Predicting Adherent Perinephric Fat Using Preoperative Clinical and Radiological Factors in Patients Undergoing Partial Nephrectomy.

BACKGROUND: The decision to perform a partial nephrectomy (PN) relies largely upon the complexity of the renal mass and its surrounding anatomy. The presence of adherent perinephric fat (APF) can increase surgical complexity and extend operative times. The accurate prediction of APF may improve surgical planning and aid in decision making for the surgical approach. OBJECTIVE: We sought to develop and externally validate a score that predicts APF based on preoperative clinical and radiological prognostic factors. DESIGN, SETTING, AND PARTICIPANTS: We retrospectively analyzed 495 consecutive patients who underwent open or minimally invasive PN. APF was defined as the presence of "dense," "adherent," or "sticky" perinephric fat at the time of dissection by the surgeon, and this did not require subcapsular dissection. Additionally, we analyzed an independent cohort of 285 patients for external validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A score model was developed using multivariate logistic regression analysis. Calibration of the fitted model was assessed graphically with a plot of the predicted versus the actual probability of APF, and discrimination was assessed by calculating the area under the receiver operating characteristic curve. RESULTS AND LIMITATIONS: Of the 495 patients, 95 (19%) had APF. Patients with APF had longer operative (p=0.02) and arterial clamp (p=0.01) times than non-APF patients. On multivariate analyses, diabetes mellitus (p=0.009), posterior perinephric fat thickness (p<0.001), and perinephric stranding (p<0.001) were predictors of encountering APF in PN. A risk score ranging from 0 to 4 was developed based on these three variables to predict APF. The scoring system demonstrated good discrimination of 0.82 and 0.84 for the development and external validation cohorts, respectively. CONCLUSIONS: The APF score can accurately predict the presence of APF in patients with a small renal mass who are planning to undergo PN. This score could aid in pre- and intraoperative planning and impact the surgical approach. PATIENT SUMMARY: The presence of "sticky" fat surrounding the kidney in patients undergoing partial nephrectomy has previously been linked to longer operative times, intraoperative complications, and surgical conversion. In our study, we found that this feature is more often presented in patients with diabetes mellitus, and thicker and more inflammatory fat on renal imaging. Based on these findings, we developed a risk score that can accurately predict this feature before surgery, in order to improve surgical planning and better counsel the patients.

Surveyed dermatologists are less likely to curette invasive squamous cell carcinoma in solid organ transplant recipients.

BACKGROUND: The risk of squamous cell carcinoma (SCC) is increased in solid organ transplant recipients (OTRs), and preferential treatment modalities vary among clinicians. OBJECTIVES: The purpose of this study was to survey dermatologists regarding practice patterns for electrodesiccation and curettage (EDC) of SCC in OTRs and nontransplant patients. METHODS: An 18-question survey was sent to dermatologist members of the International Transplant Skin Cancer Collaborative, Association of Professors of Dermatology, and American College of Mohs Surgery. Differences in EDC practice patterns for treatment of SCC in OTRs and nontransplant patients were evaluated. RESULTS: Dermatologists in this study (N = 227) were more likely to treat SCC with EDC in nontransplant patients (67.4%) than in OTRs (48.0%; P = .0003).Dermatologists who perform EDC in both groups (n = 108) were unlikely to use EDC on the H-zone of the face; they were more likely to EDC tumors on non-H-zone areas of the face and neck in nontransplant patients compared to OTRs (P = .0007). Dermatologists were more likely to use EDC over surgery in nontransplant patients compared to OTRs with the following demographics: dementia or psychiatric disease (P = .04), multiple medical comorbidities (P = .03), or anticoagulation medications (P = .02). CONCLUSIONS: In OTRs with SCC, 48% of clinicians would consider EDC. The main factors that affect the decision to perform EDC include tumor location and patient comorbidities.

Incidence of androgen receptor and androgen receptor variant 7 coexpression in prostate cancer.

BACKGROUND: Prostate cancer (PRCA) is an androgen-driven disease, where androgens act through the androgen receptor (AR) to induce proliferation and survival of tumor cells. Recently, AR splice variant 7 (ARv7) has been implicated in advanced stages of PRCA and clinical recurrence. With the widespread use of AR-targeted therapies, there has been a rising interest in the expression of full-length AR and ARv7 in PRCA progression and how these receptors, both independently and together, contribute to adverse clinicopathologic outcomes. METHODS: Despite a multitude of studies measuring the expression levels of AR and ARv7 in PRCA progression, the results have been inconsistent and sometimes contradictory due to technical and analytical discrepancies. To circumvent these inconsistencies, we used an automated multiplexed immunostaining platform for full-length AR and ARv7 in human PRCA samples and objectively quantified expression changes with machine learning-based software. With this technology, we can assess receptor prevalence both independently, and coexpressed, within specific tissue and cellular compartments. RESULTS: Full-length AR and ARv7 expression increased in epithelial nuclei of metastatic samples compared to benign. Interestingly, a population of cells with undetectable AR persisted through all stages of PRCA progression. Coexpression analyses showed an increase of the double-positive (AR+ /ARv7+ ) population in metastases compared to benign, and an increase of the double-negative population in PRCA samples compared to benign. Importantly, analysis of clinicopathologic outcomes associated with AR/ARv7 coexpression showed a significant decrease in the double-positive population with higher Gleason score (GS), as well as in samples with recurrence in under 5 years. Conversely, the double-negative population was significantly increased in samples with higher GS and in samples with recurrence in under 5 years. CONCLUSIONS: Changes in AR and ARv7 coexpression may have prognostic value in PRCA progression and recurrence. A better understanding of the prevalence and clinicopathologic outcomes associated with changes in these receptors' coexpression may provide a foundation for improved diagnosis and therapy for men with PRCA.

Tissue-specific quantification and localization of androgen and estrogen receptors in prostate cancer.

Androgens and estrogens, working together, promote prostate cancer (PRCA) initiation and progression, with androgens acting via androgen receptor (AR) and estrogens acting primarily through estrogen receptor α (ERα). While the interplay between these steroid hormones has been established, the interaction between steroid hormone receptors in prostatic disease remains unstudied. The goal of this study was to objectively determine the incidence, stage specificity, and tissue/cell type specificity of AR and ERα expression, both independently and simultaneously, during the progression of PRCA. Using multiplexed immunohistochemistry and multispectral imaging analysis, AR, ERα, and smooth muscle α-actin expression was detected and quantitated in benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), PRCA, and metastasis (MET) from patient specimens (n=340). Epithelial AR expression was significantly increased in HGPIN, PRCA, and MET compared with BPT, whereas ERα expression in epithelial and stromal cells was highest in HGPIN. With analysis of AR and ERα coexpression, we identified a unique population of double-positive (AR+/ERα+) cells that increased in HGPIN specimens in both the stroma and the epithelium. Double-negative (AR-/ERα-) cells significantly decreased across PRCA progression, from 65% in BPT to 30% in MET. Preliminary analysis of this AR+/ERα+ population indicates potential cell type specificity in smooth muscle α-actin-negative stromal cells. This study demonstrates stage-, tissue-, and cell type-specific AR and ERα expression changes during PRCA progression, both independently and coexpressed. A more complete understanding of steroid hormones and their receptors in the initiation and progression of prostatic disease may elucidate improved strategies for PRCA prevention or therapy.

Risk Factors for Basal Cell Carcinoma in Men Younger Than 40 Years: A Case-Control Study.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignancy in the United States and is more prevalent in older populations. OBJECTIVE: The aim of this study was to investigate BCC risk factors in male patients younger than 40 years. MATERIALS AND METHODS: A consecutive series of male patients with pathology-proven BCC and younger than 40 years at time of diagnosis were retrospectively identified along with matched controls. Phone interviews were conducted using a structured questionnaire, and differences between patients with and without BCC were investigated. RESULTS: A total of 50 patients with BCC and 27 controls were included in this study. Compared with controls, patients with BCC worked outdoor jobs for longer lengths of time (43.2 vs 15.6 months; p = .04), were more likely to have a family history of skin cancer (66% vs 44%; p = .02), and were more likely to use sunscreen heavily after biopsy (p = .02). Patients with multiple BCCs (n = 20) were more likely to have a history of substantial recreational sun exposure (p = .01) than patients with solitary lesions (n = 30). CONCLUSION: The authors conclude that outdoor sun exposure in patients with underlying genetic susceptibility is the most likely mechanism of BCC formation in young male patients.

A tale of two clones: Caldesmon staining in the differentiation of cutaneous spindle cell neoplasms.

BACKGROUND: We sought to compare the sensitivity and specificity of 2 different caldesmon antibodies in differentiating leiomyosarcoma from other cutaneous spindle cell neoplasms. METHODS: Representative cutaneous spindle cell neoplasms were identified, including leiomyosarcoma, atypical fibroxanthoma, dermatomyofibroma and spindle cell squamous cell carcinoma. Immunohistochemistry was performed with antibodies directed toward caldesmon, smooth-muscle actin (SMA) and desmin. Sensitivity and specificity were calculated using grades from 3 independent observers. RESULTS: The sensitivity of caldesmon (Ventana) was 100% (95% CI 78.2%-100%) and the specificity was 8.3% (2.8%-18.4%). Because this stain appeared to be non-specific, additional testing was performed on the same set of specimens using a second caldesmon clone (H-caldesmon, Dako), which had a sensitivity of 53.9% (25.1%-80.8%) and specificity of 96.6% (88.1%-99.6%). The sensitivity and specificity of SMA were 85.7% (57.2%-98.2%) and 84.5% (72.6%-92.7%), respectively. The sensitivity of desmin was 53.3% (26.6%-78.7%) with a specificity of 100% (94.0%-100%). CONCLUSIONS: The Ventana caldesmon clone is not specific to smooth muscle, a potential pitfall to laboratories using this clone. The staining pattern, sensitivity and specificity of the Dako H-caldesmon antibody clone are similar to results from prior studies. The sensitivity and specificity of the Dako clone support its use in smooth muscle identification as an additional marker in challenging cases.

Extramammary Paget's disease of the scrotum with complete response to imiquimod and photodynamic therapy.

Extramammary Paget's disease (EMPD) is a rare intraepithelial neoplasm with an extremely variable clinical course. The objective of this study was to determine if combination imiquimod and photodynamic therapy could induce remission of EMPD. A 69-year-old man with EMPD was treated with topical imiquimod 5% cream at night for 5 days per week for 1 month, followed by 2 months of 5% imiquimod for three nights a week. For the following 6 months, monthly 5-aminolevulinic acid photodynamic therapy was added. After 6 months, imiquimod was discontinued and the patient continued to be treated with quarterly photodynamic therapy. Treatment resulted in significant improvement in the appearance of the lesion, and pathology revealed no evidence of residual disease. The patient has had no clinical signs of disease for >5 years. We conclude that topical imiquimod 5% cream and photodynamic therapy may aid in the treatment of some patients with EMPD.

Catheterization alters bladder ecology to potentiate Staphylococcus aureus infection of the urinary tract.

Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of catheter-associated urinary tract infection (CAUTI), which frequently progresses to more serious invasive infections. We adapted a mouse model of CAUTI to investigate how catheterization increases an individual's susceptibility to MRSA UTI. This analysis revealed that catheterization was required for MRSA to achieve high-level, persistent infection in the bladder. As shown previously, catheter placement induced an inflammatory response resulting in the release of the host protein fibrinogen (Fg), which coated the bladder and implant. Following infection, we showed that MRSA attached to the urothelium and implant in patterns that colocalized with deposited Fg. Furthermore, MRSA exacerbated the host inflammatory response to stimulate the additional release and accumulation of Fg in the urinary tract, which facilitated MRSA colonization. Consistent with this model, analysis of catheters from patients with S. aureus-positive cultures revealed colocalization of Fg, which was deposited on the catheter, with S. aureus Clumping Factors A and B (ClfA and ClfB) have been shown to contribute to MRSA-Fg interactions in other models of disease. We found that mutants in clfA had significantly greater Fg-binding defects than mutants in clfB in several in vitro assays. Paradoxically, only the ClfB- strain was significantly attenuated in the CAUTI model. Together, these data suggest that catheterization alters the urinary tract environment to promote MRSA CAUTI pathogenesis by inducing the release of Fg, which the pathogen enhances to persist in the urinary tract despite the host's robust immune response.

Hoarseness as a presentation of mycosis fungoides infiltrating the larynx.

Laryngeal involvement is a rare manifestation of mycosis fungoides (MF), with only nine reported cases of cutaneous T cell lymphoma with laryngeal or vocal cord involvement. Herein, we report the case of a patient with a 7-year history of MF who presented to the emergency department with hoarseness, throat tightness and cough, as well as erythroderma and skin tumours. Laryngoscopy and CT imaging were concerning for lymphomatous involvement of the left false vocal cord. A biopsy was taken of the false vocal cord lesion, which revealed an aberrant immunophenotype consistent with MF. The patient was started on doxorubicin with initial rapid improvement in symptoms. Within 2 months, her respiratory status and skin involvement worsened. Subsequent studies showed bone marrow involvement. The patient expired 4 months after original presentation. This report describes the patient's presentation and clinical course, and reviews the literature on vocal cord and laryngeal involvement of MF.

Patient and nonradiographic tumor characteristics predicting lipid-poor angiomyolipoma in small renal masses: Introducing the BEARS index.

PURPOSE: To create a simple model using clinical variables for predicting lipid-poor angiomyolipoma (AML) in patients with small renal masses presumed to be renal cell carcinoma (RCC) from preoperative imaging. MATERIALS AND METHODS: A series of patients undergoing partial nephrectomy (PN) for renal masses ≤4 cm was identified using a prospectively maintained database. Patients were excluded if standard preoperative imaging was not consistent with RCC. Chi square and Mann-Whitney U analyses were used to evaluate differences in characteristics between patients with AML and other types of pathology. A logistic regression model was constructed for multivariable analysis of predictors of lipid-poor AML. RESULTS: A total of 730 patients were identified that underwent PN for renal masses ≤4 cm between 2007-2015, including 35 with lipid-poor AML and 620 with RCC. In multivariable analysis, the following features predicted AML: female sex (odds ratio, 6.89; 95% confidence interval, 2.35-20.92; p<0.001), age <56 years (2.84; 1.21-6.66; p=0.02), and tumor size <2 cm (5.87; 2.70-12.77; p<0.001). Sex, age, and tumor size were used to construct the BEnign Angiomyolipoma Renal Susceptibility (BEARS) index with the following point values for each particular risk factor: female sex (2 points), age <56 years (1 point), and tumor size <2 cm (2 points). Within the study population, the BEARS index distinguished AML from malignant lesions with an area under the curve of 0.84. CONCLUSIONS: Young female patients with small tumors are at risk for having lipid-poor AML despite preoperative imaging consistent with RCC. Identification of these patients may reduce the incidence of unnecessary PN for benign renal lesions.

Predictive Nomogram for Recurrence following Surgery for Nonmetastatic Renal Cell Cancer with Tumor Thrombus.

PURPOSE: Following surgery for nonmetastatic renal cell carcinoma with tumor thrombus the risk of recurrence is significant but variable among patients. The purpose of this study was to develop and validate a predictive nomogram for individual estimation of recurrence risk following surgery for renal cell carcinoma with venous tumor thrombus. MATERIALS AND METHODS: Comprehensive data were collected on patients with nonmetastatic renal cell carcinoma and thrombus treated at a total of 5 institutions from 2000 to 2013. Independent predictors of recurrent renal cell carcinoma from a competing risks analysis were developed into a nomogram. Predictive accuracy was compared between the development and validation cohorts, and between the nomogram and the UISS (UCLA Integrated Staging System, SSIGN (Stage, Size, Grade and Necrosis) and Sorbellini models. RESULTS: A total of 636 patients were analyzed, including the development cohort of 465 and the validation cohort of 171. Independent predictors, including tumor diameter, body mass index, preoperative hemoglobin less than the lower limit of normal, thrombus level, perinephric fat invasion and nonclear cell histology, were developed into a nomogram. Estimated 5-year recurrence-free survival was 49% overall. Five-year recurrence-free survival in patients with 0, 1, 2 and more than 2 risk factors was 77%, 53%, 47% and 20%, respectively. Predictive accuracy was similar in the development and validation cohorts (AUC 0.726 and 0.724, respectively). Predictive accuracy of the thrombus nomogram was higher than that of the UISS (AUC 0.726 vs 0.595, p = 0.001), SSIGN (AUC 0.713 vs 0.612, p = 0.04) and Sorbellini models (AUC 0.709 vs 0.638, p = 0.02). CONCLUSIONS: We present a predictive nomogram for postoperative recurrence in patients with nonmetastatic renal cell carcinoma with venous thrombus. Improving individual postoperative risk assessment may allow for better design and analysis of future adjuvant clinical trials.

Partial Nephrectomy for Presumed Renal-Cell Carcinoma: Incidence, Predictors, and Perioperative Outcomes of Benign Lesions.

BACKGROUND: The aim of this study was to investigate the incidence of benign histology after partial nephrectomy (PN) in patients with presumed malignancy from preoperative imaging. Furthermore, preoperative predictors of benign lesions and perioperative outcomes were also assessed. METHODS: A series of patients undergoing PN for renal masses was identified using a prospectively maintained database. Patients were excluded for known genetic conditions, if more than one renal mass was resected, or if standard preoperative imaging was not suspicious for renal-cell carcinoma (RCC). Differences in characteristics between patients with benign and malignant pathology were assessed. RESULTS: A total of 916 patients were identified who underwent PN between 2007 and 2015, including 129 (14.1%) patients with a final diagnosis of benign disease. The most common types of benign pathology were oncocytoma (n = 66, 51.2%), angiomyolipoma (n = 37, 28.7%), and complex cysts (n = 10, 7.8%). Low body mass index (BMI) [0.96 (0.92-0.99) p = 0.02], low R.E.N.A.L. score [0.86 (0.76-0.96) p = 0.007], and low preoperative creatinine [0.37 (0.14-0.91) p = 0.04] predicted benign histology in multivariate analysis. Tumor size was a significant predictor in additional modeling [0.81 (0.69-0.94) p = 0.008]. Patients with benign histology had significantly shorter operative times (p < 0.001) and less estimated blood loss (p < 0.001), and there was no difference in complication (p = 0.93) or blood transfusion (0.24) rates. CONCLUSIONS: In this study, the rate of benign pathology after PN for presumed RCC is 14.1%. BMI, R.E.N.A.L. score, and preoperative creatinine are predictive of benign histology, but the ability of different variables to predict benign lesions may be influenced by the distribution of benign tumor subtypes, reflecting potential unidentified selection bias.

Computed Tomography and Magnetic Resonance Findings of Fat-Poor Angiomyolipomas.

INTRODUCTION: Approximately 5% of angiomyolipomas (AMLs) are classified as "fat poor" due to lack of visually detectable fat on imaging, making them difficult to distinguish from renal cell carcinoma. Recent investigations have proposed CT and MR imaging features suggestive of fat-poor AML (fp-AML). Herein, we determined the frequency of these features in a cohort of fp-AMLs by retrospective review of preoperative imaging. METHODS: A pathology database query from January 2005 to August 2013 identified 49 renal specimens of AML with available imaging. A retrospective review of all CT and MR images of these 49 cases was conducted. Cases with visually detectable fat on imaging were excluded. RESULTS: A total of 26 fp-AMLs were identified. Thirteen lesions had available unenhanced CT images, of which eight (62%) were hyperdense compared to the adjacent renal parenchyma, while five (38%) were isodense. Twenty lesions had enhanced CT images: 14 (70%) and 6 (30%) with homogeneous and heterogeneous enhancement, respectively. Of the nine lesions with enhanced MR sequences, five (56%) were homogeneously enhancing, and four (44%) were heterogeneously enhancing. Eight of nine (89%) lesions had hypointense signal intensity (SI) on T2-weighted MR sequences, while one (11%) had hyperintense SI. None of the eight lesions displayed a decrease in signal on fat-suppressed sequences. CONCLUSIONS: In this study, we confirmed common imaging features of fp-AML: high attenuation on unenhanced CT sequences, homogeneous enhancement on CT, and hypointensity on T2-weighted MR. When these features are present, a renal mass biopsy may be prudent.

Is extended preoperative antibiotic prophylaxis for high-risk patients necessary before percutaneous nephrolithotomy?

PURPOSE: The goal of this study was to compare the rate of systemic inflammatory response syndrome (SIRS) in high-risk patients undergoing percutaneous nephrolithotomy (PCNL) between patients who received 7, 2, or 0 days of preoperative antibiotics. MATERIALS AND METHODS: We retrospectively reviewed a series of consecutive PCNLs performed at our institution. Patients with infected preoperative urine cultures were excluded. High-risk patients were defined as those with a history of previous urinary tract infection (UTI), hydronephrosis, or stone size ≥2 cm. Patients were treated with 7, 2, or 0 days of preoperative antibiotic prophylaxis prior to PCNL. All patients received a single preoperative dose of antibiotics within 60 minutes of the start of surgery. Fisher exact test was used to compare the rate of SIRS by preoperative antibiotic length. RESULTS: Of the 292 patients identified, 138 (47.3%) had sterile urine and met high-risk criteria, of which 27 (19.6%), 39 (28.3%), and 72 (52.2%) received 7, 2, and 0 days of preoperative antibiotics, respectively. The 3 groups were similar in age, sex, and duration of surgery (p>0.05). There was no difference in the rate of SIRS between the groups, with 1 of 27 (3.7%), 2 of 39 (5.1%) and 3 of 72 patients (4.2%) meeting criteria in the 7, 2, and 0 days antibiotic groups (p=~1). CONCLUSIONS: Extended preoperative antibiotic prophylaxis was not found to reduce the risk of SIRS after PCNL in our institutional experience of high-risk patients. For these patients, a single preoperative dose of antibiotics is sufficient.

Antibody-Based Therapy for Enterococcal Catheter-Associated Urinary Tract Infections.

Gram-positive bacteria in the genus Enterococcus are a frequent cause of catheter-associated urinary tract infection (CAUTI), a disease whose treatment is increasingly challenged by multiantibiotic-resistant strains. We have recently shown that E. faecalis uses the Ebp pilus, a heteropolymeric surface fiber, to bind the host protein fibrinogen as a critical step in CAUTI pathogenesis. Fibrinogen is deposited on catheters due to catheter-induced inflammation and is recognized by the N-terminal domain of EbpA (EbpANTD), the Ebp pilus's adhesin. In a murine model, vaccination with EbpANTD confers significant protection against CAUTI. Here, we explored the mechanism of protection using passive transfer of immune sera to show that antisera blocking EbpANTD-fibrinogen interactions not only is prophylactic but also can act therapeutically to reduce bacterial titers of an existing infection. Analysis of 55 clinical CAUTI, bloodstream, and gastrointestinal isolates, including E. faecalis, E. faecium, and vancomycin-resistant enterococci (VRE), revealed a diversity of levels of EbpA expression and fibrinogen-binding efficiency in vitro Strikingly, analysis of 10 strains representative of fibrinogen-binding diversity demonstrated that, irrespective of EbpA levels, EbpANTD antibodies were universally protective. The results indicate that, despite diversity in levels of fibrinogen binding, strategies that target the disruption of EbpANTD-fibrinogen interactions have considerable promise for treatment of CAUTI. IMPORTANCE: Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections.

Neovascularity as a prognostic marker in renal cell carcinoma.

Endothelial markers platelet and endothelial cell adhesion molecule (PECAM-1), cluster of differentiation (CD31) and endoglin (CD105) may be used to identify endothelium and activated endothelium, respectively, with the CD105/CD31 ratio used to measure neovascularity. This study investigated the hypothesis that neovascularity in renal cell carcinoma (RCC) is associated with more aggressive RCC tumors and can be used to predict oncological outcomes. Multiplexed immunohistochemistry using antibodies to detect endoglin and PECAM-1 was performed on tissue microarray of benign kidney samples and RCC tumors including clear cell, papillary, chromophobe, and collecting duct and unclassified tumors (combined for statistics), and multispectral imaging was used for analysis. The CD105/CD31 ratio was compared with clinical and pathologic features of RCC as well as clinical outcomes after surgery using Cox proportional hazards regression and Kaplan-Meier analysis. A total of 502 tumor samples and 122 normal kidney samples from 251 RCC patients were analyzed. The average CD105/CD31 expression ratio, an indicator of neovascularization, was increased in higher pathologic stage tumors (P< .0001). Among RCC morphotypes, the ratio was lower in papillary RCC morphotype tumors (P= .001) and higher in collecting duct/unclassified tumors (P= .0001) compared with clear cell RCC. Among nuclear grades, grade 4 RCC displayed a significantly elevated CD105/CD31 ratio (P< .0001). In multivariable analysis, increased neovascularity was associated with decreased overall survival (hazard ratio, 1.54 [95% confidence interval, 1.06-2.23]; P= .02). In patients receiving anti-vascular endothelial growth factor therapy (VEGF, n = 13) for metastatic RCC, a low CD105/CD31 ratio was associated with increased survival (P= .02). We conclude that higher neovascularity is associated with worse outcomes after surgery for RCC. The ratio of CD105/CD31 expression is a potential indicator of response to anti-VEGF therapy.

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Expression and colocalization of ß-catenin and lymphoid enhancing factor-1 in prostate cancer progression.

The purpose of this study was to objectively investigate ß-catenin and LEF1 abundance, subcellular localization, and colocalization across benign and staged prostate cancer (PCa) specimens. A tissue microarray containing tumor-adjacent histologically benign prostate tissue (BPT; n = 48 patients), high-grade prostatic intraepithelial neoplasia (HGPIN; n = 25), localized PCa (n = 42), aggressive PCa (n = 31), and metastases (n = 22) was stained using multiplexed immunohistochemistry with antibodies toward E-cadherin, ß-catenin, and LEF1. Multispectral imaging was used for quantitation, and protein expression and colocalization was evaluated across PCa progression. Stromal nuclear ß-catenin abundance was greater in HGPIN and PCa compared with BPT (P < .05 for both), and epithelial nuclear ß-catenin abundance was lower in metastatic PCa than in BPT (P < .05 for both). Epithelial and stromal nuclear LEF1 abundance was greater in HGPIN compared with BPT, whereas epithelial nuclear LEF1 was also greater in metastases. The proportion of epithelial and stromal nuclear double-positive ß-catenin(+)/LEF1(+) cells was greater in HGPIN compared with BPT. In addition, the proportion of epithelial ß-catenin(+)/LEF1(+) cells was greater in localized PCa and metastases compared with BPT. A significant amount of stromal cells were positive for LEF1 but not ß-catenin. ß-Catenin and LEF1 abundance were negatively correlated in the epithelium (P < .0001) but not the stroma (P > .05). We conclude that ß-catenin and LEF1 colocalization is increased in HGPIN and metastasis relative to BPT, suggesting a role for ß-catenin/LEF1-mediated transcription in both malignant transformation and metastasis of PCa. Furthermore, our results suggest that LEF1 abundance alone is not a reliable readout for ß-catenin activity in prostate tissues.