Diagnosis and treatment of small renal masses: the role for molecular biology.

Renal cell carcinoma (RCC), the most common type of kidney cancer, is increasing in incidence and is the most lethal genitourinary cancer. Due to the increasing use of abdominal imaging, incidentally detected, asymptomatic small renal masses (SRMs), most of which are RCC, have become the most common presentation of kidney cancer. Most RCC SRMs initially grow slowly or not at all, but others progress to advanced and metastatic cancer. Several diagnostic and prognostic genomic, transcriptomic and proteomic studies have been completed in RCC, however signatures for SRM progression have not been identified. In the absence of useful factors to distinguish those tumors requiring treatment for progression from those that can be managed by active surveillance alone, most SRMs are treated as RCC with surgery. Currently, the only prognostic factor at diagnosis is tumor size. Tumor growth rate also appears to identify potential progressive tumours. Identifying signatures for progression and the utilization of needle biopsies will be important for SRM patients and will guide therapy.

The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the beta-actin mRNA in a U-rich-element-dependent manner.

A high expression level of the beta-actin protein is required for important biological mechanisms, such as maintaining cell shape, growth, and motility. Although the elevated cellular level of the beta-actin protein is directly linked to the long half-life of its mRNA, the molecular mechanisms responsible for this effect are unknown. Here we show that the RNA-binding protein HuR stabilizes the beta-actin mRNA by associating with a uridine-rich element within its 3' untranslated region. Using RNA interference to knock down the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the stabilization but not in the nuclear/cytoplasmic distribution of the beta-actin mRNA. HuR depletion in HeLa cells alters key beta-actin-based cytoskeleton functions, such as cell adhesion, migration, and invasion, and these defects correlate with a loss of the actin stress fiber network. Together our data establish that the posttranscriptional event involving HuR-mediated beta-actin mRNA stabilization could be a part of the regulatory mechanisms responsible for maintaining cell integrity, which is a prerequisite for avoiding transformation and tumor formation.

Characterization of a von Hippel Lindau pathway involved in extracellular matrix remodeling, cell invasion, and angiogenesis.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene results in highly vascularized tumors, making the VHL tumor syndrome an ideal system to study the mechanisms of angiogenesis. VHL operates along two pathways with the first involving hypoxia-inducible factor-alpha degradation and down-regulation of its proangiogenic target genes vascular endothelial growth factor and platelet-derived growth factor-beta, and the second pathway promoting extracellular matrix (ECM) assembly. Secretion of proangiogenic factors was shown to be a primary inducer of angiogenesis. Here, we show that loss of ECM assembly correlates with tumor angiogenesis in VHL disease. Upon inactivation of the VHL-ECM assembly pathway, we observe tumors that are highly vascularized, have a disrupted ECM, and show increased matrix metalloproteinase-2 activity. Loss of the VHL pathway leading to hypoxia-inducible factor-alpha degradation results in tumors with increased vascular endothelial growth factor levels but with surprisingly low microvessel density, a tightly assembled ECM and low invasive ability. We conclude that loss of ECM integrity could promote and maintain tumor angiogenesis by providing a route for blood vessels to infiltrate tumors.

Needle core biopsies provide ample material for genomic and proteomic studies of kidney cancer: observations on DNA, RNA, protein extractions and VHL mutation detection.

The use of needle biopsies in basic research is increasing, and our study provides a comprehensive analysis of their adequacy in genomic and proteomic studies of kidney cancer. Frozen clear cell renal cell carcinoma (ccRCC) needle core biopsies and sections from core biopsies embedded in optimal cutting temperature (OCT) compound were used to extract DNA, RNA and protein. Their integrity was determined using genomic and proteomic analyses. VHL mutation testing was performed on ccRCC biopsies and corresponding tumors using bulk and laser capture microdissection (LCM) extractions for comparison. Adequate amounts of good quality DNA (5.8-13.3 µg/whole core, 0.6-2.7 µg/20 sections), RNA (2.9-11.9 µg/whole core, 0.5-1.3 µg/20 sections) and protein (137.4-444 µg/whole core, 39.9-74.1 µg/20 sections) were obtained from whole core and frozen sections of ccRCC needle biopsies, respectively. We observed VHL sequence mutations in 75% of ccRCC tumors and, in most cases, the same mutations were detected in both tumors and corresponding biopsies. Mutations observed by bulk extractions from tumors and biopsies were also detected by LCM without significant differences between both methodologies. ccRCC needle biopsies provide ample material for genomic and proteomic studies of kidney cancer. They are good representatives of their corresponding tumors for VHL mutation detection using both bulk and LCM extractions. LCM does not increase sensitivity of VHL mutation detection.

Structured electronic operative reporting: comparison with dictation in kidney cancer surgery.

PURPOSE: The purpose of this study was to evaluate the functionality of eKidney as a structured reporting tool in operative note generation. To do this, we compared completeness and timeliness of eKidney template-generated nephrectomy OR notes with standard narrative dictation. METHODS: A group of academic uro-oncologists and medical informaticians at the University Health Network designed and adopted an electronic online, point-of-care clinical documentation tool, eCancerCare(Kidney) (eKidney) for kidney cancer patient care. The optimal components of clinic and operative note templates, including those for nephrectomy, were agreed upon by expert consensus of the uro-oncologists. Clinician nephrectomy OR reports were analyzed for completeness, comparing those generated in eKidney with conventionally dictated notes. Patterns of missing information from both dictated and eKidney-generated reports were analyzed. The procedure, note completion and transcription dates were recorded which generated time intervals between these events. The records of 189 procedures were included in the analysis. RESULTS: Comparison of clinicians who used both note generation modalities, revealed a mean completion rate of 92% for eKidney/structured notes and 68% for dictated notes (p<0.0001). There was no significant difference in completion rates between attending staff and trainees (residents and fellows) (p=0.131). Most notes were dictated/entered on the day of surgery. Dictated notes were transcribed to EPR a median of 2 days after dictation, however roughly 30% of dictated notes took 5 days or more to get transcribed. All notes generated using eKidney were uploaded to the EPR immediately. LIMITATIONS: Our study has three significant limitations. Firstly, our study was not randomized: physicians could elect to dictate or use eKidney. Secondly, we did not identify data from dictated notes that were not captured by eKidney. Third, we did not compare the time it took physicians to complete the fields in eKidney with the time it takes to dictate a note. CONCLUSIONS: We have demonstrated that the use of structured reporting improves the completeness and timeliness of documentation in kidney cancer surgery. eKidney is an example of the power of templates in ensuring that important details of a procedure are recorded. Future studies looking at user satisfaction, and research and educational potential of eKidney would be valuable.

State of the science: an update on renal cell carcinoma.

Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication.

Active surveillance of small renal masses: progression patterns of early stage kidney cancer.

BACKGROUND: Most early stage kidney cancers are renal cell carcinomas (RCCs), and most are diagnosed incidentally by imaging as small renal masses (SRMs). Indirect evidence suggests that most small RCCs grow slowly and rarely metastasize. OBJECTIVE: To determine the progression and growth rates for newly diagnosed SRMs stratified by needle core biopsy pathology. DESIGN, SETTING, AND PARTICIPANTS: A multicenter prospective phase 2 clinical trial of active surveillance of 209 SRMs in 178 elderly and/or infirm patients was conducted from 2004 until 2009 with treatment delayed until progression. INTERVENTION: Patients underwent serial imaging and needle core biopsies. MEASUREMENTS: We measured rates of change in tumor diameter (growth measured by imaging) and progression to ≥ 4 cm, doubling of tumor volume, or metastasis with histology on biopsy. RESULTS AND LIMITATIONS: Local progression occurred in 25 patients (12%), plus 2 progressed with metastases (1.1%). Of the 178 subjects with 209 SRMs, 127 with 151 SRMs had>12 mo of follow-up with two or more images, with a mean follow-up of 28 mo. Their tumor diameters increased by an average of 0.13 cm/yr. Needle core biopsy in 101 SRMs demonstrated that the presence of RCC did not significantly change growth rate. Limitations included no central review of imaging and pathology and a short follow-up. CONCLUSIONS: This is the first SRM active surveillance study to correlate growth with histology prospectively. In the first 2 yr, the rate of local progression to higher stage is low, and metastases are rare. SRMs appear to grow very slowly, even if biopsy proven to be RCC. Many patients with SRMs can therefore be initially managed conservatively with serial imaging, avoiding the morbidity of surgical or ablative treatment.

Multiple acquired renal carcinoma tumor capabilities abolished upon silencing of ADAM17.

Malignancy is a manifestation of acquired defects in regulatory circuits that direct normal cell proliferation and homeostasis. Most of these circuits operate through cell autonomous pathways, whereas others potentially involve the neighboring microenvironment. We report that the metalloprotease ADAM17 plays a pivotal role in several acquired tumor cell capabilities by mediating the availability of soluble transforming growth factor-alpha, an epidermal growth factor receptor (EGFR) ligand, and thus the establishment of a key autocrine signaling pathway. Silencing of ADAM17 in human renal carcinoma cell lines corrects critical features associated with cancer cells, including growth autonomy, tumor inflammation, and tissue invasion. Highly malignant renal carcinoma cancer cells fail to form in vivo tumors in the absence of ADAM17, confirming the essential function of this molecule in tumorigenesis. These data show that ligand shedding is a crucial step in endogenous EGFR activation and endorse prospective therapeutic strategies targeting ADAM17 in human cancer.

Activation of sarcolemma and nuclear membranes ET-1 receptors regulates transcellular calcium levels in heart and vascular smooth muscle cells.

The use of an ET-1 fluorescent probe in human heart and vascular smooth muscle cells showed that ET-1 receptors are present at both the sarcolemma and nuclear envelope membranes. The use of immunofluorescence studies showed that the ETA receptor was mainly present at the sarcolemma and cytosolic levels. However, the ETB receptor was present at the sarcolemma and the cytosol, as well as the nuclear envelope membranes and the nucleoplasm. In addition, ET-1 immunoreactivity was seen in the cytosol and the nucleus. Using Ca2+ fluorescent probes such as Fluo-3, Indo 1, and yellow cameleon, as well as confocal microscopy three-dimensional image measurement technique, stimulation of ET-1 receptors at the sarcolemma membranes induced an increase of cytosolic and nuclear free Ca2+ levels. This effect of extracellular ET-1 was blocked by removal of extracellular calcium. Direct stimulation of ET-1 receptors at the nuclear envelope membranes also induced an increase of intranuclear free Ca2+ level. Our results suggest that the stimulation of sarcolemmal Ca2+ influx by ET-1 seems to be due to the activation of ETA and ETB receptors. However, the increase of nucleoplasmic Ca2+ levels by cytosolic ET-1 seems to be mediated via the activation of ETB receptors. Activation of nuclear membranes ETB receptors seems to prevent nuclear Ca2+ overload and may protect the cell from apoptosis.