESULTS: Miltuximab®-IR700-mediated PIT caused 67.3-92.3% reduction in viability of cells with medium-high GPC-1 expression and did not affect the viability of GPC-1-low cells. Cytotoxicity was attributed to the targeted binding of the conjugate with subsequent photoactivation, as the conjugate or light exposure alone had no effect on the cell viability. Miltuximab®-IR700 did not induce cytotoxicity in cells blocked by unconjugated Miltuximab®. CONCLUSIONS: PIT with Miltuximab®-IR700 appears to be highly specific and effective against GPC-1-expressing cancer cells, indicating that it holds promise for an effective and safe treatment of early stage solid tumors or as adjuvant therapy following surgical resection. These findings necessitate further investigation of PIT with Miltuximab®-IR700 in other GPC-1-expressing cancer cell lines in vitro and in vivo in xenograft tumor models.

Near-Infrared Molecular Imaging of Glioblastoma by Miltuximab®-IRDye800CW as a Potential Tool for Fluorescence-Guided Surgery.

Glioblastoma (GBM) is one of the most aggressive tumors and its 5-year survival is approximately 5%. Fluorescence-guided surgery (FGS) improves the extent of resection and leads to better prognosis. Molecular near-infrared (NIR) imaging appears to outperform conventional FGS, however, novel molecular targets need to be identified in GBM. Proteoglycan glypican-1 (GPC-1) is believed to be such a target as it is highly expressed in GBM and is associated with poor prognosis. We hypothesize that an anti-GPC-1 antibody, Miltuximab®, conjugated with the NIR dye, IRDye800CW (IR800), can specifically accumulate in a GBM xenograft and provide high-contrast in vivo fluorescent imaging in rodents following systemic administration. Miltuximab® was conjugated with IR800 and intravenously administered to BALB/c nude mice bearing a subcutaneous U-87 GBM hind leg xenograft. Specific accumulation of Miltuximab®-IR800 in subcutaneous xenograft tumor was detected 24 h later using an in vivo fluorescence imager. The conjugate did not cause any adverse events in mice and caused strong fluorescence of the tumor with tumor-to-background ratio (TBR) reaching 10.1 ± 2.8. The average TBR over the 10-day period was 5.8 ± 0.6 in mice injected with Miltuximab®-IR800 versus 2.4 ± 0.1 for the control group injected with IgG-IR800 (p = 0.001). Ex vivo assessment of Miltuximab®-IR800 biodistribution confirmed its highly specific accumulation in the tumor. The results of this study confirm that Miltuximab®-IR800 holds promise for intraoperative fluorescence molecular imaging of GBM and warrants further studies.

Computer-assisted cystoscopy diagnosis of bladder cancer.

OBJECTIVES: One of the most reliable methods for diagnosing bladder cancer is cystoscopy. Depending on the findings, this may be followed by a referral to a more experienced urologist or a biopsy and histological analysis of suspicious lesion. In this work, we explore whether computer-assisted triage of cystoscopy findings can identify low-risk lesions and reduce the number of referrals or biopsies, associated complications, and costs, although reducing subjectivity of the procedure and indicating when the risk of a lesion being malignant is minimal. MATERIALS AND METHODS: Cystoscopy images taken during routine clinical patient evaluation and supported by biopsy were interpreted by an expert clinician. They were further subjected to an automated image analysis developed to best capture cancer characteristics. The images were transformed and divided into segments, using a specialised color segmentation system. After the selection of a set of highly informative features, the segments were separated into 4 classes: healthy, veins, inflammation, and cancerous. The images were then classified as healthy and diseased, using a linear discriminant, the naïve Bayes, and the quadratic linear classifiers. Performance of the classifiers was measured by using receiver operation characteristic curves. RESULTS: The classification system developed here, with the quadratic classifier, yielded 50% false-positive rate and zero false-negative rate, which means, that no malignant lesions would be missed by this classifier. CONCLUSIONS: Based on criteria used for assessment of cystoscopy images by medical specialists and features that human visual system is less sensitive to, we developed a computer program that carries out automated analysis of cystoscopy images. Our program could be used as a triage to identify patients who do not require referral or further testing.

Trends in operative caseload and mortality rates after radical cystectomy for bladder cancer in England for 1998-2010.

BACKGROUND: The Improving Outcomes in Urological Cancers guidelines recommended centralisation of cystectomy services to improve outcomes for bladder cancer (BCa) patients. OBJECTIVE: To investigate trends in all-cause and cause-specific survival to see if there was an improvement in survival after centralisation was implemented. To analyse trends in the number of acute hospital trusts undertaking cystectomy. DESIGN, SETTING, AND PARTICIPANTS: We used routine data to capture information on radical cystectomy (RC) in BCa patients aged 20 yr and older between 1998 and 2010 (n=16,033). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We calculated 30-d and 90-d mortality, and 30-d, 90-d, 1-yr, and 5-yr survival. The average number of RCs per trust was derived. Trends were identified using regression analysis. RESULTS AND LIMITATIONS: The 30-d crude mortality decreased from 5.2% to 2.1% (p<0.001) and 90-d crude mortality decreased from 10.3% to 5.1% (p<0.001). There was an increase in 30-d relative survival from 96% to 98% (p<0.001), in 90-d relative survival from 91% to 96% (p<0.001), in 1-yr relative survival from 71% to 80% (p<0.001), and in 5-yr relative survival from 49% to 56% (2004-2006 data; p<0.001). The mean number of RCs performed by trusts in England increased from six to 24 (p<0.001). Smoking status and stage at diagnosis were not available. CONCLUSIONS: Survival after RC has increased alongside decreases in short-term mortality. There is little evidence of a cohort effect. The trends in survival are linear and we conclude that the continued survival improvements are a result of a combination of service improvements that include service reconfiguration, improved surgical training, neoadjuvant chemotherapy, enhanced recovery principles, and continued improvements in perioperative care. PATIENT SUMMARY: We analysed routinely collected hospital data. Outcomes for patients who undergo cystectomy have improved for all age groups. This is likely to be due to a combination of changes in practice.

The prevalence and natural history of urinary symptoms among recreational ketamine users.

UNLABELLED: Study Type--Symptom prevalence (prospective cohort) Level of Evidence 1b. What's known on the subject? and What does the study add? Case series have described lower urinary tract symptoms associated with ketamine use including severe pain, frequency, haematuria and dysuria. Little is known regarding the frequency of symptoms, relationship of symptoms with dose and frequency of use and natural history of symptoms once the ketamine user has stopped. This study describes the prevalence of ketamine use in a population of recreational drug users in a dance music setting. It shows a dose-frequency relationship with ketamine use. It shows that urinary symptoms associated with recreational ketamine use may lead to a considerable demand on health resources in the primary-, secondary- and emergency-care settings. It shows that symptoms may improve once ketamine use is decreased. OBJECTIVE: • To investigate the prevalence and natural history of urinary symptoms in a cohort of recreational ketamine users. PATIENTS AND METHODS: • A purposeful sampling technique was used. • Between November 2009 and January 2010 participants were invited to undertake an on-line questionnaire promoted by a national dance music magazine and website. • Data regarding demographics and illicit drug-use were collected. • Among respondents reporting recent ketamine use, additional information detailing their ketamine use, experience of urinary symptoms and use of related healthcare services was obtained. RESULTS: • In all, 3806 surveys were completed, of which 1285 (33.8%) participants reported ketamine use within the last year. • Of the ketamine users, 17% were found to be dependent on the drug; 26.6% (340) of recent ketamine users reported experiencing urinary symptoms. • Urinary symptoms were significantly related to both dose of ketamine used and frequency of ketamine use. • Of 251 users reporting their experience of symptoms over time in relationship to their use of ketamine, 51% reported improvement in urinary symptoms upon cessation of use with only eight (3.8%) reporting deterioration after stopping use. CONCLUSIONS: • Urinary tract symptoms are reported in over a quarter of regular ketamine users. • A dose and frequency response relationship has been shown between ketamine use and urinary symptoms. • Both users and primary-care providers need to be educated about urinary symptoms that may arise in ketamine users. A multi-disciplinary approach promoting harm reduction, cessation and early referral is needed to manage individuals with ketamine-associated urinary tract symptoms to avoid progression to severe and irreversible urological pathologies.

Introduction of an enhanced recovery protocol for radical cystectomy.

OBJECTIVE: To describe and assess an enhanced recovery protocol (ERP) for the peri-operative management of patients undergoing radical cystectomy (RC), which was started at our institution on 1 October 2005, as RC is associated with increased morbidity and longer inpatient stays than other major urological procedures. PATIENTS AND METHODS: An ERP was introduced in our institution that focused on reduced bowel preparation, and standardized feeding and analgesic regimens. In all, 112 consecutive patients were compared, i.e. 56 before implementing the ERP and 56 since introducing the ERP. The primary outcome measures were duration of total inpatient stay and interval from surgery to discharge, and the morbidity and mortality. Data were analysed retrospectively from cancer network and hospital records. RESULTS: The demographics of the two groups showed no significant difference in age, gender distribution, American Society of Anesthesiologists grade, or type of urinary diversion. Re-admission, mortality and morbidity rates showed no statistically significant difference between the groups. The median (interquartile range) duration of hospital stay was 17 (15-23) days in the no-ERP group, and 13 (11-17) days in the ERP group (significantly different, P < 0.001, Wilcoxon rank-sum test). The median duration of recovery after RC was 15 (13-21) days in the no-ERP group and 12 (10-15) days in the ERP group (significantly different, P = 0.001, Wilcoxon rank-sum test). CONCLUSION: The introduction of an ERP was associated with significantly reduced hospital stay, with no deleterious effect on morbidity or mortality.

Differences and commonalities in the management of locally advanced prostate cancer: results from a survey of oncologists and urologists in the UK.

OBJECTIVE: To determine the management practices used by UK oncologists and urologists for patients with locally advanced (non-metastatic) prostate cancer. METHODS: Using a postal questionnaire, 155 practising specialist oncologists and urologists were surveyed in the UK. Their views were sought on a multidisciplinary approach to the management of locally advanced prostate cancer and their current management practices. RESULTS: Over half of respondents recognized the need for both oncologists and urologists to take the lead in management decisions, but almost as many still expected the sole responsibility to lie within their own speciality. Radical radiotherapy (RT) was considered the current optimum treatment by most respondents, but 22% of urologists thought that radical prostatectomy is optimal. Most responders would use luteinizing hormone-releasing hormone agonists as neoadjuvant and adjuvant to RT but there was significant variation in the favoured duration of treatment of these drugs, and in the dose of RT. CONCLUSION: This survey suggests that there are still wide variations in the management practices for locally advanced prostate cancer in the UK, and between urologists and oncologists. Improved consensus guidelines are required.

Hereditary urological cancer syndromes.

Hereditary urological cancer syndromes are rare, but it is important that they are recognized because they have important prognostic implications; prompt diagnosis can dramatically improve patient outcomes. The urologist or urological oncologist should, therefore, ascertain which tumors of the many seen in clinical practice warrant referral for the opinion of a clinical geneticist. Despite the aggressive natural history of most inherited urological cancer syndromes, organ-preserving treatments are desirable because these syndromes predispose affected patients to the formation of multifocal and metachronous tumors. Identification of the molecular mechanisms that underlie carcinogenesis in both familial and sporadic urological cancers has, in some cases, resulted in novel and specifically targeted approaches to therapy. Patients who present with early-onset or multiple tumors should be carefully investigated for the presence of a hereditary cancer syndrome, and once a diagnosis is made, appropriate screening should be instigated for family members to enable early detection of tumors both within and outside the urogenital tract.

VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression.

Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF(165)b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF(165)b binds VEGF receptor 2 with the same affinity as VEGF(165) but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF(165)-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF(165)b is not angiogenic and that it inhibits VEGF(165)-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF(165)b expressing tumors grow significantly more slowly than VEGF(165)-expressing tumors, indicating that a switch in splicing from VEGF(165) to VEGF(165)b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein.

Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF165b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123-4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF165 (stimulatory) and VEGF165b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF189 mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF165 to VEGF165b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF165b siRNA to 20+/-11% of the level of mock-transfected cells (P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109+/-8% of mismatch siRNA, P > 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF165b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.

Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes.

Vascular endothelial growth factor (VEGF) is expressed by renal glomerular epithelial cells (podocytes) and is thought to be protective against nephrotoxic agents. VEGF has been shown to be an autocrine survival factor in neuropilin-1-positive, VEGF receptor-negative breast carcinoma cells. Normal human podocytes are also known to express neuropilin-1, VEGF, and are VEGF-R2 negative. Here, we investigated whether a similar VEGF autocrine loop may exist in podocytes. Podocyte cytosolic calcium concentration ([Ca(2+)](i)) was analyzed in primary cultured and conditionally immortalized podocytes using ratiometric fluorescence measurement. Cytotoxicity was determined by lactate dehydrogenase assay, proliferation by [(3)H]-thymidine incorporation, and cell counts by hemocytometric assay. VEGF decreased [Ca(2+)](i) in primary podocytes (from 179 +/- 36 to 121 +/- 25 nM, P < 0.05) and conditionally immortalized podocytes (from 95 +/- 10 to 66 +/- 8 nM, P < 0.02) in the absence of extracellular calcium. The type III receptor tyrosine-kinase inhibitor PTK787/ZK222584 abolished this reduction. VEGF increased podocyte [(3)H]-thymidine incorporation (3,349 +/- 283 cpm, control 2,364 +/- 301 cpm, P < 0.05) and cell number (4.5 +/- 0.7 x 10(4)/ml, control 2.6 +/- 0.5 x 10(4)/ml, P < 0.05) and decreased cytotoxicity (5.9 +/- 0.7%, control 12 +/- 3%, P < 0.05), whereas a monoclonal antibody to VEGF increased cytotoxicity. Electron microscopy of normal human glomeruli demonstrated that the glomerular VEGF is mostly podocyte cell membrane associated. These results indicate that one of the functions of VEGF secreted from podocytes may be to act as an autocrine factor on calcium homeostasis and cell survival.

Beware the BCG failures: a review of one institution's results.

OBJECTIVE: To review the outcome of all superficial transitional cell (TCC) bladder cancer treated with intravesical Bacille Calmette-Guerin (BCG) at one institution and, in particular, the prognosis for those patients who gained little benefit from BCG therapy. PATIENTS AND METHODS: The notes of 122 patients treated with BCG over a nine-year period were reviewed. The following details were recorded: time of diagnosis; time of decision to treat with BCG; results of cystoscopies before and after BCG; duration of follow up; time of progression if occurred, mortality and cause of death. RESULTS: Complete follow up data was available for 112 patients. At a median follow up of 23 months (range 3-107) 57 patients (51%) remained free of tumour, 30 (27%) had progressed and 18 (16%) had died of transitional cell carcinoma. There was a significant association between a positive initial check cystoscopy and subsequent progression (p<0.001) and disease specific mortality (p<0.001). Of the 35 patients who had a positive cystoscopy after BCG treatment 21 (60%) progressed and 14 (40%) died of transitional cell carcinoma compared with 9 (12%) and 4 (5%) of the 77 with a negative cystoscopy. Adjusted odds ratios for progression and death from TCC for patients with a positive initial check cystoscopy were 21 and 13, respectively. CONCLUSION: In our series the patients found to have tumour at the initial check cystoscopy following intravesical BCG had a poor prognosis. This should be remembered when considering treatment options and counselling patients. Follow up of all BCG patients need to be rigorous and protocols would help to unify the treatment patients receive.