Alexander Laird

5-hydroxymethylcytosine profiling as an indicator of cellular state

DNA methylation is widely studied in the context of cancer. However, the rediscovery of 5-hydroxymethylation of DNA adds a new layer of complexity to understanding the epigenetic basis of development and disease, including carcinogenesis. There have been significant advances in techniques for the detection of 5-hydroxymethylcytosine and, with this, greater insight into the distribution, regulation and function of this mark, which are reviewed here. Better understanding of the associated pathways involved in regulation of, and by, 5-hydroxymethylcytosine may give promise to new therapeutic targets. We discuss evidence to support the view of 5-hydroxymethylcytosine as a unique and dynamic mark of cellular state. These 5-hydroxymethylcytosine profiles may offer optimism for the development of diagnostic, prognostic and predictive biomarkers.

Differential Expression of Prognostic Proteomic Markers in Primary Tumour, Venous Tumour Thrombus and Metastatic Renal Cell Cancer Tissue and Correlation with Patient Outcome

Renal cell carcinoma (RCC) is the most deadly of urological malignancies. Metastatic disease affects one third of patients at diagnosis with a further third developing metastatic disease after extirpative surgery. Heterogeneity in the clinical course ensures predicting metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been shown to have prognostic significance, including Ki67, p53, vascular endothelial growth factor receptor 1 (VEGFR1) and ligand D (VEGFD), SNAIL and SLUG. Previous pathway analysis has been from study of the primary tumour, with little attention to the metastatic tumours which are the focus of targeted molecular therapies. As such, in this study a tissue microarray from 177 patients with primary renal tumour, renal vein tumour thrombus and/or RCC metastasis has been created and used with Automated Quantitative Analysis (AQUA) of immunofluorescence to study the prognostic significance of these markers in locally advanced and metastatic disease. Furthermore, this has allowed assessment of differential protein expression between the primary tumours, renal vein tumour thrombi and metastases. The results demonstrate that clinico-pathological parameters remain the most significant predictors of cancer specific survival; however, high VEGFR1 or VEGFD can predict poor cancer specific survival on univariate analysis for locally advanced and metastatic disease. There was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared with the primary tumours and renal vein tumour thrombi. With the exception of p53, these differences in protein expression have not been shown previously in RCC. This confirms the importance of proliferation, angiogenesis and epithelial to mesenchymal transition in the pathogenesis and metastasis of RCC. Importantly, this work highlights the need for further pathway analysis of metastatic tumours for overcoming drug resistance and developing new therapies.

Carbonic Anhydrase 9 Expression Increases with Vascular Endothelial Growth Factor–Targeted Therapy and Is Predictive of Outcome in Metastatic Clear Cell Renal Cancer

Background There is a lack of biomarkers to predict outcome with targeted therapy in metastatic clear cell renal cancer (mccRCC). This may be because dynamic molecular changes occur with therapy. Objective To explore if dynamic, targeted-therapy-driven molecular changes correlate with mccRCC outcome. Design, setting, and participants Multiple frozen samples from primary tumours were taken from sunitinib-naïve (n = 22) and sunitinib-treated mccRCC patients (n = 23) for protein analysis. A cohort (n = 86) of paired, untreated and sunitinib/pazopanib-treated mccRCC samples was used for validation. Array comparative genomic hybridisation (CGH) analysis and RNA interference (RNAi) was used to support the findings. Intervention Three cycles of sunitinib 50 mg (4 wk on, 2 wk off). Outcome measurements and statistical analysis Reverse phase protein arrays (training set) and immunofluorescence automated quantitative analysis (validation set) assessed protein expression. Results and limitations Differential expression between sunitinib-naïve and treated samples was seen in 30 of 55 proteins (p < 0.05 for each). The proteins B-cell CLL/lymphoma 2 (BCL2), mutL homolog 1 (MLH1), carbonic anhydrase 9 (CA9), and mechanistic target of rapamycin (mTOR) (serine/threonine kinase) had both increased intratumoural variance and significant differential expression with therapy. The validation cohort confirmed increased CA9 expression with therapy. Multivariate analysis showed high CA9 expression after treatment was associated with longer survival (hazard ratio: 0.48; 95% confidence interval, 0.26–0.87; p = 0.02). Array CGH profiles revealed sunitinib was associated with significant CA9 region loss. RNAi CA9 silencing in two cell lines inhibited the antiproliferative effects of sunitinib. Shortcomings of the study include selection of a specific protein for analysis, and the specific time points at which the treated tissue was analysed. Conclusions CA9 levels increase with targeted therapy in mccRCC. Lower CA9 levels are associated with a poor prognosis and possible resistance, as indicated by the validation cohort. Patient summary Drug treatment of advanced kidney cancer alters molecular markers of treatment resistance. Measuring carbonic anhydrase 9 levels may be helpful in determining which patients benefit from therapy.

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.

A Critical Analysis of the Learning Curve and Postlearning Curve Outcomes of Two Experience- and Volume-Matched Surgeons for Laparoscopic and Robot-Assisted Radical Prostatectomy

BACKGROUND AND PURPOSEnThere remains equipoise with regard to whether laparoscopic radical prostatectomy (LRP) or robot-assisted radical prostatectomy (RARP) has any benefit over the other. Despite this, there is a trend for the increasing adoption of RARP at great cost to health services across the world. The aim was to critically analyze the learning curve and outcomes for LRP and RARP for two experience- and volume-matched surgeons who have completed the learning curve for LRP and RARP.nnnPATIENTS AND METHODSnTwo experience- and volume-matched LRP and RARP surgeons who have completed the learning curve were compared with respect to their learning curve and outcomes for RARP and LRP. There were 531 RARP and 550 LRPs analyzed from April 2003 until January 2012 at two relatively high-volume United Kingdom centers. Outcome measures included operative time, blood loss, complication rate (Clavien-Dindo grade III), positive surgical margin (PSM) rate, and early continence rate.nnnRESULTSnLearning curves for blood loss, operative times, and complication rate were similar between groups. The overall PSM rate and pT2 PSM rate learning curves were longer for RARP compared with LRP but shorter for early continence. Apical PSM showed no learning curve for RARP; however, a long learning curve for LRP and the rate was lower for RARP than for LRP (P=<0.001).nnnCONCLUSIONSnThis study of RARP and LRP identified that both modalities had long learning curves. Despite the long learning curve for RARP, significant benefits in lower PSM rates and better early continence in comparison with LRP exist. There are benefits to patients with RARP over LRP, especially those linked to better apical dissection (apical PSM and early continence).

Matched pair analysis of laparoscopic versus open radical nephrectomy for the treatment of T3 renal cell carcinoma.

AbstractPurposenThe perioperative and oncological outcomes of laparoscopic radical nephrectomy (LRN) for T1–T2 renal cell carcinoma (RCC) are well established. We aim to determine whether LRN is a comparable alternative to open radical nephrectomy (ORN) in the treatment of T3 RCC using a matched pair analysis study design.MethodsA review of a prospectively collected database at the Western General Hospital, Edinburgh, between 2000 and 2011 was conducted. Patient pairs were matched based on age at operation, gender, histological subgroup, maximal tumour diameter, TNM stage and grade. Patient demographics, operative and post-operative outcomes were compared. Overall, cancer-specific and progression-free survival [overall survival, cancer-specific survival (CSS) and progression-free survival (PFS)] were estimated using the Kaplan–Meier method.ResultsFrom 252 patients with T3 disease, 25 pairs were matched. Patients were of median age 66.2xa0years, 64xa0% male. Tumours were all clear cell RCC, were stage pT3a (32xa0%) or pT3b and had maximal tumour diameters of 8.7xa0cm for LRN and 10.0xa0cm for ORN. Estimated blood loss (100xa0ml LRN; 650xa0ml ORN, pxa0<xa00.001) and length of post-operative hospital stay (4xa0days LRN: 9xa0days ORN, pxa0<xa00.001) were lower in the LRN group. Operation time and post-operative complication rates were comparable. CSS and PFS were comparable with a mean CSS of 91.3xa0months for LRN and 88.7xa0months for ORN.ConclusionThis study reports the longest median follow-up in a T3 LRN cohort. In matched patients, LRN has been shown to have a superior perioperative profile to ORN for the treatment of pT3a/b RCC, with no adverse effect on midterm oncological outcomes.

Consultation audio-recording reduces long-term decision regret after prostate cancer treatment: A non-randomised comparative cohort study

INTRODUCTIONnThe life expectancy of prostate patients is long and patients will spend many years carrying the burdens & benefits of the treatment decisions they have made, therefore, it is vital that decisions on treatments are shared between patient and physician. The objective was to determine if consultation audio-recording improves quality of life, reduces regret or improves patient satisfaction in comparison to standard counselling.nnnPATIENTS AND METHODSnIn 2012 we initiated consultation audio-recordings, where patients are given a CD of their consultation to keep and replay at home. We conducted a prospective non-randomised study of patient satisfaction, quality of life (QOL) and decision regret at 12 months follow-up using posted validated questionnaires for the audio-recording (AR) patients and a control cohort. Qualitative and thematic analyses were used.nnnRESULTSnForty of 59 patients in the AR group, and 27 of 45 patients in the control group returned the questionnaires. Patient demographics were similar in both groups with no statistically significant differences between the two groups. Decision regret was lower in the audio-recording group (11/100) vs control group (19/100) (pxa0=xa00.04). The risk ratio for not having any long-term decision regret was 5.539 (CI 1.643-18.674), with NNT to prevent regret being 4. Regression analysis showed that receiving audio-recording was strongest predictor for absence of regret even greater than potency and incontinence.nnnCONCLUSIONnThe study has shown that audio-recording clinic consultation reduces long-term decision regret, increases patient information recall, understanding and confidence in their decision. There is great potential for further expansion of this low-cost intervention.

Dynamic epigenetic changes to VHL occur with sunitinib in metastatic clear cell renal cancer

Background Genetic intratumoral heterogeneity (ITH) hinders biomarker development in metastatic clear cell renal cancer (mccRCC). Epigenetic relative to genetic ITH or the presence of consistent epigenetic changes following targeted therapy in mccRCC have not been evaluated. The aim of this study was to determine methylome/genetic ITH and to evaluate specific epigenetic and genetic changes associated with sunitinib therapy. Patients and methods Multi-region DNA sampling performed on sequential frozen pairs of primary tumor tissue from 14 metastatic ccRCC patients, in the Upfront Sunitinib (SU011248) Therapy Followed by Surgery in Patients with Metastatic Renal Cancer: a Pilot Phase II Study (SuMR; ClinicalTrials.gov identifier: NCT01024205), at presentation (biopsy) and after 3-cycles of 50mg sunitinib (nephrectomy). Untreated biopsy and nephrectomy samples before and after renal artery ligation were controls. Ion Proton sequencing of 48 key ccRCC genes, and MethylCap-seq DNA methylation analysis was performed, data was analysed using the statistical computing environment R. Results Unsupervised hierarchical clustering revealed complete methylome clustering of biopsy and three nephrectomy samples for each patient (14/14 patients). For mutational status, untreated biopsy and all treated nephrectomy samples clustered together in 8/13 (61.5%) patients. The only methylation target significantly altered following sunitinib therapy was VHL promoter region 7896829 which was hypermethylated with treatment (FDR=0.077, P<0.001) and consistent for all patients (pre-treatment 50% patients had VHL mutations, 14% patients VHL hypermethylation). Renal artery ligation did not affect this result. No significant differences in driver or private mutation count was found with sunitinib treatment. Conclusions Demonstration of relative methylome homogeneity and consistent VHL hypermethylation, after sunitinib, may overcome the hurdle of ITH present at other molecular levels for biomarker research.

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.

A novel bovine model for training urological surgeons in laparoscopic radical nephrectomy.

PURPOSEnTo design a training phantom to allow trainees to demonstrate and practice the key steps of a laparoscopic nephrectomy, while demonstrating the anatomical landmarks, together with correct instrumentation and safe ergonomic layout.nnnMATERIALS AND METHODSnCalves, prepared according to the strict standards of the meat industry, were purchased from a local abattoir. The skinned torso of a 30-kg calf was prepared by opening the abdomen and removing most of the ribs and the pelvis to create a larger working space. The small intestine and the majority of the large intestine were removed, leaving 30u2009cm of ascending colon, the liver, and spleen as internal landmarks and placed inside a standard laparoscopic abdominal trainer. This model was then used as part of a structured training course.nnnRESULTSnFrom April 2005 to April 2010, 104 urologists have worked on this phantom. Evaluation forms were completed by these participants, and analysis of the feedback shows all attendees found the model to be very realistic in terms of anatomical conditions, tissue color and consistency, and organ tactility, compared with human nephrectomy (average score of 4.8 on a Likert scale of 1 (unrealistic/poor) to 5 (realistic/useful)). Participants found the model useful for developing dissection techniques, electrosurgery, coagulation, and suturing skills.nnnCONCLUSIONnThis model can be easily established and is valuable as part of a multimodal training program for laparoscopy.