Marie O'Donnell

Clinical Utility of an Epigenetic Assay to Detect Occult Prostate Cancer in Histopathologically Negative Biopsies: Results of the MATLOC Study

PURPOSE Concern about possible false-negative prostate biopsy histopathology findings often leads to rebiopsy. A quantitative methylation specific polymerase chain reaction assay panel, including GSTP1, APC and RASSF1, could increase the sensitivity of detecting cancer over that of pathological review alone, leading to a high negative predictive value and a decrease in unnecessary repeat biopsies. MATERIALS AND METHODS The MATLOC study blindly tested archived prostate biopsy needle core tissue samples of 498 subjects from the United Kingdom and Belgium with histopathologically negative prostate biopsies, followed by positive (cases) or negative (controls) repeat biopsy within 30 months. Clinical performance of the epigenetic marker panel, emphasizing negative predictive value, was assessed and cross-validated. Multivariate logistic regression was used to evaluate all risk factors. RESULTS The epigenetic assay performed on the first negative biopsies of this retrospective review cohort resulted in a negative predictive value of 90% (95% CI 87-93). In a multivariate model correcting for patient age, prostate specific antigen, digital rectal examination and first biopsy histopathological characteristics the epigenetic assay was a significant independent predictor of patient outcome (OR 3.17, 95% CI 1.81-5.53). CONCLUSIONS A multiplex quantitative methylation specific polymerase chain reaction assay determining the methylation status of GSTP1, APC and RASSF1 was strongly associated with repeat biopsy outcome up to 30 months after initial negative biopsy in men with suspicion of prostate cancer. Adding this epigenetic assay could improve the prostate cancer diagnostic process and decrease unnecessary repeat biopsies.

The Effect of VEGF-Targeted Therapy on Biomarker Expression in Sequential Tissue from Patients with Metastatic Clear Cell Renal Cancer

Purpose: To investigate how biologically relevant markers change in response to antiangiogenic therapy in metastatic clear cell renal cancer (mRCC) and correlate these changes with outcome. Experimental Design: The study used sequential tumor tissue and functional imaging (taken at baseline and 12–16 weeks) obtained from three similar phase II studies. All three studies investigated the role of VEGF tyrosine kinase inhibitors (TKI) before planned nephrectomy in untreated mRCC (n = 85). The effect of targeted therapy on ten biomarkers was measured from sequential tissue. Comparative genomic hybridization (CGH) array and DNA methylation profiling (MethylCap-seq) was performed in matched frozen pairs. Biomarker expression was correlated with early progression (progression as best response) and delayed progression (between 12–16 weeks). Results: VEGF TKI treatment caused a significant reduction in vessel density (CD31), phospho-S6K expression, PDL-1 expression, and FOXP3 expression (P < 0.05 for each). It also caused a significant increase in cytoplasmic FGF-2, MET receptor expression in vessels, Fuhrman tumor grade, and Ki-67 (P < 0.05 for each). Higher levels of Ki-67 and CD31 were associated with delayed progression (P < 0.05). Multiple samples (n = 5) from the same tumor showed marked heterogeneity of tumor grade, which increased significantly with treatment. Array CGH showed extensive intrapatient variability, which did not occur in DNA methylation analysis. Conclusion: TKI treatment is associated with dynamic changes in relevant biomarkers, despite significant heterogeneity in chromosomal and protein, but not epigenetic expression. Changes to Ki-67 expression and tumor grade indicate that treatment is associated with an increase in the aggressive phenotype of the tumor. Clin Cancer Res; 19(24); 6924–34. ©2013 AACR.

REVIEW The renal pathology of mushroom poisoning

We present the histology and electronmicroscopy of renal failure caused by ingestion of the mushroom species Cortinarius speciosissimus. Light microscopy revealed acute tubular necrosis, multinucleated tubular epithelial cells  and concentric  medial muscular hyperplasia of arteries. Electronmicroscopy showed the displaced and abnormal architecture of actin filaments at the periphery of the cytoplasm in renal tubular epithelial cells.

Sunitinib treatment exacerbates intratumoral heterogeneity in metastatic renal cancer

Purpose: The aim of this study was to investigate the effect of VEGF-targeted therapy (sunitinib) on molecular intratumoral heterogeneity (ITH) in metastatic clear cell renal cancer (mccRCC). Experimental Design: Multiple tumor samples (n = 187 samples) were taken from the primary renal tumors of patients with mccRCC who were sunitinib treated (n = 23, SuMR clinical trial) or untreated (n = 23, SCOTRRCC study). ITH of pathologic grade, DNA (aCGH), mRNA (Illumina Beadarray) and candidate proteins (reverse phase protein array) were evaluated using unsupervised and supervised analyses (driver mutations, hypoxia, and stromal-related genes). ITH was analyzed using intratumoral protein variance distributions and distribution of individual patient aCGH and gene-expression clustering. Results: Tumor grade heterogeneity was greater in treated compared with untreated tumors (P = 0.002). In unsupervised analysis, sunitinib therapy was not associated with increased ITH in DNA or mRNA. However, there was an increase in ITH for the driver mutation gene signature (DNA and mRNA) as well as increasing variability of protein expression with treatment (P < 0.05). Despite this variability, significant chromosomal and transcript changes to key targets of sunitinib, such as VHL, PBRM1, and CAIX, occurred in the treated samples. Conclusions: These findings suggest that sunitinib treatment has significant effects on the expression and ITH of key tumor and treatment specific genes/proteins in mccRCC. The results, based on primary tumor analysis, do not support the hypothesis that resistant clones are selected and predominate following targeted therapy. Clin Cancer Res; 21(18); 4212–23. ©2015 AACR.

Intracytoplasmic lumina and mucinous inclusions in ovarian carcinomas

Intracytoplasmic mucinous inclusions and lumina have been previously described in non‐glandular neoplasms such as urothelial carcinoma. We describe their presence in 93% of non‐mucinous ovarian carcinomas. They were found in abundance in all 25 cases (100%) of clear cell carcinoma, in 48 of 50 cases (96%) of serous carcinoma and 20 of 25 cases (80%) of endometrioid carcinoma. The degree of the differentiation of the tumour did not influence the number of inclusions or lumina observed. These results suggest that the presence of intracytoplasmic lumina and mucinous inclusions is more widespread than hitherto appreciated. Their presence in an otherwise poorly differentiated metastatic carcinoma might, at the very least, prompt one to consider the ovary as a possible primary site. In addition, an abundance of intracytoplasmic mucinous inclusions and lumina with microcyst formation, in an otherwise poorly differentiated malignant primary ovarian epithelial tumour, might suggest the possibility of a clear cell carcinoma.

The use of reverse phase protein arrays (RPPA) to explore protein expression variation within individual renal cell cancers.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant. In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma as well as other solid tumors. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis. Protein based analysis of RCC is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

Estrogen secretion from a malignant sex cord stromal tumor in a patient with complete androgen insensitivity

We report on a 68-year-old patient with complete androgen insensitivity syndrome (testicular feminization syndrome) in whom an estrogen-secreting malignant sex cord stromal tumor developed in an intraabdominal testis. We believe this to be the first such case to document estrogen secretion by the tumor.

Proteomic analysis of pre- and post-sunitinib treated renal cancer tissue to assess tumor heterogeneity and differential protein expression.

388 Background: To investigate acquired resistance of clear cell renal cell cancer (ccRCC) patients to sunitinib and develop personalised treatment strategies, sequential tissue after a specific period of targeted therapy is required. This approach has proven successful with targeted therapy in chronic myeloid leukaemia; however, we are concerned that extensive tumour heterogeneity occurs in ccRCC. In this study we evaluated heterogeneity and differential protein expression in sunitinib treated and untreated ccRCC samples using high-throughput proteomics. METHODS Fresh frozen tissue was obtained from 27 sunitinib naïve ccRCC specimens and 27 nephrectomy samples from patients treated with neoadjuvant sunitinib (18 weeks) as part of the SuMR trial. From each tumour frozen sections were performed and up to 5 protein lysates obtained from each morphologically differing region of each tumour as well as matched normal kidney. Reverse phase protein arrays (RPPA) were performed to assess the levels of multiple proteins relevant to ccRCC pathogenesis and sunitinib activity. Appropriate statistical tests were used to examine protein heterogeneity and differential expression, including false discovery rate (FDR) correction. Kaplan-Meier method was used to correlate changes in protein expression with outcome. RESULTS Expression of 20 proteins has been examined to date. The range of expression in tumours normalised by matched normal renal tissue had >2-fold differences in untreated (n=8 proteins) and treated samples (n=4 proteins). Four markers displayed significantly increased inter-tumoural variance in sunitinib treated tumours compared with untreated tissue (e.g. VEGFR1, FDR P<0.05). Despite this heterogeneity, sunitinib was associated with significant expression changes for several key proteins (e.g. VEGFR2, CyclinD2; FDR P<0.05). CONCLUSIONS Protein expression in ccRCC is heterogenous and key proteins showed significantly increased variance of expression with sunitinib therapy. Despite heterogeneity, significant changes in protein expression can be identified with sunitinib treatment and have been correlated with outcome.

Translational research will fail without surgical leadership: SCOTRRCC a successful surgeon-led Nationwide translational research infrastructure in renal cancer

BACKGROUND High quality human biosamples with associated high quality clinical data are essential for successful translational research. Despite this, the traditional approach is for the surgeon to act as a technician in the tissue collection act. Biomarker research presents multiple challenges and the field is littered with failures. Tissue quality, poor clinical information, small sample numbers and lack of validation cohorts are just a few reasons for failure. It is clear that the surgeon involved in tissue acquisition must be fully engaged in the process of biosampling for a specific condition, as this will negate many of the issues for translational research failure due to an inadequate bioresource. APPROACH In this Matter for Debate paper, the Scottish Collaboration On Translational Research into Renal Cell Cancer (SCOTRRCC) is discussed as an example of a urological surgery lead bioresource which has resulted in a National collection of renal cancer tissue and blood (from over 900 patients to date), negating all of the traditional issues with biobanks because of close enagagement and acknowledgement of urologists and uropathologists from seven centres around Scotland. SCOTRRCC has leveraged renal cancer research in Scotland resulting in several high impact publications and providing a springboard for future research in this disease in Scotland and beyond. CONCLUSIONS The SCOTRRCC model presented here can be transferred to other surgical disciplines for success in translational research.

Renal cell carcinoma needle biopsy

Pathological examination of a radical nephrectomy for papillary renal cell carcinoma showed tumour present within the perinephric fat, with a morphology indicative of iatrogenic tumour cell tracking caused by previous biopsy of the mass. This is a rare complication of percutaneous biopsy, and as this procedure is becoming more common in investigating renal masses, it is important that the professionals are aware of the potential risks posed by renal mass biopsy.