Overcoming intratumoural heterogeneity for reproducible molecular risk stratification: a case study in advanced kidney cancer.

BACKGROUND: Metastatic clear cell renal cell cancer (mccRCC) portends a poor prognosis and urgently requires better clinical tools for prognostication as well as for prediction of response to treatment. Considerable investment in molecular risk stratification has sought to overcome the performance ceiling encountered by methods restricted to traditional clinical parameters. However, replication of results has proven challenging, and intratumoural heterogeneity (ITH) may confound attempts at tissue-based stratification. METHODS: We investigated the influence of confounding ITH on the performance of a novel molecular prognostic model, enabled by pathologist-guided multiregion sampling (n = 183) of geographically separated mccRCC cohorts from the SuMR trial (development, n = 22) and the SCOTRRCC study (validation, n = 22). Tumour protein levels quantified by reverse phase protein array (RPPA) were investigated alongside clinical variables. Regularised wrapper selection identified features for Cox multivariate analysis with overall survival as the primary endpoint. RESULTS: The optimal subset of variables in the final stratification model consisted of N-cadherin, EPCAM, Age, mTOR (NEAT). Risk groups from NEAT had a markedly different prognosis in the validation cohort (log-rank p = 7.62 × 10-7; hazard ratio (HR) 37.9, 95% confidence interval 4.1-353.8) and 2-year survival rates (accuracy = 82%, Matthews correlation coefficient = 0.62). Comparisons with established clinico-pathological scores suggest favourable performance for NEAT (Net reclassification improvement 7.1% vs International Metastatic Database Consortium score, 25.4% vs Memorial Sloan Kettering Cancer Center score). Limitations include the relatively small cohorts and associated wide confidence intervals on predictive performance. Our multiregion sampling approach enabled investigation of NEAT validation when limiting the number of samples analysed per tumour, which significantly degraded performance. Indeed, sample selection could change risk group assignment for 64% of patients, and prognostication with one sample per patient performed only slightly better than random expectation (median logHR = 0.109). Low grade tissue was associated with 3.5-fold greater variation in predicted risk than high grade (p = 0.044). CONCLUSIONS: This case study in mccRCC quantitatively demonstrates the critical importance of tumour sampling for the success of molecular biomarker studies research where ITH is a factor. The NEAT model shows promise for mccRCC prognostication and warrants follow-up in larger cohorts. Our work evidences actionable parameters to guide sample collection (tumour coverage, size, grade) to inform the development of reproducible molecular risk stratification methods.

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.

The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro.

Anti-angiogenic agents, such as the multi-tyrosine kinase inhibitor sunitinib, are key first line therapies for metastatic clear cell renal cell carcinoma (ccRCC), but their mechanism of action is not fully understood. Here, we take steps towards validating a computational prediction based on differential transcriptome network analysis that phosphorylated adapter RNA export protein (PHAX) is associated with sunitinib drug treatment. The regulatory impact factor differential network algorithm run on patient tissue samples suggests PHAX is likely an important regulator through changes in genome-wide network connectivity. Immunofluorescence staining of patient tumours showed strong localisation of PHAX to the microvasculature consistent with the anti-angiogenic effect of sunitinib. In normal kidney tissue, PHAX protein abundance was low but increased with tumour grade (G1 vs. G3/4; p < 0.01), consistent with a possible role in cancer progression. In organ culture, ccRCC cells had higher levels of PHAX protein expression than normal kidney cells, and sunitinib increased PHAX protein expression in a dose dependent manner (untreated vs. 100 µM; p < 0.05). PHAX knockdown in a ccRCC organ culture model impacted the ability of sunitinib to cause cancer cell death (p < 0.0001 untreated vs. treated), suggesting a role for PHAX in mediating the efficacy of sunitinib.

Toxicological profiling of chemical and environmental samples using panels of test organisms and optical oxygen respirometry.

A simple and versatile methodology for high throughput toxicological assessment of chemical and environmental samples is presented. It uses panels of test organisms ranging from prokaryotic (E. coli, V. fischeri) and eukaryotic (Jurkat) cells to invertebrate (Artemia salina) and vertebrate (Danio rerio) organisms, to analyze alterations in their oxygen consumption by optical oxygen respirometry. All the assays are carried out in a convenient microtiter plate format using commercial reagents (phosphorescent oxygen probe, microplates) and detection on a standard fluorescent plate reader. Simple experimental set-up and mix-and-measure procedure allow parallel assessment of up to 96 samples (or assay points) in 2 h, easy generation of dose- and time-dependent responses, and EC(50) values. The methodology was demonstrated with several different classes of chemicals including heavy metal ions, PAHs, pesticides, their mixtures, and also validated with complex environmental samples such as wastewater from a wastewater treatment plant. It has been shown to provide high sensitivity, sample throughput and information content, flexibility and general robustness. It allows ranking and profiling of samples, compares favorably with alternative methods such as MicroTox and mortality tests with animal models, and is well suited for large-scale monitoring programs such as CWA and WFD.

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.

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 50mg (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.

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.

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.

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.

The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) has recently been implicated in the initiation and progression of renal cell carcinoma (RCC). Some mRNA gene expression studies have suggested a link between the EMT phenotype and poorer clinical outcome from RCC. This study evaluated expression of EMT-associated proteins in RCC using in situ automated quantitative analysis immunofluorescence (AQUA) and compared expression levels with clinical outcome. METHODS/PRINCIPAL FINDINGS: Unsupervised hierarchical cluster analysis of pre-existing RCC gene expression array data (GSE16449) from 36 patients revealed the presence of an EMT transcriptional signature in RCC [E-cadherin high/SLUG low/SNAIL low]. As automated immunofluorescence technology is dependent on accurate definition of the tumour cells in which measurements take place is critical, extensive optimisation was carried out resulting in a novel pan-cadherin based tumour mask that distinguishes renal cancer cells from stromal components. 61 patients with ccRCC and clinical follow-up were subsequently assessed for expression of EMT-associated proteins (WT1, SNAIL, SLUG, E-cadherin and phospho-ß-catenin) on tissue microarrays. Using Kaplan-Meier analysis both SLUG (p = 0.029) and SNAIL (p = 0.024) (log rank Mantel-Cox) were significantly associated with prolonged progression free survival (PFS). Using Cox regression univariate and multivariate analysis none of the biomarkers were significantly correlated with outcome. 14 of the 61 patients expressed the gene expression analysis predicted EMT-protein signature [E-cadherin high/SLUG low/SNAIL low], which was not found to be associated to PFS when measured at the protein level. A combination of high expression of SNAIL and low stage was able to stratify patients with greater significance (p = 0.001) then either variable alone (high SNAIL p = 0.024, low stage p = 0.029). CONCLUSIONS: AQUA has been shown to have the potential to identify EMT related protein targets in RCC allowing for stratification of patients into high and low risk groups, as well the ability to assess the association of reputed EMT signatures to progression of the disease.

What can molecular pathology contribute to the management of renal cell carcinoma?

The incidence of renal cell carcinoma (RCC) is increasing and outcomes remain poor. One-third of patients with localized disease will relapse, and 5-year survival for patients with metastatic disease is less than 10%. No molecular test is currently available to identify which patients who have undergone 'curative' surgery will relapse, and which patients will respond to targeted therapy. Some well characterized biochemical pathways, such as those associated with von Hippel-Lindau disease, are aberrantly regulated in RCC and are associated with histological subtype, but the understanding of these pathways contributes little to the clinical management of patients with RCC. Gene expression and sequencing studies have increased our understanding of the genetic basis of the disease but have failed to establish any unified classification to improve molecular stratification or to predict which patients are likely to relapse or respond to targeted therapy. Instead, they have served to highlight that RCC is heterogeneous at histological, morphological, and molecular levels, and that novel approaches are required to resolve the complexity of RCC prognostication and prediction of treatment response.

Rapid high-throughput assessment of aerobic bacteria in complex samples by fluorescence-based oxygen respirometry.

A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given.

Optical oxygen microrespirometry as a platform for environmental toxicology and animal model studies.

We present a new methodology for testing physiological responses of small organisms (size 70-500 microm) via changes in their oxygen respiration monitored by quenched-phosphorescence oxygen sensing on a scale of a single organism. The method is demonstrated using three different formats of respirometric assays, Artemia salina and mouse embryos as model animals, and various effectors including compounds that induce and prevent superoxide-mediated and heavy metal ion toxicity. These assays, which employ soluble oxygen probes, standard fluorescent readers, and accessorytools, provide sensitive, noninvasive, real-time monitoring of animal respiration, and rapid assessment of EC50, sublethal effects, and metabolic alterations. Applications include screening for acute toxicity of compound libraries and environmental samples, and the study of animal physiology and metabolism.