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Frontiers in clinical and molecular diagnostics and staging of metastatic clear cell renal cell carcinoma Anna M Czarnecka*,1, Anna Kornakiewicz1,2, Wojciech Kukwa3 & Cezary Szczylik1

ABSTRACT: The last few years have brought advances in the understanding of the molecular biology of metastatic clear cell renal cell carcinoma (RCC). Both preclinical research and clinical trials brought together results from the latest advancements in RCC diagnostic and staging. Understanding of the complex molecular alterations involved in the development and progression of RCC enables development of immunohistochemical and genetic diagnostic tools and is also opening the doors for experimental targeted therapies. At the same time, improvements of medical and molecular imaging improves the sensitivity and specificity of metastatic disease diagnosis. Moreover, independent validation of molecular profiles across high-throughput platforms, methods, laboratories and cancer populations has recently been successfully performed in RCC. Generation of informative, clinical diagnostic tools is likely to contribute to development of novel personalized diagnostic and treatment protocols and ensure prolonged survival of RCC patient in the near future. The last 20 years have brought considerable advances in the understanding of the molecular abnormalities that promote the development of renal epithelial tumors including clear cell renal cell carcinoma (ccRCC). Biological studies have shown a strong correlation between histopathological anatomy and morphology with genetic alterations in renal cancers [1] . Accurate histopathologic classification has historically been of great practical value in the clinical management of renal cell neoplasms, but in recent years, the discovery of new molecular and cytogenetic markers has led to the recognition and classification of several novel subtypes of renal cell neoplasms. Over the last decade, the number of diagnostic and staging possibilities has increased and the importance of distinguishing different tumor types and stages and has been followed by development of targeted therapies [2–6] . In spite of the large and growing number of renal cell carcinoma (RCC) subtypes, classification of well-differentiated, low-grade tumors is relatively routine [2] . The diagnosis of ccRCC on morphologic grounds alone is generally straightforward; however, challenging cases are not infrequent. A misdiagnosis of ccRCC has clinical consequences, particularly in the current era of targeted therapies [7–9] . Since early stages of renal cancer are often asymptomatic, 25–30% of patients display metastatic spread by the time they are diagnosed with RCC. The important challenge in the case of metastatic RCC is to establish effective staging protocol to detect metastases at very early stage. This article presents and discusses the current state of the art in diagnostic and staging tools and clinical methods developed recently, and gives insight into novel approaches to validate in clinical practice. The authors also describe the use of molecular and cytogenetic techniques in


• HIF • immunohistochemistry • molecular imaging • PBRM1 • PET • renal cell cancer • VHL

Department of Oncology with Laboratory of Molecular Oncology, Military Institute of Medicine, Szaserow 128, 04–141 Warsaw, Poland First Faculty of Medicine, Medical University of Warsaw, Zwirki i Wigury 61, 02-091 Warsaw, Poland 3 Department of Otolaryngology, Czerniakowski Hospital, Medical University of Warsaw, Stepinska 19/25, 00-739 Warsaw, Poland *Author for correspondence: Tel.: +48 22 68 17 106; Fax: +48 22 61 03 098; [email protected] 1 2

10.2217/FON.13.258 © Cezary Szczylik

Future Oncol. (2014) 10(6), 1095–1111

part of

ISSN 1479-6694


Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik establishing an accurate diagnosis in difficult cases and their potential usefulness in accurately classifying renal neoplasms [10–12] . Laboratory test in ccRCC diagnostics Over the last decade, more than 50% of renal cell cancer (RCC), including the most common ccRCC cases, are detected incidentally owing to the widespread use of abdominal imaging, including ultrasonography and computed tomography (CT). Nevertheless, every suspicion of RCC should prompt first of all careful physical examination accompanied by laboratory examinations [13] . Preliminary tests performed within the diagnostic process of RCC include urine analysis in search for hematuria; however, there are still no other cancer type- or stagespecific biomarkers convenient for clinical usage in urine analysis. Other initial laboratory studies in the evaluation of suspected RCC (Box 1) include: complete blood cell count with white blood cell types differential; renal profile (creatinine, calcium, sodium, chloride, carbon dioxide, albumin, blood urea nitrogen, protein, phosphorus, glucose and potassium); liver function tests (AST and ALT); calcium; erythrocyte sedimentation rate (ESR); and prothrombin time abd activated partial thromboplastin time. Other tests are to be performed as indicated by the patient’s presenting symptoms. For treatment selection and prognosis evaluation, Memorial Sloan–Kettering Cancer Center risk factors including serum LDH level (natural log scale), corrected serum calcium level and hemoglobin level should be evaluated [14] and accompanied by reanalysis of albumin level, alkaline phosphatase level, creatinine level, absolute neutrophil count and platelet count for prognosis of progression-free survival, overall survival and long-term overall survival (≥30 months) [15,16] . Moreover, already in 1997 international consensus conference on ccRCC prognosis suggested ESR, ALP and anaemia as prognostic biomarkers for this group of patients and stated that these parameters should be evaluated in every case of ccRCC diagnostics (Box 1) . Recent reanalysis in Korean population confirmed that preoperative high ESR is indeed a significant predictor for cancer-specific survival among ccRCC patients and that anemia increases the risk of death from comorbidities [17] , which confirms the need for routine performance of those tests. According to the European Association of Urology guidelines renal function (Box 1) should always be estimated


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if there is a solitary kidney tumor or bilateral tumors are found and also when serum creatinine level is highly increased or when there is a risk of future renal impairment because of associated disorders [18] . As shown by all analyzed guidelines there is space for classical biochemical blood tests in ccRCC diagnostics and those test should not be underestimated [13,19] . Multiple molecular urine biomarkers are under development in the ccRCC field. The only marker with significant clinical data is the urinary NMP-22, which is a biomarker approved by the US FDA for bladder cancer screening and monitoring and it was also reported with levels higher in RCC patients compared with those in control subjects (all p ≤ 0.005) [20] . At the same time urine AQP1 and adipophilin (ADFP) concentrations were shown to be sensitive and specific biomarkers of kidney cancers of proximal tubule origin. It was proposed that these biomarkers may be useful to diagnose an imaged renal mass and screen for kidney cancer at an early stage [21] . Recently, two new candidates for urine biomarkers have been proposed by the screening of proteins in urine of RCC patients by the SELDI-TOF method. It has been shown that downexpression of protein K-12 (secreted and transmembrane 1 precursor) and MASP-2 is characteristic for RCC [22] . A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UEs), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. UE protein content is substantially and reproducibly different from control UE, and proteins with substantial difference in ccRCC include: carrier protein, AQP1, DKK4, CAIX, PODXL and MMP9 [23] . None of the mentioned biomarkers has been approved by the FDA so far; however, since the phenomenon was observed in two histological subtypes of renal cancer (clear cell and papillary), authors suggest that subtype independent RCC urine markers may be on the way. It is important to emphasize clinical utility of those potential biomarkers in urological diseases since any change in concentration of proteins in plasma will reflect in the urine. Moreover, recently, latest column technology – hydrophilic interaction ­chromatography-mass spectrometry (MS) coupled to electrospray MS enabled the detection of highly polar compounds that appear in urine. The utility of urine metabolites

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Diagnostics & staging of renal cell carcinoma  analysis for diagnosis of RCC has been proposed with the usage of multivariate data analysis for data generated by MS/nuclear magnetic resonance (NMR) Spectroscopy ClinProt™ technique (McGill University, Quebec, Canada). The efficacy of cluster of signals to distinguish RCC patients grouped by tumor stage with this method showed a sensibility of 100% for patients at the primary tumor 1 stage [24] . This approach was based on the idea that a large group of potential biomarkers is more likely to show pattern for disease recognition rather than any single-compound analysis [25] . All in all a systematic study of multiple biomarker analysis determining their interrelationships and power to detect tumor is lacking. Medical imaging in ccRCC diagnostics Most cases of RCC including ccRCC are discovered by medical imaging (Figure 1) . Diagnosis is usually suggested by routine ultrasonography and confirmed by x-ray CT scan. CT enables assessment of local invasiveness, lymph node involvement and distant metastases description. At the same time, MRI may provide additional information on local advancement, and presence of venous tumor thrombus. MRI should also be used and in situations where intravenous contrast cannot be used [13] . The American Urological Association guideline for the management of the clinical T1 renal mass recommended a high-quality cross-sectional CT or MRI, with and without intravenous contrast to rule out angiomyolipoma, evaluate for locally invasive features, study the involved anatomy, and determine status of the uninvolved kidney and its vasculature [26] . The National Comprehensive Cancer Network guidelines for renal cancer stated that abdominal and pelvic CT with and without contrast and chest CT or radiograph are necessary imaging for RCC diagnostics [19] . One of the challenges in the diagnostics of metastatic renal cancer refers to accurate examination of tumor thrombus invasion in veins. With regard to the extent of renal vein thrombi, a three-phase helical CT scan is most appropriate; for vena caval thrombi only a MRI examination is able to accurately identify any infra- or supra-hepatic, as well as intracardial extension of the thrombus [27] . There is a study that has shown that multidetector CT and MRI are comparable and more effective than abdominal ultrasound (US) in diagnosing inferior vena cava tumor thrombus in RCC, but none of the

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Box 1. Initial laboratory studies in the renal cell carcinoma diagnostics and treatment enrollment. Urine analysis ●● Complete blood cell count with differential: ●● Red blood cells ●● Platelet count ●● White blood cells: ūū Neutrophils ●● Hemoglobin: ūū Mean cell volume ūū Mean corpuscular hemoglobin Renal profile ●● Creatinine ●● Calcium (Ca2+) ●● Sodium (Na+) ●● Potassium (K+) ●● Chloride (Cl−) ●● Phosphorus (HPO42−) ●● Albumin ●● Blood urea nitrogen ●● Total protein ●● Glucose Liver function tests ●● AST ●● ALT ●● Erythrocyte sedimentation rate ●● Prothrombin time ●● Activated partial thromboplastin time ●● Other tests are to be performed as indicated by the patient’s presenting symptoms For treatment selection & prognosis evaluation Memorial Sloan–Kettering Cancer Center risk factors ●● Serum LDH level ●● Corrected serum calcium level ●● Hemoglobin level

three methods can detect inferior vena cava wall invasion [28] . All standard imaging tools and methods are suboptimal at evaluating patients disease status because of poor sensitivity, which is the most noticeable when comparing results obtained using different methods (Table 1) [29] . CT accurately predicts the tumor size with only a 0.5-cm resolution compared with the pathological size of the lesion [18] . However, tumor diameter determined by CT may be greater than by US and histopathological measurements. The US scan tends to underestimate tumor size in relation to histopathological assessment obtained by patients after radical nephrectomy and


Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik Preliminary diagnosis Basic laboratory panel: – Urine analysis – Complete blood cell – Renal profile – Liver function tests – Calcium – Erythrocyte sedimentation rate – Prothrombin time – aPTT

USG Molecular USG Diagnosis of primary tumor



Molecular CT

Molecular MRI



Future tests (biomarkers): – AQP1 – ADFP – Protein K-12 – MASP-2 – NMP-22 – Urine metabolites analysis–multiple biomarker

Final diagnosis


Diagnosis of metastatic disease Optional laboratory tests: – LDH level – Corrected serum calcium level – Hemoglobin level


Chest CT/RTG Pelvic/abdominal CT

DWI–whole-body MRI



Immunohistochemistry, cytogenetic, molecular analysis and high-throughput gene-expression profiling

Molecular PET

Methods used in current diagnostic protocol Future methods to validate in clinic

Figure 1. Medical and molecular imaging techniques in clear cell renal cell carcinoma diagnostics and staging. aPTT: Activated partial thromboplastin time; CT: Computed tomography; CTC: Circulating tumor cell; DWI: Diffusion-weighted imaging; OCT: Optical coherence tomography; ORS: Optical reflectance spectroscopy; RTG: x-ray imaging; USG: Ultrasonography.

nephron-sparing surgery [30] . At this point it should be underestimated that minimally invasive modalities of tumor resection has shown promising results and favorable safety profile, but they must be reserved only for selected patients. The identification of lymph node metastases still remains a problem since the limiting size is 4 mm and CT will result in a false-negative rate of approximately 10%, especially in the presence of micrometastases; the false-positive rate of 3–43% is mainly owing to reactive hyperplasia. New technologies, such as the multidetector CT with thin collimation and multiplanar reformatting, may result in a diagnostic improvement [18] . In particular, lymphotrophic nanoparticle-enhanced MRI identify malignant


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nodal involvement in patients with renal carcinoma demonstrated with high sensitivity (100%) and specificity (95.7%) [31] . Reported problems of accurate diagnosis of micrometastases and small tumors further underestimates the need for advancement in medical imaging t­echnologies  [32] . New promising imaging approaches have been in development over last decade. First of all multiparametric MRI comprising diffusion-weighted imaging (DWI), blood oxygendependent (BOLD) and dynamic contrastenhanced (DCE)-MRI have been tested [33] . The goal of a multiparametric approach to MRI is to increase the accuracy of the identification of tumors [34] . BOLD moderately correlates to

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Diagnostics & staging of renal cell carcinoma  DCE-MRI-derived perfusion-weighted imaging. Finally, in the preliminary study DCE-MRI appeared superior to BOLD and DWI for histological assessment of tumors [35] . DWI and perfusion-weighted imaging increase the role of MRI in management of renal cell cancer as they ensure not only renal lesion detection and characterization as benign or malignant, but also allow for assessment of RCC subtype and nuclear grade [33] . The great challenge that remains is the standardization of above mentioned modalities [36] as diffusion-weighted whole-body MRI is discussed as an alternative offered by MRI probes to screen for distant metastases [37] . DWI may be an interesting tool for detecting renal cancer regarding the difference of apparent diffusion coefficient (ADC) values between the tumors and surrounding healthy tissues [38] . In multiparametric MRI, ADC displays moderate correlation to the extracellular volume, but does not correlate to tumor oxygenation or perfusion [35] . In regard to low number of renal carcinoma cases screened, the association of ADC values and histological characteristics need further investigations in a large prospective multi-institutional study [38] . Another diagnostic imaging approach is to use exponential ADC (EADC) as an indicator of DWI. The advantage is that EADC reflects the pathological changes of tissues quantitatively. The first study has evaluated the diagnostic role of EADC values at a high


magnetic field strength (3.0 T) in kidney neoplastic lesions compared with that of the ADC values, and it has been suggested that EADC map shows the internal structure of the kidney tumor more intuitively than the ADC map dose, and is also in line with the observation habits of the clinicians. The authors conclude that EADC can be used as an effective imaging method for tumor diagnosis [39] . All described new imaging modalities require further technical development and clinical evaluation. Large-scale studies are needed to normalize the results and define reference values. PET is not a standard imaging method in the diagnosis and staging of RCC and remains controversial in renal cancer since this technique has a higher sensitivity for detecting metastatic lesions than for determining the presence of cancer in the renal primary site [13] . At the same time, PET with 2-deoxy-2-[fluorine-18]fluorod-glucose integrated with CT has the potential for estimation of the patient’s survival according to the 2-deoxy-2-[fluorine-18]fluoro-d-glucose accumulation measured in maximum standardized uptake value [40] .Nevertheless, PET has the potential to extend the sensitivity of examinations in the diagnosis of metastatic RCC and display some advantages over US, CT and PET. This imaging method is powerful to show metastases in unusual locations as in the muscles of the trunk, as well as upper and lower extremities [41] .

Table 1. Application of medical imaging in diagnostic process of metastatic clear cell renal cell carcinoma. Basic application

Extended application

USG First indication of diagnosis CT Confirmation of diagnosis Local invasiveness, lymph node involvement and distant metastases description Rule out angiomyolipoma, study the involved anatomy and determine status of the uninvolved kidney and its vasculature

Three-phase helical CT: examination of tumor thrombus invasion in veins Multidetector CT: examination of vena caval thrombi

MRI Additional information on local advancement and presence of Examination of vena caval thrombi, identication of infra- or venous tumor thrombus suprahepatic, as well as intracardial extension of the thrombus Confirmation of diagnosis when intravenous contrast cannot be used Rule out angiomyolipoma, study the involved anatomy and determine status of the uninvolved kidney and its vasculature Abdominal/pelvic CT Detection of metastases Chest CT/RTG Detection of metastases CT: Computed tomography; RTG: x-ray imaging; USG: Ultrasonography.

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Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik What seems to be important, PET has proven to resolve the cases of the venous migration of the tumor as a malignant thrombus arising from a remnant stump of the left renal vein, passing through hemiazygos vein further into the azygos vein and finally into the superior vena cava just before entering into the right atrium [27] . In order to enhance RCC imaging, new methods are being proposed recently. Integrated optical coherence tomography (OCT) and optical coherence microscopy imaging may provide coregistered, multiscale images of renal pathology in real time without additional contrast or histological procedure. High sensitivity and specificity of the method has potential application for guiding renal mass biopsy or evaluating surgical margins [42] . In the first in vivo study on OCT for differentiation of renal tumors in humans, the attenuation coefficients (as a quantitative assessment) differed significantly between normal renal tissue and cancer. Tumor surface and internal tumor did not differ significantly, suggesting that a superficial OCT attenuation coefficient reliably assesses tissue composition inside the tumor. These results justify further research on OCT for various clinical applications in the diagnosis of renal tumors [43] . Moreover benign and malignant renal tumors can be accurately distinguished by a combination of Raman spectroscopy and optical reflectance spectroscopy; however, no in vivo studies has been launched so far [44] . Molecular imaging in ccRCC diagnostics Molecular radiology refers to all previously described methods: US, CT, MRI, PET/CT, PET/MRI, SPECT and optical imaging (Figure 1) . The perspective of molecular imaging lies in multimodality and nanoparticle-based approaches, imaging of protein–protein interactions and quantitative molecular imaging. Monitoring angiogenesis is potentially an effective strategy for the early detection of cancer [45] , The future imaging of RCC may be able to reveal the complex tumor functioning as metabolism, glycolysis, angiogenesis, cell proliferation, metastatic potential, hypoxia, apoptosis and specific receptors expressed by tumor cells and diagnostic p­rototocols and tools are to develop [45] . Molecular imaging with US relies on microbubble contrast agents that display property of selective adherence to a ligand-specific target [46] . It has been shown that exposition of US contrast agent of given concentration to a


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continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation [47] . Prior studies have revealed that only small quantities of microbubbles are retained at their target site. Improving contrast sensitivity to low concentrations of microbubbles is essential to enhance molecular US [46] . Furthermore, it has been determined that VEGFR2-targeted US contrast agents, such as BR55, may prove to be useful in human for the early detection of cancer, as well as monitoring of treatment [48] . In another study, early detection of metastatic breast cancer was achieved by evaluating blood vessel density in the liver using a 3D contrast-enhanced highfrequency US system and Sonazoid microbubbles [49] . 3D contrast-enhanced high-frequency US detected an increase in blood vessel density in the liver after intrasplenic injection of breast tumor cells into mice. The results were confirmed by immunohistochemical analysis of blood vessel density [49] . It is to consider if the similar approach might be apply to the diagnosis of metastatic renal cancer. Imaging biomarkers are important tools for the detection and characterization of cancers. The first clinically validated molecular imaging biomarker for malignancy for renal cancer 124 I-girentuximab ensures an accurate and noninvasive identification of RCC. It recognizes cellular carbonic anhydrase IX, which is overexpressed on cells of renal cancer and allows the acquisition of diagnostic PET/CT images of RCC without biopsy. This antibody was used as a therapy in metastatic ccRCC [45] . However, the application of this molecular diagnostic tool has encountered the problem that the 124I label is rapidly excreted from the tumor cells after internalization of the radiolabeled monoclonal antibody. It has been suggested that labeling chimeric antirenal cell cancer monoclonal antibody G250 with the residualizing positron emitter 89Zr would lead to higher tumor uptake and more sensitive detection of ccRCC lesions [50] . Target-specific molecular imaging probes for tumor invasiveness have been developed for PET examination and optical imaging, but advancement in MRI has been slower. For example, proteases associated with tumor invasion (metalloproteinases or cathepsins), can be targeted in vivo using optical methods or PET, but it is not applicable in case of MRI. However, novel MRI contrast agents based on iron oxide and dendrimer nanomaterials allow for better

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Diagnostics & staging of renal cell carcinoma  characterization of tumor metastases [37] ; therefore, molecular MRI methods may potentially have greater application in case of metastatic RCC (Table 1) . Surgery in ccRCC diagnostics Final diagnosis is based on pathology from either the primary umor or a metastatic site [13] . Renal tumor biopsy (or nephrectomy) is always indicated before first systemic therapy [5,19,51] . However, as nephrectomy is performed as part of a treatment algorithm, the era of targeted therapy in metastatic ccRCC has posed doubts to necessity of surgery procedure. In one current review, the authors discuss the emerging controversy that cytoreductive nephrectomy is believed by many physicians at best be reserved only for patients responding to targeted therapy; however, current level of evidence only supports the use of nephrectomy in patients where IFN-α therapy is planned. As IFN-α monotherapy is largely replaced by targeted therapy, it has been highlighted that repetition of clinical trials is necessary in order to have level 1 evidence to support surgery [52] . There is a consensus based on R AND Corporation and University of California, Los Angeles (CA, USA) appropriateness ratings that cytoreductive nephrectomy should be considered appropriate in patients with good surgical risks, tumor-related symptoms and limited metastatic burden. All other clinical conditions were indicated of uncertain benefit [52,53] . Nephrectomy can be performed as a standard open surgery; however, there are some novel advancements in surgical technique and roboticassisted partial nephrectomy may also be performed using the Simon’s clamp (Aesculap®, B Braun Melsungen AG, Melsungen, Germany). It provides selective parenchymal compression without the need of vascular clamping. Preliminary results provide an alternative to vascular control in selected patients with polar renal tumors [54] . Nephrectomy can be performed also as laparoscopy procedure. In one study, a large clinical series of pure laparoscopic radical nephrectomy for left RCC with differential extensions of level I renal vein tumor thrombus using a retroperitoneal approach has been described. It has been concluded that despite the technical difficulties, pure laparoscopic radical nephrectomy for patients with this characteristic is safe and feasible. The surgery will be more difficult for patients with higher grades of thrombus

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[55] .

In second study, it has been shown that also laparoscopic partial nephrectomy for small-sized renal tumors is a safe and viable alternative to open partial nephrectomy and provides equivalent oncologic outcomes and comparable morbidity to the traditional approach in experienced centers [56] . Another point to consider is that nephrectomy has not only a treatment goal but also diagnostic value. In most cases, ccRCC is diagnosed with high specificity and sensitivity with standard methods, although 10–20% of biopsies are not diagnostic [57] . There is increasing evidence that further diagnostic and prognostic information can be obtained from renal tumor biopsies with the use of immunohistochemistry (IHC), cytogenetic and molecular analysis, and high-throughput gene-expression profiling; however, in all cases, feasible nephrectomy or nephron-sparing surgery should be considered as ­treatment and diagnostic option of choice [4,8,9,58,59] . General pathologic examination of ccRCC A reporting system of surgically resected specimens of RCC, including gross description and diagnostic information has been proposed by The Association of Directors of Anatomic and Surgical Pathology [2] . The morbid anatomic appearances of ccRCC are well recognized by most pathologists. The ccRCC tumors are frequently soft, yellow and haemorrhagic with areas of necrosis. The typical yellow tumor surface is due to the lipid content of the cells, but cholesterol, neutral lipids and phospholipids are also abundant. Clear cell tumors have very vascular tumor stroma, and therefore frequently show hemorrhagic areas. ccRCC tumor shows a highly characteristic histology. Cells present with optically clear cytoplasm and a very welldefined cytoplasmic membrane. Occasionally, the cells show faintly eosinophilic cytoplasm around the nucleus, a circumstance observed in high nucleargrade cases. In a minority of cases ccRCC has a white sclerotic appearance, with or without grossly evident calcifications or even ossification. The ccRCC tumor may be composed of a mixture of clear, eosinophilic and granular cells. The cells are usually of cuboidal shape and supported by a rich sinusoidal vascular architecture [1] . Most ccRCC tumors are well circumscribed and infiltrate with a pushing margin and with a fibrous pseudocapsule. Less well-defined ccRCC cases present infiltrative margins and this is often an ominous


Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik feature suggesting sarcomatoid transformation. ccRCC can also have a significant component that grossly appears fleshy and tan, also reflecting microscopic sarcomatoid differentiation [7] . The nucleus may show variable morphologies and the Fuhrman system was developed to grade the carcinoma on the basis of its size, nuclear irregularities and the presence of a nucleolus. This grading system is used worldwide and has been shown to significantly correlate with clinical outcomes, although recently, there have been attempts to partially change this way of assigning the histological grade of renal carcinomas [60,61] . Finally it should be noted that based on pathology results a postoperative prognostic nomogram for RCC has been developed by Memorial Sloan–Kettering Cancer Center. This nomogram has been developed to predict the 5-year probability of treatment failure among patients with newly diagnosed RCC and may be useful for patient counseling, clinical trial design and patient follow-up planning [62] . Although standard pathology examination is satisfactory in most of the cases there research is still being conducted in order to develop more advanced techniques. Recently, there are at least dozen of preclinical trials and techniques in development. The combination of coherent anti-Stokes Raman scattering spectroscopy, two-photon excited fluorescence and second harmonic generation enables a high resolution imaging, gives strong information on tissue characteristic and this way provide useful information for tumor diagnosis [63] . In one of the studies, the potential of multimodal nonlinear microscopy for imaging of renal tumors has been shown. Using cryosections of renal carcinoma, the method gave a detailed insight into cancer tissue morphology and composition what enables more precisely distinguish between normal kidney tissue, tumor and necrosis. It has been demonstrated that several features significant for the diagnosis were visualized without use of any staining. Authors imply that translation of this method in clinical pathology will greatly improve speed and quality of the analyses [10,63] . IHC in ccRCC diagnostics IHC distinguishes the most common histological subtypes of RCC. However, difficulties still remain. There is no single marker that is specific for ccRCC and therefore it is generally recommended that a panel of markers be used if IHC is necessary in unclear case [7] . IHC findings in


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conventional ccRCC are cytokeratin, vimentin, glutathione-S-transferase-α and epithelial membrane antigen being positive [1] . Sporadic (and VHL-associated) ccRCC express low-molecularweight cytokeratins (CAM 5.2) and vimentin. Such double staining is characteristic, and may be helpful if it comes to assessing the tumor origin in a distant metastasis of unknown primary site. RCC of the conventional type is usually not reactive with monoclonal antibodies to cytokeratin 7, a reaction which is helpful in distinguishing it from papillary RCC [1] . In ccRCC, c-kit and AMACR are also negative [64] . On the other hand, IHC with epithelial membrane antigen, CD10, CD13, CD15, CD110, PAX-8and MUC-1 is most often positive in ccRCC. As a result of the genetic change on chromosome 3 that underlies practically all ccRCC cases (see below), staining with CAIX and with cyclin D1 is also positive. TFE3 expression is essential to confirming the diagnosis of Xp11 translocation in specific RCC subtype. Thrombomodulin, uroplakin III, p63 and S100P are useful markers for urothelialcarcinoma [7,60] . Vast panel of markers has the diagnostic utility in differential diagnosis contexts and those include: cytokeratins, vimentin, AMACR, carbonic anhydrase IX, PAX2, PAX8, RCC marker, CD10, E-cadherin, kidney-specific cadherin, parvalbumin, claudin-7, claudin-8, S100A1, CD82, CD117, TFE3, thrombomodulin, uroplakin III, p63 and S100P (Table 2) , but some discrepancies between ­percentage of positive cases exist [1,64–67] . More specific markers have been developed over the last 5 years and one of these – the G250 monoclonal antibody antigen - has been shown to be identical to the human MN/CAIX member of the carbonic anhydrase family [1] . Marker most frequently used to support a diagnosis of ccRCC is CAIX, a membrane-bound protein that functions in intracellular and extracellular pH regulation and whose expression is driven by hypoxia. In the case of ccRCC, CAIX expression is due to increased hypoxia-inducible factor (HIF)-driven transcription that is secondary to loss of pVHL function. The pattern of CAIX staining in ccRCC is membranous and typically diffuse. One major pitfall of CAIX IHC is that any tissue or tumor that is hypoxic or necrotic can exhibit membranous labeling. This physiologic expression of CAIX is usually not diffuse in hypoxic tissue and when microscopic necrosis is evident, membranous expression of CAIX is usually only present in tissue immediately

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Diagnostics & staging of renal cell carcinoma 


Table 2. Immunohistochemical staining pattern of renal cell carcinomasubtypes. Marker

Clear cell Papillary RCC RCC

Chromophobe RCC


Xp11.2 translocation RCC


+ + + +/+ + + + + +

+/+ +/+/+ + + + + + -

+/+ + + + + +/+ +/+ + -

+ + + ND ND +/ND ND ND ND + ND +/+ + + ND +

+ +/+ +/-  + + + + + +

EMA: Epithelial membrane antigen; EpCAM: Epithelial cell adhesion molecule; Ksp-cad: Kidney-specific cadherin; ND: No reported data known; PVALB: Parvalbumin; RCC: Renal cell carcinoma; RCC Ma: Renal cell carcinoma marker; SMA: Smooth muscle action; VIM: Vimentin.

surrounding the areas of necrosis [7] . Although pathologists still requires new markers and more research is to be carried out, histology remains the cornerstone for diagnosis and IHC serves as a useful adjunct to the diagnosis in selected circumstances. When considering IHC in differential diagnosis, profiles of antigens are required since any type of RCC may does not express single specific protein type [66] . Circulating tumor cells in ccRCC diagnostics In the future, the clinical value of circulating tumor cells (CTCs) as a biomarker for early cancer detection may be widely used [68] . CTCs in the blood play a critical role for initiation of metastases. It has been highlighted that CTCs tests are especially promising tool to give new insights in the complex biology of micrometastasis and further bring important clinical repercussions. It may ensure not only better monitoring of cancer disease, but also early identification of metastasis [69] . CTC could potentially help in the diagnosis of metastatic cancers, especially in cases where the primary tumor is difficult to visualize with scans, or in cases where neither the metastatic nor the primary lesion is accessible for biopsy

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[69] .

CTC detection is sometimes compared with ‘liquid biopsies’. It ensures a noninvasive, repeatable window into each patient’s tumor, facilitating early cancer diagnosis. Results of enumeration of CTCs should be analyzed together with all clinical i­nformation derived from standard diagnostic tests. To enumerate CTCs of epithelial origin (CD45-, EpCAM+ and cytokeratins 8, 18+ and/or 19+) in whole blood the CELLSEARCH® Circulating Tumor Cell (Janssen Diagnostics, LLC, NJ, USA) assay has been developed. Recent studies indicate that CELLSEARCH may underestimate the number of CTCs, especially in tumors, such as RCC, frequently lacking cytokeratin expression [70] . It has been suggested that the low number of cells detected through CELLSEARCH in RCC may be due to the presence of a CTC population with atypical characteristics and gene-expression profile, characterized by lack of cytokeratin expression and gain of CD44 [70] . This observation needs to be clarified before the tool gain strong clinical value both in diagnosis and prognosis for metastatic RCC. Another challenge in the process of validation of CTC tests in clinical practice is that the rarity of CTCs, approximated at one CTC for


Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik every billion peripheral blood cells. Recently, a new μNMR platform has been developed for biosensing. Through the synergistic integration of microfabrication, nanosensors and novel chemistries, the μNMR platform offers high detection sensitivity and point-of-care operation, overcoming technical barriers in CTC research [71] . Using hybrid nanoparticles, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs [72] . Another study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme–nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles functionalized with the horseradish peroxidases and DNA probes were used as labels. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest ­incidence [73] . Karyotype abnormalities in ccRCC diagnostics The pattern of somatic mutations in renal tumors has been extensively investigated over last decades and has become a major criterion for classification. RCC cases may be classified with respect to the presence or absence of chromosomal imbalances, as well as possible karyotypic signatures and cytogenetic subtypes since RCC subtypes present characteristic chromosomal abnormalities. Therefore, cytogenetic analysis has a distinct role in diagnosis of conventional RCC, but it must be emphasized that also alterations of the tumor karyotype may contribute to determining prognosis of patients with ccRCC. Several cell biology techniques including karyotyping, FISH, comparative genomic hybridization, restriction fragment length polymorphism analysis and loss of heterozygosity (LOH) analysis on related microsatellite loci can all be used in the detection of genomic alterations of renal neoplasms including ccRCC [10] . Cytogenetic data have clarified the morphologic features in some of the cases, but such data is not routinely available to assist most practicing pathologists and oncologists attempting to classify individual tumors cases.


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Nevertheless cytogenetic evaluation should be used in tumors of unusual morphologic appearance and tumors in young patients [2] . The deletion of chromosome 3p is the most typical genetic abnormality in sporadic ccRCC. LOH 3p is considered to be one of the primary events in the carcinogenesis of ccRCC – an important step in tumor initiation and is found in approximately 70–90% cases, but are rarely seen in other types of RCC. Regions frequently lost or inactivated on chromosome 3p include 3p12–14, 3p21 and 3p25. Functional inactivation of genes through the loss of chromosome fragments or mutation in these distinct regions were investigated. Introduction of normal chromosome 3p fragments into a renal cancer cell line resulted in the suppression of tumorigenicity, which has proven that loss of function of one or more tumor suppressor genes on this fragment is the underlying event in the genesis of ccRCC. Since that time, different genes have been located on the short arm of chromosome 3. The most important of them is the von Hippel–Lindau disease tumor suppressor gene in 3p25–26. Other putative genes at 3p are PBRM, RASSF1a and NRC-1 [10,61] . An additional site on the short arm of chromosome 3 – 3p14 – encompasses the FHIT gene [1] . This year LOH of chromosome 3p, was shown to include four very commonly mutated genes (VHL, PBRM1, BAP1 and SETD2) between ­segments 3p25 and 3p21 [12] . Genetic alteration of chromosome 5q is the second most frequent chromosomal change in ccRCCs. Chromosome 5q alterations frequently manifest as trisomy or partial trisomy of chromosome 5, including 5q22-qter. Allelic duplications at 5q31.1 are commonly seen in ccRCC [74] . LOH studies may be performed using five microsatellite polymorphic markers and deletion status was determined by dual color interphase FISH analysis [10] . Translocations that involve chromosomes 3 and 5 leading to the loss of 3p13pter and the duplication of 5q22-qter are also observed in ccRCC. In addition to the loss of chromosomes 3p and 5q, chromosome arm losses of 6q, 8p, 9p and 14q have also been detected in sporadic ccRCC. Moreover besides abnormalities of 5p, loss of 14q and chromosome 16 have also been described [1] . Losses of DNA from 9q, 10q, 13p, 17p and 14q is observed less frequently that in the case of chromosomes 3p and 5q [75] . All the lost chromosomal loci are expected to harbor important tumor suppressor genes. Loss of chromosomal material from 8p, 9p and 14q in ccRCC

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Diagnostics & staging of renal cell carcinoma  has been reported to be associated with higher histologic grade and poor clinical outcomes [76] . Stage and grade were also associated with losses on 14q harboring HIF1A (14q23.2) and PHD1EGLN genes (14q13.2) [77] . High-density single nucleotide polymorphism arrays have been used recently to profile chromosomal aberrations in ccRCC. Illumina’s 317K array (Illumina Inc., CA, USA) confirmed that the most common ccRCC LOH were 3p including whole 3p arm losses, and large fragment LOHs (spanning 3p21–36). Other identified loses included those at 8p12-pter, 6q23.3–27, 14q24.1-qter, 9q32qter, 10q22.3-qter, 9p13.3-pter, 4q28.3-qter and 13q12.1–21.1. In this study, significant associations of LOH at 9p, 9q, 14q and 18q with higher nuclear grade and 14q, 18p and 21q with tumor stage were observed [78] . All the identified regions and genes may become diagnostic and prognostic biomarkers, as well as potential targets of therapy. Genomic copy number alterations in ccRCC is still under investigation and array comparative genomic hybridization is now used for that purpose. Studies are designed to examined associations between chromosomal copy number variation with stage and grade or expression profiles between cases with/without bi-allelic VHL loss [77] . Gene-sequence & -expression analysis in ccRCC diagnostics The pattern of somatic mutations in kidney tumors has been extensively investigated and has become an important criterion for tumor classification. The clinical use of gene-expression profiles could result in more accurate and objective diagnoses of ccRCC, as well as prognoses of disease and treatment selection. The gene definitively shown to be involved in the development of ccRCC is the tumor suppressor von Hippel-Lindau VHL gene. The VHL tumor suppressor gene is located at chromosomal region 3p25 (see above) and is consistently inactivated in both sporadic and hereditary renal cancers. The VHL protein is involved in cell-cycle control and plays a pivotal role in the regulation of the transcription factor, HIF, which is a key regulator of hypoxia-inducible genes (Figure 2) . Inactivation of the VHL gene has been demonstrated in 57% of sporadic ccRCCs and 100% of familial ccRCCs. VHL gene inactivation occur through different mechanism including genomic mutation (50–80%), deletion (60–80%) or abnormal DNA methylation (20–25%) [10] . Inactivation of

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the VHL gene promote tumor cell survival and growth through the mTOR pathway and downstream PI3K pathways [10] . In turn, accumulated HIFs induces the transcriptional activation of a broad range of factors that regulate angiogenesis (VEGF), cell cycles and cell growth (PDGF), pH balance (CAIX), and erythropoietin [60,61] . Recently, genetic and functional studies reported that HIF1A is a target of 14q loss and that HIF1A activity is diminished in ccRCC cases [77] . In 2013, new hotspot TCEB1 mutations, which abolishes Elongin C-VHL binding and leads to HIF accumulation was described. Other new biomarkers were identified by array-based gene-expression. Copy number and/or methylation analyses of ccRCC cells also confirmed disturbances in PI3K–AKT–mTOR signaling, the KEAP1-NRF2-CUL3 apparatus, DNA methylation, p53-related pathways and mRNA processing. In whole-genome sequencing analysis that compared ccRCC tumor and normal kidney DNA, a total number of 71,424 somatic mutations, including 68,273 single nucleotide polymorphisms were reported; the average number of nonsilent mutations observed in the tumor samples was 47 and, as expected, VHL was the most frequently mutated gene; total of 28 genes found to be significantly mutated in ccRCC. Mutations in BAP1 - protease that cleaves ubiquitin–-protein bonds - were found to be mutually exclusive with mutations in PBRM1 - chromatin remodeling gene - and to be associated with poor prognosis. Also frequently mutated gene was TCEB1 – gene for elongin C - part of a protein complex that induces mRNA elongation, and component of the VHL complex. Other genes found to be frequently mutated in this tumor type included TET2 - DNA demethylationezyme – and tumor suppressor p53, KEAP1 and NRF2. Of those, KEAP1 and NRF2 encode proteins that form a complex that is involved in the oxidative stress response [12] . In another study, significant distinction in gene-expression profile between patients with a relatively nonaggressive form of the disease (100% survival after 5 years) versus patients with a relatively aggressive form of the disease (0% 5-year survival rate) was described as 40 gene profile and revealed great variation in upregulated and downregulated genes among the tumors, but all clear cell cancers have shown a remarkable similarity in gene-expression profiles [79,80] . Integrative genome-wide gene-expression profiling of ccRCC revealed that all RCCs cases are tightly clustered together suggesting homogeneity


Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik

Ub Ub Ub

VHLmut HIF-1α









p300/CBP HIF-1α



HIF-1 target genes Proteolysis

Angiogenesis factors (VEGF, PDGF) Erythropoiesis Apoptosis

Cell proliferation (PI3K, mTOR)

Glucose metabolism

pH regulation (CAIX)

Figure 2. Role of VHL protein in target gene-expression regulation.

of ccRCC with 630 upregulated and 720 downregulated genes. The majority of genes downregulated in ccRCC are involved in metabolic processes (organic, carboxylic, amino acid and lipid), oxidative reduction, excretion, ion transport, response to chemical stimulus and cellular localization. On the other hand, genes upregulated in ccRCC are associated with regulation of immune and inflammatory responses, response to stimuli, stress, wounding and hypoxia, regulation of cell proliferation, cell activation, DNA methylation, angiogenesis, TCA cycle, cell adhesion and motility and up to deregulation of approximately 7% of genes could be explained by epigenetic changes [81,82] . Proteomic analysis has revealed that eleven proteins are either underexpressed or undetected in the ccRCC cells – most of all prohibitin and peroxiredoxin-3 - whereas five proteins are overexpressed - αβ crystalin and heat shock protein 27 [83] . Differentially expressed proteins identified were: glucose-6-phosphate isomerase, UGDH pentose phosphate pathway protein, and HSP60 downregulation and, on the other hand, mitochondrial MDH2, ATP5A1 and ATP5B upregulation [84] . In subsequent tissue microarray analysis vimentin, histone 2A.X and α-enolase were found of high expression in ccRCC [85] . Finally, 29 proteins were found differentially expressed


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(12 overexpressed and 17 underexpressed) in metastatic versus primary RCC and of those 14-3-3 zeta/delta and Gal-1 were shown to be associated with poor prognosis [86] . Conclusion Approximately 2–3% of all cancers in adults occur in the kidney. Several types of kidney cancer are known, and the most prevalent of these is ccRCC accounting for 70–80% of all cases. For accurate staging of RCC, abdominal and chest CT or MRI is mandatory. A renal tumor biopsy and nephrectomy provides the histopathological confirmation of malignancy with high sensitivity and specificity. For ccRCC typical histological feature is the clear aspect of the cells due to glycogen and lipids in their cytoplasm. The molecular mechanisms that lead to the development of this tumor are not yet completely understood. Over 90% of ccRCCs tested have been found to have deletions of part of chromosome 3p or other lesions that cause inactivation of the Von Hippel–Lindau tumor suppressor gene VHL. VHL gene mutations that lead to stabilization of HIFs (HIF-1α and HIF-2α). PBRM1, a subunit of the PBAF SWI/SNF chromatin remodelling complex, as well as histone deubiquitinase BAP1 and histone methyltransferase SETD2,

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Diagnostics & staging of renal cell carcinoma  were recently found to be altered in ccRCC. Molecularly distinct forms of ccRCC have been found and the integration of expression profile experiments with clinical parameters analysis could result in the enhancement of the diagnosis and prognosis of ccRCC patients. Moreover, any identified biomarkers could provide insight into the molecular mechanisms of aggressive ccRCC and suggest intervention strategies and new drug targets since clear cell cancers show a remarkable similarity in gene-expression profiles [9,12,13,80–82] . Future perspective Significant advances in our understanding of the biology of RCC have been achieved in recent years, but even more is to be carried out in next decade. Although patients with ccRCC are now offered a better diagnostic tool, several questions remain: how do we optimize clinical use of these novel techniques and biomarkers, how do we identify biomarkers most likely to show good candidates for targeted therapies, and which combination of diagnostic protocols further accelerate clinical practice. The development of high-throughput molecular profiling technologies including genomics, transcriptomics, proteomics and metabolomics may lead to a revolution in our understanding of the biology of ccRCC and refine diagnostics and optimize the development of predictive and prognostic markers. Molecular profiling may result in multiple clinical applications including not only diagnosis, but also prognosis, prediction of treatment efficiency, and patient follow-up after surgery for early detection of recurrence and, finally, subgrouping of patients into smaller categories, thus allowing for individualization of treatment options. Nevertheless, in parallel to molecular studies, a number of large-scale, collaborative clinical trial are necessary to validate promising biomarker candidates. In 2013, two major genomic papers have shown 19 significantly mutated genes in ccRCC which opens new field for further pharmacogenomic research and drug design [78,81] . In RCC, the PI3K/AKT pathway was shown to be mutated and indicated as a new important therapeutic target. The second therapeutic novel approach may be on the other hand suggested based on genome-wide DNA hypomethylation associated with mutation of the H3K36 methyltransferase SETD2, and SWI/SNF chromatin remodeling complex genes including PBRM1, ARID1A and SMARCA4.

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The subset of aggressive RCCs are show characterized by metabolic shift, involving downregulation of genes involved in the tricarboxylic acid cycle, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl coenzyme A carboxylase protein. Finally, this subset of tumors is characterized by decreased AMPK and phosphatase and tensin homolog protein levels and altered promoter methylation of miR-21 (MIR21) and GRB10 genes. In the field of ccRCC diagnostics functional imaging techniques including DCE-MRI, DWI, arterial spin labeling and magnetic resonance spectroscopy are currently being investigated in preclinical and clinical trials. Moreover, functional imaging techniques, especially DCE-CT, seem promising also for assessing response of metastatic ccRCC to treatment and should be implemented in clinics in the future. In 2013, preliminary first radiogenomic analysis of ccRCC revealed associations between CT features and underlying VHL mutations, which proves the need for further multidisciplinary investigations in this field. It is expected that metaanalysis of studies, will identify biomarkers of ccRCC that could be implemented in diagnostic process in clinics and for prognostic or predictive application. Equal focus must be applied to the clinical validation phase for such studies, technology development, patient recruitment and professional education on new methods. A large concerted effort is required to advance the field of ccRCC diagnostics and staging through systematic discovery, verification, and validation. Financial & competing interests disclosure The authors were supported with the Military Institute of Medicine statutory founding number 1/1744 (101). C  Szczylik and AM Czarnecka have been supported by the National Science Centre project numbers UMO2011/01/B/NZ5/02822. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Open Access This work is licensed under the Creative Commons Attribution-NonCommercial 3.0 Unported License. To view a copy of this license, visit licenses/by-nc-nd/3.0/


Review  Czarnecka, Kornakiewicz, Kukwa & Szczylik EXECUTIVE SUMMARY Laboratory & pathology test in clear cell renal cell carcinoma diagnostics ●●

Initial laboratory studies in the evaluation of suspected renal cell carcinoma (RCC) must include urine analysis, complete blood cell count and renal profile.


Immunohistochemical examination in clear cell RCC (ccRCC) differential diagnosis should cover CK7, RCC Ma, AMACR, epithelial membrane antigen, CD10, CD117, PAX8, vimentin and TFE.

Established & novel medical imaging techniques in ccRCC diagnostics & staging ●●

Renal cancer diagnostics must cover abdominal and pelvic computed tomography (CT) and MRI for vena caval thrombus identification and extension evaluation.


Molecular imaging based on ultrasound, CT, MRI, PET-CT, PET-MRI, single-photon emission computed tomography and optical imaging is under extensive development.


Further optimization of targeted imaging agents, along with the integration of new imaging modalities, is necessary to enhance RCC pre- and intra-operative diagnostics.

Genetic & genomic characterization of ccRCC ●●

LOH 3p is considered one of the primary events in the carcinogenesis of ccRCC with important genes lost including the von Hippel–Lindau disease tumor suppressor, and PBRM, RASSF1a and NRC-1.


VHL gene inactivation occur in 80% of ccRCCs and is driven by different mechanism including genomic mutation (50–80%), deletion (60–80%) or abnormal DNA methylation (20–25%).


Genomic analysis identified 19 significantly mutated genes in ccRCC with VHL, PBRM1, SETD2, KDM5C, phosphatase and tensin homolog, BAP1, MTOR and TP53 of considerably high significance.


The majority of genes downregulated in ccRCC are involved in metabolic processes, oxidative reduction, excretion, ion transport, response to chemical stimulus and cellular localization.


The majority of genes upregulated in ccRCC are associated with regulation of immune and inflammatory responses, response to stimuli, stress, wounding and hypoxia, regulation of cell proliferation, cell activation, angiogenesis, cell adhesion and motility.

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Frontiers in clinical and molecular diagnostics and staging of metastatic clear cell renal cell carcinoma.

The last few years have brought advances in the understanding of the molecular biology of metastatic clear cell renal cell carcinoma (RCC). Both precl...
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