Trevor A. Flood

Association of the Ste20-like Kinase (SLK) with the Microtubule ROLE IN Rac1-MEDIATED REGULATION OF ACTIN DYNAMICS DURING CELL ADHESION AND SPREADING

Cytoskeletal remodeling events are tightly regulated by signal transduction systems that impinge on adhesion components and modulators of cellular architecture. We have previously shown that the Ste20-like kinase (SLK) can induce apoptosis through the induction of actin disassembly and cellular retraction (Sabourin, L. A., Tamai, K., Seale, P., Wagner, J., and Rudnicki, M. A. (2000) Mol. Cell. Biol. 20, 684–696). Using immunofluorescence studies, we report that SLK is redistributed with adhesion components at large podosome-like adhesion sites in fibronectin-stimulated fibroblasts. However, in vitrokinase assays demonstrate that its activity is not modulated following fibronectin stimulation. Double immunofluorescence studies in exponentially growing or spreading fibroblasts show that SLK is associated with the microtubule network and can be coprecipitated with α-tubulin. Furthermore, the stimulation of adhesion site formation by microtubule-disrupting agents induces the relocalization of SLK with unpolymerized α-tubulin to large vinculin-containing adhesion complexes. Using microinjection studies, we show that ectopic expression of activated SLK induces the disassembly of actin stress fibers, a process that can be inhibited by dominant negative Rac1. Significantly, endogenous SLK can be colocalized with Rac1 in spreading cells on FN. Finally, the overexpression of SLK by adenoviral infection inhibits cell spreading on fibronectin. These results suggest that SLK is part of a microtubule-associated complex that is targeted to adhesion sites and implicated in the regulation of cytoskeletal dynamics.

Multiparametric MRI of solid renal masses: pearls and pitfalls.

Functional imaging [diffusion-weighted imaging (DWI) and dynamic contrast enhancement (DCE)] techniques combined with T2-weighted (T2W) and chemical-shift imaging (CSI), with or without urography, constitutes a comprehensive multiparametric (MP) MRI protocol of the kidneys. MP-MRI of the kidneys can be performed in a time-efficient manner. Breath-hold sequences and parallel imaging should be used to reduce examination time and improve image quality. Increased T2 signal intensity (SI) in a solid renal nodule is specific for renal cell carcinoma (RCC); whereas, low T2 SI can be seen in RCC, angiomyolipoma (AML), and haemorrhagic cysts. Low b-value DWI can replace conventional fat-suppressed T2W. DWI can be performed free-breathing (FB) with two b-values to reduce acquisition time without compromising imaging quality. RCC demonstrates restricted diffusion; however, restricted diffusion is commonly seen in AML and in chronic haemorrhage. CSI must be performed using the correct echo combination at 3 T or T2* effects can mimic intra-lesional fat. Two-dimensional (2D)-CSI has better image quality compared to three-dimensional (3D)-CSI, but volume averaging in small lesions can simulate intra-lesional fat using 2D techniques. SI decrease on CSI is present in both AML and clear cell RCC. Verification of internal enhancement with MRI can be challenging and is improved with image subtraction. Subtraction imaging is prone to errors related to spatial misregistration, which is ameliorated with expiratory phase imaging. SI ratios can be used to confirm subtle internal enhancement and enhancement curves are predictive of RCC subtype. MR urography using conventional extracellular gadolinium must account for T2* effects; however, gadoxetic acid enhanced urography is an alternative. The purpose of this review it to highlight important technical and interpretive pearls and pitfalls encountered with MP-MRI of solid renal masses.

Whole-Tumor Quantitative Apparent Diffusion Coefficient Histogram and Texture Analysis to Predict Gleason Score Upgrading in Intermediate-Risk 3 + 4 = 7 Prostate Cancer

OBJECTIVE The objective of our study was to evaluate whole-lesion quantitative apparent diffusion coefficient (ADC) for the prediction of Gleason score (GS) upgrading in 3 + 4 = 7 prostate cancer. MATERIALS AND METHODS Fifty-four patients with GS 3 + 4 = 7 prostate cancer diagnosed at systematic transrectal ultrasound (TRUS)-guided biopsy underwent 3-T MRI and radical prostatectomy (RP) between 2012 and 2014. A blinded radiologist contoured dominant tumors on ADC maps using histopathologic correlation. The whole-lesion mean ADC, ADC ratio (normalized to peripheral zone), ADC histogram, and texture analysis were compared between tumors with GS upgrading and those without GS upgrading using multivariate ROC analyses and logistic regression modeling. RESULTS Tumors were upgraded to GS 4 + 3 = 7 after RP in 26% (n = 14) of the 54 patients, and tumors were downgraded after RP in none of the patients. The mean ADC, ADC ratio, 10th-centile ADC, 25th-centile ADC, and 50th-centile ADC were similar between patients with GS 3 + 4 = 7 tumors (0.99 ± 0.22, 0.58 ± 0.15, 0.77 ± 0.31, 0.94 ± 0.28, and 1.15 ± 0.24, respectively) and patients with upgraded GS 4 + 3 = 7 tumors (1.02 ± 0.18, 0.55 ± 0.11, 0.71 ± 0.26, 0.89 ± 0.20, and 1.11 ± 0.16) (p > 0.05). Regression models combining texture features improved the prediction of GS upgrading. The combination of kurtosis, entropy, and skewness yielded an AUC of 0.76 (SE = 0.07) (p < 0.001), a sensitivity of 71%, and a specificity of 73%. The combination of kurtosis, heterogeneity, entropy, and skewness yielded an AUC of 0.77 (SE = 0.07) (p < 0.001), a sensitivity of 71%, and a specificity of 78%. CONCLUSION In this study, whole-lesion mean ADC, ADC ratio, and ADC histogram analysis were not predictive of pathologic upgrading of GS 3 + 4 = 7 prostate cancer after RP. ADC texture analysis improved accuracy.

Diagnosis of Sarcomatoid Renal Cell Carcinoma With CT: Evaluation by Qualitative Imaging Features and Texture Analysis.

OBJECTIVE The objective of our study was to determine whether CT findings, including texture analysis, can differentiate sarcomatoid renal cell carcinoma (RCC) from clear cell RCC. MATERIALS AND METHODS A retrospective case-control study was performed of consecutive patients with a histologic diagnosis of sarcomatoid RCC (n = 20) and clear cell RCC (n = 25) who underwent preoperative CT over a 3-year period. The CT images were independently reviewed by two blinded abdominal radiologists; they evaluated the following: tumor heterogeneity, tumor margin, calcification, intratumoral neovascularity, peritumoral neovascularity, renal sinus invasion, renal vein invasion, and adjacent organ invasion. Interobserver agreement was assessed using the Cohen kappa coefficient, and results were compared between groups using an independent Student t test and the chi-square test with a Bonferroni correction. For texture analysis, gray-level co-occurrence and run-length matrix features were extracted and compared using Mann-Whitney U tests. ROC curves for each tumor were constructed, and AUCs were calculated. RESULTS Overall, sarcomatoid RCCs were larger than clear cell RCCs, measuring 77 ± 27 mm (mean ± SD) compared with 50 ± 29 mm (p = 0.003), respectively; however, there was no difference in tumor size between the tumors that were compared using texture analysis or subjective analysis (p = 0.06 and 0.03, respectively). From the subjective analysis, only peritumoral neovascularity (readers 1 and 2: 70% and 70% sarcomatoid RCCs vs 0% and 41.6% clear cell RCCs, respectively; p = 0.001) and the size of the peritumoral vessels (p < 0.001) differed between sarcomatoid RCCs and clear cell RCCs, and interobserver agreement was fair (κ = 0.38). Other subjective imaging features did not differ between the tumors (p > 0.005). There was greater run-length nonuniformity and greater gray-level nonuniformity in sarcomatoid RCCs than in clear cell RCCs (p = 0.03 and p = 0.04, respectively). The combined textural features identified sarcomatoid RCC with an AUC of 0.81 ± 0.08 (standard error) (p < 0.0001). CONCLUSION Large tumor size, the presence of peritumoral neovascularity, and larger peritumoral vessels are features that are more commonly associated with sarcomatoid RCCs than with clear cell RCCs. Sarcomatoid RCCs are also more heterogeneous by texture analysis than clear cell RCCs.

Evaluation of the European Society of Urogenital Radiology (ESUR) PI-RADS scoring system for assessment of extra-prostatic extension in prostatic carcinoma

INTRODUCTION To evaluate extra-prostatic extension (EPE) comparing PI-RADS to non-standardized reporting. MATERIALS AND METHODS With IRB approval, 145 consecutive patients underwent radical prostatectomy (RP) and multi-parametric (T2W+DWI+DCE) MRI between 2012 and 2013. Eighty patients (66.3% with EPE) were staged without PI-RADS and 65 patients (64.6% with EPE) were staged using a 5-point PI-RADS scoring system. Studies were reported by fellowship-trained radiologists in routine clinical practice. Individual PIRADS scores were assessed using ROC to determine the score which optimized sensitivity/specificity. Diagnostic accuracy for EPE was compared with/without PI-RADS using the McNemar test. Subgroup analysis by radiologist experience was performed using Spearman correlation and chi-square. RESULTS Area under ROC curve for EPE using PI-RADS was 0.62 and optimal sensitivity/specificity was achieved with PI-RADS score ≥ 3. Compared to non-standardized reporting, sensitivity for EPE improved with PI-RADS (59.5% [49.1-68.2] vs. 24.5% [16.7-31.2]), p=0.01; with no difference in specificity (68.0% [50.5-82.6]) vs. (75.0% [60.1-87.6]), p=0.06. Overall accuracy improved with PI-RADS (62.7% [49.6-73.6] vs. 42.0% [31.7-50.7%]), p=0.006. Diagnostic accuracy was better among experienced radiologists without PI-RADS (p=0.005); however, there was no difference in accuracy by reader experience using PI-RADS (p=0.24). CONCLUSION The PI-RADS criteria for EPE improves sensitivity without reducing specificity. PI-RADS may reduce differences in accuracy by reader experience.

Quantitative pharmacokinetic analysis of prostate cancer DCE-MRI at 3T: comparison of two arterial input functions on cancer detection with digitized whole mount histopathological validation.

Accurate pharmacokinetic (PK) modeling of dynamic contrast enhanced MRI (DCE-MRI) in prostate cancer (PCa) requires knowledge of the concentration time course of the contrast agent in the feeding vasculature, the so-called arterial input function (AIF). The purpose of this study was to compare AIF choice in differentiating peripheral zone PCa from non-neoplastic prostatic tissue (NNPT), using PK analysis of high temporal resolution prostate DCE-MRI data and whole-mount pathology (WMP) validation. This prospective study was performed in 30 patients who underwent multiparametric endorectal prostate MRI at 3.0T and WMP validation. PCa foci were annotated on WMP slides and MR images using 3D Slicer. Foci ≥0.5cm(3) were contoured as tumor regions of interest (TROIs) on subtraction DCE (early-arterial - pre-contrast) images. PK analyses of TROI and NNPT data were performed using automatic AIF (aAIF) and model AIF (mAIF) methods. A paired t-test compared mean and 90th percentile (p90) PK parameters obtained with the two AIF approaches. Receiver operating characteristic (ROC) analysis determined diagnostic accuracy (DA) of PK parameters. Logistic regression determined correlation between PK parameters and histopathology. Mean TROI and NNPT PK parameters were higher using aAIF vs. mAIF (p<0.05). There was no significant difference in DA between AIF methods: highest for p90 volume transfer constant (K(trans)) (aAIF differences in the area under the ROC curve (Az) = 0.827; mAIF Az=0.93). Tumor cell density correlated with aAIF K(trans) (p=0.03). Our results indicate that DCE-MRI using both AIF methods is excellent in discriminating PCa from NNPT. If quantitative DCE-MRI is to be used as a biomarker in PCa, the same AIF method should be used consistently throughout the study.

Identification of succinate dehydrogenase-deficient bladder paragangliomas.

A significant number of patients with paragangliomas harbor germline mutations in one of the succinate dehydrogenase (SDH) genes (SDHA, B, C, or D). Tumors with mutations in SDH genes can be identified using immunohistochemistry. Loss of SDHB staining is seen in tumors with a mutation in any one of the SDH genes, whereas loss of both SDHB and SDHA expression is seen only in the context of an SDHA mutation. Identifying an SDH-deficient tumor can be prognostically significant, as tumors with SDHB mutations are more likely to pursue a malignant course. Although the rate of SDH deficiency in paragangliomas in general is known to be approximately 30%, there are only rare reports of SDH-deficient bladder paragangliomas. Therefore, the aim of this study was to determine the rate of SDH deficiency in bladder paragangliomas. Eleven cases of bladder paragangliomas were identified. Hematoxylin and eosin-stained slides of all tumors were reviewed, and immunohistochemical analysis for SDHB and SDHA was performed. For cases with loss of SDHA expression by immunohistochemistry, mutation analysis of the SDHA gene was performed. Loss of SDHB staining was seen in 3 (27%) cases (2 with loss of SDHB only, 1 with loss of SDHB and SDHA). Patients with SDH-deficient tumors were younger than those with tumors with intact SDH expression (mean age at presentation 39 y and 58 y, respectively). Of the 2 patients with SDHB-deficient and SDHA-intact tumors, one was found to have a germline SDHB mutation, and the other had a family history of a malignant paraganglioma. Both patients developed metastatic disease. The one patient with a tumor that was deficient for both SDHB and SDHA had no family history of paragangliomas and no evidence of metastatic disease. Sequencing of this tumor revealed a deleterious heterozygous single–base pair substitution in exon 10 of SDHA (c.1340 A>G; p.His447Arg) in both the tumor and normal tissue, indicative of a germline SDHA mutation, and a deleterious single–base pair substitution in exon 5 of SDHA (c.484 A>T; p.Arg162*) in 1 allele of the tumor only. No patients with intact SDH expression had a family history of paragangliomas; 1 had a synchronous paraganglioma, but none developed metastatic disease. A significant subset of bladder paragangliomas is SDH deficient. It is essential to identify SDH-deficient tumors, as the presence of an SDH mutation has prognostic implications and is important in guiding genetic counseling.

Comparison of Contrast-Enhanced Multiphase Renal Protocol CT Versus MRI for Diagnosis of Papillary Renal Cell Carcinoma

OBJECTIVE The objective of this study was to compare contrast-enhanced (CE) CT with MRI for the diagnosis of papillary renal cell carcinoma (pRCC). MATERIALS AND METHODS Between 2006 and 2013, a total of 27 pRCCs were assessed using CECT or CE-MRI. A blinded radiologist placed ROIs that measured attenuation on unenhanced CT; corticomedullary and nephrographic phase CECT images, with an attenuation difference of 20 HU or more denoting enhancing lesions, 10-19 HU indicating indeterminate findings, and less than 10 HU denoting nonenhancing lesions. MRI enhancement ratios were calculated as follows: (signal intensity on gadolinium-enhanced image minus signal intensity) / (signal intensity on unenhanced image × 100) for phase 1 (acquired at 30 s), phase 2 (acquired at 70 s), and phase 3 (acquired at 180 s), where a difference of 15% or more denoted enhancement. Two additional blinded radiologists qualitatively assessed tumor margin, homogeneity, and calcification with the use of CT, and they also assessed enhancement with the use of subtraction MRI. A fourth radiologist established consensus. Twenty consecutive hemorrhagic/proteinaceous cysts served as a control group. Statistical analyses were performed using a chi-square test and multivariate regression. RESULTS There was no statistically significant difference in patient age (p = 0.22), patient sex (p = 0.36), or tumor size (p = 0.29), when pRCCs were compared with hemorrhagic/proteinaceous cysts. On unenhanced CT, attenuation of pRCCs (mean ± SD, 35.7 ± 12.9 HU; range, 19-66 HU) was similar to that of hemorrhagic/proteinaceous cysts (mean, 38.9 ± 16.9; range, 8-71 HU) (p = 0.48). A total of 51.9% of pRCCs (14/27) had either absent or indeterminate enhancement on corticomedullary phase CECT images (mean attenuation difference, 23.2 ± 20.3 HU; range, 6-105 HU), and 14.8% of pRCCs (4/27) had indeterminate enhancement on nephrographic phase CECT images (mean attenuation difference, 36.4 ± 24.9; range, 10-128 HU). No pRCC was nonenhancing on nephrographic phase CECT. Qualitatively, pRCCs were more heterogeneous (80% vs 45%; p = 0.02; κ = 0.24), irregular (50% vs 5%; p < 0.001; κ = 0.21), and calcified (25% vs 0%; p = 0.004; κ = 0.67), with overlap existing between hemorrhagic/proteinaceous cysts. On CE-MRI, all pRCCs were quantitatively enhanced by phase 2 (95.4 ± 83.1; percentage change in signal intensity ratio, 16-450%) and qualitatively enhanced after consensus review. No hemorrhagic/proteinaceous cyst enhanced on MRI when quantitative or subjective analysis was performed. CONCLUSION A small number of pRCCs have indeterminate enhancement when renal protocol CT is used. Heterogeneity, irregular margins, and calcification are suggestive diagnostic features; however, quantitative and qualitative CE-MRI can accurately differentiate hemorrhagic/proteinaceous cysts from pRCC.

The Role of Pathology Correlation Approach in Prostate Cancer Index Lesion Detection and Quantitative Analysis with Multiparametric MRI

Rationale and Objectives Development of imaging biomarkers often relies on their correlation with histopathology. Our aim was to compare two approaches for correlating pathology to multiparametric magnetic resonance (MR) imaging (mpMRI) for localization and quantitative assessment of prostate cancer (PCa) index tumor using whole mount (WM) pathology (WMP) as the reference. Materials and Methods Patients (N = 30) underwent mpMRI that included diffusion-weighted imaging and dynamic contrast-enhanced (DCE) MRI at 3 T before radical prostatectomy (RP). RP specimens were processed using WM technique (WMP) and findings summarized in a standard surgical pathology report (SPR). Histology index tumor volumes (HTVs) were compared to MR tumor volumes (MRTVs) using two approaches for index lesion identification on mpMRI using annotated WMP slides as the reference (WMP) and using routine SPR as the reference. Consistency of index tumor localization, tumor volume, and mean values of the derived quantitative parameters (mean apparent diffusion coefficient [ADC], Ktrans, and ve) were compared. Results Index lesions from 16 of 30 patients met the selection criteria. There was WMP/SRP agreement in index tumor in 13 of 16 patients. ADC-based MRTVs were larger (P < .05) than DCE-based MRTVs. ADC MRTVs were smaller than HTV (P < .005). There was a strong correlation between HTV and MRTV (Pearson r > 0.8; P < .05). No significant differences were observed in the mean values of Ktrans and ADC between the WMP and SPR. Conclusions WMP correlation is superior to SPR for accurate localization of all index lesions. The use of WMP is however not required to distinguish significant differences of mean values of quantitative MRI parameters within tumor volume.

Pitfalls of adrenal imaging with chemical shift MRI

Chemical shift (CS) MRI of the adrenal glands exploits the different precessional frequencies of fat and water protons to differentiate the intracytoplasmic lipid-containing adrenal adenoma from other adrenal lesions. The purpose of this review is to illustrate both technical and interpretive pitfalls of adrenal imaging with CS MRI and emphasize the importance of adherence to strict technical specifications and errors that may occur when other imaging features and clinical factors are not incorporated into the diagnosis. When performed properly, the specificity of CS MRI for the diagnosis of adrenal adenoma is over 90%. Sampling the in-phase and opposed-phase echoes in the correct order and during the same breath-hold are essential requirements, and using the first echo pair is preferred, if possible. CS MRI characterizes more adrenal adenomas then unenhanced CT but may be non-diagnostic in a proportion of lipid-poor adenomas; CT washout studies may be able to diagnose these lipid-poor adenomas. Other primary and secondary adrenal tumours and supra-renal disease entities may contain lipid or gross fat and mimic adenoma or myelolipoma. Heterogeneity within an adrenal lesion that contains intracytoplasmic lipid could be due to myelolipoma, lipomatous metaplasia of adenoma, or collision tumour. Correlation with previous imaging, other imaging features, clinical history, and laboratory investigations can minimize interpretive errors.