Sporadic Clear Renal Cell Carcinoma Research Articles

The Clinical and Molecular Features in the VHL Renal Cancers; Close or Distant Relatives with Sporadic Clear Cell Renal Cell Carcinoma?

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited cancer syndrome caused by germline mutations in the VHL tumor suppressor gene, characterized by the susceptibility to a wide array of benign and malign neoplasms, including clear-cell renal cell carcinoma. Moreover, VHL somatic inactivation is a crucial molecular event also in sporadic ccRCCs tumorigenesis. While systemic biomarkers in the VHL syndrome do not currently play a role in clinical practice, a new promising class of predictive biomarkers, microRNAs, has been increasingly studied. Lots of pan-genomic studies have deeply investigated the possible biological role of microRNAs in the development and progression of sporadic ccRCC; however, few studies have investigated the miRNA profile in VHL patients. Our review summarize all the new insights related to clinical and molecular features in VHL renal cancers, with a particular focus on the overlap with sporadic ccRCC.

The combination of medullary thyroid carcinoma and sporadic clear cell renal cell carcinoma: coincidence or new syndrome?

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Deubiquitylase OTUD6B stabilizes the mutated pVHL and suppresses cell migration in clear cell renal cell carcinoma

Von Hippel-Lindau (VHL) is an important tumor suppressor, and its inactivation is a hallmark of inherited VHL disease and most sporadic clear cell renal cell carcinoma (ccRCC). VHL protein (pVHL) with missense point mutations are unstable and degraded by the proteasome because of the disruption of elongin binding. Deubiquitylase ovarian tumor domain-containing 6B (OTUD6B) had been documented to couple pVHL and elongin B to form stable VHL - elonginB - elonginC complex, which protects pVHL from degradation. However, whether OTUD6B governs the stability of pVHL wild type and the missense mutants in ccRCC remains largely elusive. Here, we reported that low OTUD6B level predicted poorer survival in ccRCC patients with VHL missense mutation, but not frameshift deletion and nonsense mutation. OTUD6B is able to interact with wild type pVHL and tumor-derived pVHL missense mutants, except for pVHL I151T, and decrease their ubiquitylation and proteasomal degradation in ccRCC cells. Functionally, we revealed that OTUD6B depletion enhanced cell migration and HIF-2α level in ccRCC cells in a pVHL dependent manner. In addition, OTUD6B depletion reduced the inhibitory effects of ectopic pVHL missense mutants on cell migration and HIF-2α level, except for pVHL I151T. Thus, we speculated that I151 residue might be one of key sites of pVHL binding to OTUD6B. These results suggested that OTUD6B is an important regulator for the stability of pVHL missense mutants, which provides a potential therapeutic strategy for ccRCC with VHL mutations.

Natural history of Von Hippel-Lindau disease-associated and sporadic clear cell renal cell carcinoma: a comparative study.

To compare the tumor growth kinetics between sporadic clear cell renal cell carcinoma (ccRCC) and Von Hippel-Lindau disease-associated renal cell carcinoma (VHL-associated RCC). To analyze predictive markers for the growth rate of these two types of RCC. The clinical data of patients with renal tumors who received active surveillance were collected retrospectively. Immunohistochemical staining was utilized to analyze the expression levels of VHL, PBRM1, H3K36me3, and BAP1 in the postoperative specimens. The age of the VHL group was significantly younger than that of the sporadic group (P < 0.0001). The mean linear growth rate (LGR) was significantly faster in the sporadic group (P = 0.0004). The tumors of those in the sporadic group tended to have a higher histologic grade (P = 0.0011). In the sporadic group, tumor histologic grade was an independent predictor for rapid mean LGR (P = 0.0022). In the VHL group, initial maximal tumor diameter (MTD) was the only independent predictor for rapid mean LGR (P < 0.0001). Tumors with low VHL expression and negative PBRM1 expression showed a faster growth rate in the sporadic group (P = 0.001 and P = 0.008, respectively). The expression levels of the four biomarkers showed no impact on the tumor growth rate in the VHL group. Sporadic ccRCC grew faster than VHL-associated RCC. High histologic grade, low VHL expression and negative PBRM1 expression were predictors of faster growth in sporadic ccRCC. A large initial MTD was a predictor of faster growth for VHL-associated RCC.

VHL suppresses RAPTOR and inhibits mTORC1 signaling in clear cell renal cell carcinoma

Inactivation of the tumor suppressor von Hippel–Lindau (VHL) gene is a key event in hereditary and sporadic clear cell renal cell carcinomas (ccRCC). The mechanistic target of rapamycin (mTOR) signaling pathway is a fundamental regulator of cell growth and proliferation, and hyperactivation of mTOR signaling is a common finding in VHL-dependent ccRCC. Deregulation of mTOR signaling correlates with tumor progression and poor outcome in patients with ccRCC. Here, we report that the regulatory-associated protein of mTOR (RAPTOR) is strikingly repressed by VHL. VHL interacts with RAPTOR and increases RAPTOR degradation by ubiquitination, thereby inhibiting mTORC1 signaling. Consistent with hyperactivation of mTORC1 signaling in VHL-deficient ccRCC, we observed that loss of vhl-1 function in C. elegans increased mTORC1 activity, supporting an evolutionary conserved mechanism. Our work reveals important new mechanistic insight into deregulation of mTORC1 signaling in ccRCC and links VHL directly to the control of RAPTOR/mTORC1. This may represent a novel mechanism whereby loss of VHL affects organ integrity and tumor behavior.

Natural history and growth kinetics of clear cell renal cell carcinoma in sporadic and von Hippel-Lindau disease.

BackgroundTo evaluate and compare the natural history and growth kinetics of sporadic clear cell renal cell carcinoma (ccRCC) with those of ccRCC in von Hippel-Lindau disease (VHL).MethodsSixty patients in the sporadic group with 61 tumors and 15 patients in the VHL group with 30 tumors whom all underwent delayed surgery after at least 12 months of active surveillance (AS) were enrolled to conduct a retrospective cohort study. The growth rate was calculated, and the growth kinetics between the sporadic and VHL groups were compared. The patient and tumor characteristics were reviewed, and their correlation with growth rate was analyzed.ResultsThe mean growth rate of sporadic ccRCC was 0.91 cm/year (ranging from 0–4.74 cm/year) and that of VHL ccRCC was 0.47 cm/year (ranging from 0.04–1.89 cm/year). The growth rate of sporadic ccRCC showed a tendency of being faster than that of VHL ccRCC but did not reach statistical significance (P=0.07). The factors affecting the growth rate were different between the two groups. For VHL ccRCC, the only factor that correlated with growth rate was initial tumor diameter (P<0.001), but for sporadic ccRCC, the only factor was pathological nuclear grade (P<0.001).ConclusionsThe growth rate of VHL-associated ccRCC might be slower than that of sporadic ccRCC. Furthermore, we identified a disparity in growth kinetics between sporadic and VHL-associated ccRCC.

Genomic Analysis Reveals Novel Specific Metastatic Mutations in Chinese Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for more than 75% of renal cell carcinoma. Nearly 25% of ccRCC patients were diagnosed with metastasis. Though the genomic profile of ccRCC has been widely studied, the difference between localized and metastatic ccRCC was not clarified. Primary tumor samples and matched whole blood were collected from 106 sporadic patients diagnosed with renal clear cell carcinoma at Qilu Hospital of Shandong University from January 2017 to November 2019, and 17 of them were diagnosed with metastasis. A hybridization capture-based next-generation sequencing of 618 cancer-related genes was performed to investigate the somatic and germline variants, tumor mutation burden (TMB), and microsatellite instability (MSI). Five genes with significantly different prevalence were identified in the metastatic group, especially TOP1 (17.65% vs. 0%) and SNCAIP (17.65% vs. 0%). The altered frequency of PBRM1 (0% vs. 27%) and BAP1 (24% vs. 10%) differed between the metastatic and nonmetastatic groups, which may relate to the prognosis. Of these 106 patients, 42 patients (39.62%) had at least one alteration in DNA damage repair (DDR) genes, including 58.82% of metastatic ccRCC patients and 35.96% of ccRCC patients without metastasis. Ten pathogenic or likely pathogenic (P/LP) variants were identified in 11 sporadic clear cell renal cell carcinoma patients (10.38%), including rarely reported ATM (n=1), MUTYH (n=1), NBN (n=1), RAD51D (n=1), and BRCA2 (n=1). No significant difference in the ratio of P/LP variant carriers or TMB was identified between the metastatic and nonmetastatic groups. We found a unique genomic feature of Chinese metastatic ccRCC patients with a higher prevalence of alterations in DDR, TOP1, and SNCAIP. Further investigated studies and drug development are needed in the future.

Birt-Hogg-Dubé syndrome-associated renal cell carcinoma: Histopathological features and diagnostic conundrum.

Birt‐Hogg‐Dubé (BHD) syndrome is associated with the development of hereditary renal cell carcinoma (RCC) and is caused by a germline mutation in the folliculin gene. Most cases of BHD syndrome‐associated RCC (BHD‐RCC) are less aggressive than sporadic clear cell RCC and multifocal. Therefore, it is critical to distinguish BHD‐RCC from its sporadic counterparts to identify and monitor affected families and to preserve renal function for as long as possible. The World Health Organization/International Society of Urological Pathology consensus classification defined distinct entities for certain hereditary RCC; however, BHD‐RCC was not included in this classification. Although the clinical features and molecular mechanisms of BHD‐RCC have been investigated intensively over the last two decades, pathologists and urologists occasionally face difficulties in the diagnosis of BHD‐RCC that require genetic testing. Affected patients usually have miscellaneous benign disorders that often precede renal carcinogenesis. In the present review, we summarize the current understanding of the histopathological features of BHD‐RCC based on our epidemiological studies of Japanese families and a literature review. Pathological diagnostic clues and differential diagnosis of BHD‐RCC from other hereditary RCC are also briefly discussed.

The Prognostic Value of Programmed Death-Ligand 1 in a Chinese Cohort With Clear Cell Renal Cell Carcinoma.

Objective: To investigate the association between tumor PD-L1 expression and patient survival to determine whether PD-L1 represents an independent prognostic feature for patients with non-metastatic clear cell renal cell carcinoma (RCC).Patients and Methods: The tissue bank of the Fudan University Shanghai Cancer Center was queried to identity tissue samples of patients treated with radical nephrectomy, for non-metastatic sporadic clear cell RCC (ccRCC) between 2008 and 2015. Real-time polymerase chain reaction and immunohistochemistry staining was performed to detect the expression level of PD-L1 in paired cancer tissue and paracancerous tissue.Results: Three-hundred-and-thirty patients were enrolled in this study, with a mean age of 55.0 years at surgery and a mean tumor size of 5.2 cm. Two-hundred-and-forty-two (73.3%) and 88 (26.7%) patients showed a high and low expression of PD-L1 mRNA, respectively, while 254 patients had positive PD-L1 immunohistochemistry staining. Two-hundred-and-ninety-two patients had consistent results for mRNA and the PD-L1 protein based on these different detection methods. Patients with high PD-L1 expression were more likely to exhibit adverse pathologic features including an advanced T stage (P = 0.002) and lymph node metastasis (P = 0.044). The Kaplan–Meier curves of PFS and OS stratified by PD-L1 expression had a statistically significant difference. PD-L1 expression maintained a significant predictive role for PFS and OS in the multivariate cox model.Conclusions: Our data suggests that PD-L1 correlates with prognosis in RCC and targeting the PD-1/PD-L1 pathway should be considered in the treatment of RCC patients.

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Kidney cancer accounts for 5% and 3% of all adult malignancies in men and women, respectively, thus representing the 7th most common cancer in men, and the 10th most common cancer in women [1.Siegel R.L. Miller K.D. Jemal A. Cancer statistics.CA Cancer J Clin. 2016; 66: 7-30Crossref PubMed Scopus (21988) Google Scholar]. However, available statistics include not only renal parenchymal tumours, but also urothelial cancer of the renal pelvis; renal cell carcinoma (RCC) accounts for ∼80% of all kidney cancers. After over two decades of increasing rates, RCC incidence trends worldwide have shown signs of plateauing in recent years. Furthermore, kidney cancer mortality rates overall have levelled. These patterns are consistent with reports of incidental diagnosis and downward shift of tumour stage and size; indeed, the widespread use of non-invasive radiological techniques [e.g. ultrasonography (US), computed tomography (CT)], allows the frequent detection of early and small RCCs, which are potentially curable. Beyond well-known risk factors for RCC, such as cigarette smoking, obesity and hypertension, evidence is accumulating to suggest an aetiological or, on the contrary, a protective role, for additional factors [2.Chow W.H. Dong L.M. Devesa S.S. Epidemiology and risk factors for kidney cancer.Nat Rev Urol. 2010; 7: 245-257Crossref PubMed Scopus (983) Google Scholar], such as trichloroethylene. In a recently published case–control study of 699 RCC patients and 1001 frequency-matched controls, consumption of caffeinated coffee was found to be associated with reduced risk of RCC; interestingly, decaffeinated coffee was associated with an increased risk for aggressive clear cell RCC (ccRCC) [3.Antwi S.O. Eckel-Passow J.E. Diehl N.D. et al.Coffee consumption and risk of renal cell carcinoma.Cancer Causes Control. 2017; 28: 857-866Crossref PubMed Scopus (10) Google Scholar]. Furthermore, RCC also appears to be more common in patients with end-stage renal failure or acquired renal cystic disease, and in patients on dialysis, those who have had kidney transplantation or those with tuberous sclerosis syndrome. Approximately 2%–3% of all RCCs are hereditary and several autosomal dominant syndromes are described, each with a distinct genetic basis and phenotype, the most common one being von Hippel–Lindau (VHL) disease. Patients with multiple and bilateral lesions and/or other related disorders should be tested for these germline mutations since it is important that they are recognised. As stated above, >50% of RCCs are currently detected incidentally, making the classical triad of flank pain, gross haematuria and palpable abdominal mass less frequent than in the past. Despite this, RCC remains the ‘Internist’s cancer’ with paraneoplastic syndromes such as hypercalcaemia, unexplained fever, erythrocytosis and Stauffer’s syndrome (signs of cholestasis unrelated to tumour infiltration of the liver or intrinsic liver disease, which typically resolve after kidney tumour resection) still being relatively frequent. Suspicion of RCC should prompt laboratory examinations of serum creatinine, haemoglobin, leukocyte and platelet counts, lymphocyte to neutrophil ratio, lactate dehydrogenase, C-reactive protein (CRP) and serum-corrected calcium [IV, B]. Some of these tests are prognostic for survival and are used for risk assessment within different prognostic score systems (see Staging and risk assessment section). Most cases of RCC are strongly suspected by imaging. Diagnosis is usually suggested by US and further investigated by CT scan, which allows for assessment of local invasiveness, eventual lymph node involvement or distant metastases. Magnetic resonance imaging (MRI) may provide additional information in investigating local advancement and venous involvement by tumour thrombus. For accurate staging of RCC, contrast-enhanced chest, abdominal and pelvic CT is mandatory [III, A]; unless indicated by clinical or laboratory signs or symptoms, the use of bone scan or brain CT (or MRI) is not recommended for routine clinical practice [III, A]. In case of an allergy to CT contrast agent or renal insufficiency, adequate staging should include a high-resolution CT scan of the chest without contrast agent, together with an abdominal MRI. Fluorodeoxyglucose positron emission tomography (FDG-PET) is not a standard investigation in the diagnosis and staging of ccRCC and should not be used. The role of new tracers is under investigation. A renal tumour core biopsy provides histopathological confirmation of malignancy with high sensitivity and specificity; it is especially recommended before treatment with ablative therapies [III, B] as well as in patients with metastatic disease before starting systemic treatment [III, B]. Complications (e.g. bleeding or tumour seeding) are rare or even exceptional (as in the case of tumour seeding) [4.Volpe A. Kachura J.R. Geddie W.R. et al.Techniques, safety and accuracy of sampling of renal tumors by fine needle aspiration and core biopsy.J Urol. 2007; 178: 379-386Crossref PubMed Scopus (284) Google Scholar], while diagnostic accuracy remains high [5.Marconi L. Dabestani S. Lam T.B. et al.Systematic review and meta-analysis of diagnostic accuracy of percutaneous renal tumour biopsy.Eur Urol. 2016; 69: 660-673Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar]. The final histopathological diagnosis, classification, grading and evaluation of prognostic factors are based on the nephrectomy specimen when available. The last edition of the World Health Organization (WHO) histological classification of renal tumours has been reported in 2016 (Table 1), and was based on tumour histology, chromosomal alterations and molecular pathways [6.Moch H. Cubilla A.L. Humphrey P.A. et al.The 2016 WHO classification of tumours of the urinary system and male genital organs-part A: renal, penile, and testicular tumours.Eur Urol. 2016; 70: 93-105Abstract Full Text Full Text PDF PubMed Scopus (1573) Google Scholar].Table 1WHO 2016 classification of renal cell tumoursReprinted with permission from [6.Moch H. Cubilla A.L. Humphrey P.A. et al.The 2016 WHO classification of tumours of the urinary system and male genital organs-part A: renal, penile, and testicular tumours.Eur Urol. 2016; 70: 93-105Abstract Full Text Full Text PDF PubMed Scopus (1573) Google Scholar].Clear cell renal cell carcinomaMultilocular cystic renal neoplasm of low malignant potentialPapillary renal cell carcinomaHereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinomaChromophobe renal cell carcinomaCollecting duct carcinomaRenal medullary carcinomaMiT family translocation renal cell carcinomasSuccinate dehydrogenase-deficient renal cell carcinomaMucinous tubular and spindle cell carcinomaTubulocystic renal cell carcinomaAcquired cystic disease-associated renal cell carcinomaClear cell papillary renal cell carcinomaRenal cell carcinoma, unclassifiedPapillary adenomaOncocytomaMiT, microphthalmia-associated transcription factor; WHO, World Health Organization. Open table in a new tab MiT, microphthalmia-associated transcription factor; WHO, World Health Organization. ccRCCs represent 80% of malignant renal tumours in adults, with the remaining 20% corresponding to several histological subtypes with different histological, molecular and cytogenetic profiles [7.Hsieh J.J. Purdue M.P. Signoretti S. et al.Renal cell carcinoma.Nat Rev Dis Primers. 2017; 3: 17009.Crossref PubMed Scopus (1204) Google Scholar]. Papillary and chromophobe RCCs account for 80% of non-ccRCCs. Papillary RCCs which represent a heterogeneous disease are characterised by:•type 1 RCCs more frequently associated to MET or epidermal growth factor receptor (EGFR) mutations and•type 2 RCCs often unique tumours with an aggressive phenotype that are associated with SETD2 mutations, CDKN2A mutations or TFE3 fusions [8.Linehan W.M. Spellman P.T. et al.Cancer Genome Atlas Research NetworkComprehensive molecular characterization of papillary renal-cell carcinomas.N Engl J Med. 2016; 374: 135-145Crossref PubMed Scopus (799) Google Scholar]. In papillary type 2 with familial history of papillary RCC, a fumarate hydratase (FH) mutation should also be investigated. The main goal in diagnosis of chromophobe RCC, especially in the eosinophilic histological subtype, is the differential diagnosis with oncocytoma. Chromophobe RCCs have diffuse positivity for cytokeratin 7 (CK7), whereas oncocytomas are negative or present focal positivity for CK7. Moreover, chromophobe RCCs display more frequent chromosome loss but fewer somatic mutations. The most frequently mutated gene is tumour suppressor protein 53 (TP53) (32%), and the most frequent oncogenic pathways involved in such tumours are mammalian target of rapamycin (mTOR) pathways (23%), including alterations of phosphatase and tensin homologue (PTEN) [9.Davis C.F. Ricketts C.J. Wang M. et al.The somatic genomic landscape of chromophobe renal cell carcinoma.Cancer Cell. 2014; 26: 319-330Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar]. Microphthalmia-associated transcription factor (MiT) family translocation RCCs (tRCCs) must be ruled out in young patients under the age of 40 years, if papillary architecture or complex architecture with clear cells and/or epithelioid cells or psammoma bodies are present. The diagnosis is based on the use of both immunohistochemistry and fluorescent in situ hybridisation (FISH) analysis to demonstrate the presence of TFE3/TFEB rearrangement. Recently, Argani et al. reported cases of TFEB-amplified RCCs that occur in older patients. These tumours presented high-grade eosinophilic cells with necrosis and papillary or pseudopapillary architecture. The expression of melanocytic markers is variable and FISH analyses revealed high levels of TFEB gene amplification. The prognosis of such tumours is poor with usually advanced stage and metastatic outcome [10.Argani P. Reuter V.E. Zhang L. et al.TFEB-amplified renal cell carcinomas: an aggressive molecular subset demonstrating variable melanocytic marker expression and morphologic heterogeneity.Am J Surg Pathol. 2016; 40: 1484-1495Crossref PubMed Scopus (82) Google Scholar]. Collecting duct carcinoma (CDC) or Bellini duct carcinoma remains a highly aggressive RCC arising from the renal collecting tubules. A recent specific gene expression signature showed that CDC appears to be a unique entity among kidney cancers [11.Becker F. Junker K. Parr M. et al.Collecting duct carcinomas represent a unique tumor entity based on genetic alterations.PLoS One. 2013; 8: e78137Crossref PubMed Scopus (27) Google Scholar]. Moreover, these tumours are characterised by an immune profile with an average of 22% of tumour-infiltrating lymphocytes [12.Malouf G.G. Compérat E. Yao H. et al.Unique transcriptomic profile of collecting duct carcinomas relative to upper tract urothelial carcinomas and other kidney carcinomas.Sci Rep. 2016; 6: 30988Crossref PubMed Scopus (32) Google Scholar]. The prognostic factors validated by the International Society of Urological Pathology (ISUP) consensus and the WHO 2016 classification of RCC to be reported in routine practice are [13.Delahunt B. Cheville J.C. Martignoni G. et al.The International Society of Urological Pathology (ISUP) grading system for renal cell carcinoma and other prognostic parameters.Am J Surg Pathol. 2013; 37: 1490-1504Crossref PubMed Scopus (511) Google Scholar]:•the tumour histological subtype;•the ISUP nucleolar grade (instead of the previous Fuhrman grade) that is only applicable to ccRCC and papillary RCC;•a sarcomatoid and/or rhabdoid differentiation that defines a grade 4 tumour;•the presence of necrosis;•the presence of microscopic vascular invasion;•the pathological tumour, node and metastasis (pTNM) staging anddescription of the non-neoplastic renal tissue. Beyond the classical one gene–one histology paradigm, a more complex biological classification of RCC (and especially of its clear cell histotype) is emerging [14.Brugarolas J. Molecular genetics of clear-cell renal cell carcinoma.J Clin Oncol. 2014; 32: 1968-1976Crossref PubMed Scopus (210) Google Scholar]. First, RCC proved to be an extremely heterogeneous disease [15.Gerlinger M. Rowan A.J. Horswell S. et al.Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.N Engl J Med. 2012; 366: 883-892Crossref PubMed Scopus (5730) Google Scholar]; beyond the seminal genetic alteration (mutation, deletion or hypermethylation) of the VHL tumour suppressor gene, which is present in the vast majority of sporadic RCCs, other genetic alterations may occur, especially over time [16.Gerlinger M. Horswell S. Larkin J. et al.Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing.Nat Genet. 2014; 46: 225-233Crossref PubMed Scopus (893) Google Scholar], contributing to worsen the prognosis of patients harbouring these tumours. Notably, three of these other genes (PBRM1, BAP1 and SETD2) are located on the same short arm of chromosome 3 where the VHL gene is also located. On the contrary, some RCCs are characterised by mutations in the mTOR pathway, and especially in the highly conserved FAT (FRAP, ATM, TTRAP) and kinase domains of the MTOR gene; these cancers have been defined as metabolic RCCs [17.Hakimi A.A. Pham C.G. Hsieh J.J. A clear picture of renal cell carcinoma.Nat Genet. 2013; 45: 849-850Crossref PubMed Scopus (84) Google Scholar]. Finally, according to another comprehensive molecular characterisation of papillary RCCs, type 1 and type 2 papillary RCCs were shown to be clinically and biologically distinct. Alterations in the MET pathway were indeed associated with type 1 and activation of the NRF2-ARE pathway was associated with type 2, while CDKN2A loss and a CpG island methylator phenotype in type 2 contributed to convey a poor prognosis [8.Linehan W.M. Spellman P.T. et al.Cancer Genome Atlas Research NetworkComprehensive molecular characterization of papillary renal-cell carcinomas.N Engl J Med. 2016; 374: 135-145Crossref PubMed Scopus (799) Google Scholar]. The Union for International Cancer Control (UICC) tumour, node and metastasis (TNM) 8 staging system should be used [18.Brierley J.D. Gospodarowicz M.K. Wittekind C. IUCC TNM Classification of Malignant Tumours. 8. John Wiley & Sons, Inc, Oxford2016: 114-115Google Scholar] (Table 2).Table 2UICC TNM 8 staging of RCC [18.Brierley J.D. Gospodarowicz M.K. Wittekind C. IUCC TNM Classification of Malignant Tumours. 8. John Wiley & Sons, Inc, Oxford2016: 114-115Google Scholar]Reprinted from [18.Brierley J.D. Gospodarowicz M.K. Wittekind C. IUCC TNM Classification of Malignant Tumours. 8. John Wiley & Sons, Inc, Oxford2016: 114-115Google Scholar] with permission from John Wiley & Sons, Inc.T—primary tumourTX Primary tumour cannot be assessedT0 No evidence of primary tumourT1 Tumour 7 cm or less in greatest dimension, limited to the kidney T1a Tumour 4 cm or less T1b Tumour more than 4 cm but not more than 7 cmT2 Tumour more than 7 cm in greatest dimension, limited to the kidney T2a Tumour more than 7 cm but not more than 10 cm T2b Tumour more than 10 cm, limited to the kidneyT3 Tumour extends into major veins or perinephric tissues but not into the ipsilateral adrenal gland and not beyond Gerota fascia T3a Tumour extends into the renal vein or its segmental branches, or tumour invades the pelvicalyceal system or tumour invades perirenal and/or renal sinus fat (peripelvic) fat but not beyond Gerota fascia T3b Tumour extends into vena cava below diaphragm T3c Tumour extends into vena cava above the diaphragm or invades the wall of the vena cavaT4 Tumour invades beyond Gerota fascia (including contiguous extension into the ipsilateral adrenal gland)N—regional lymph nodesNX Regional lymph nodes cannot be assessedN0 No regional lymph node metastasisN1 Metastasis in regional lymph node(s)M—distant metastasisM0 No distant metastasisM1 Distant metastasispTNM pathological classificationaThe pT and pN categories correspond to the T and N categories.StageStage I T1 N0 M0Stage II T2 N0 M0Stage III T3 N0 M0 T1, T2, T3 N1 M0Stage IV T4 Any N M0 Any T Any N M1pTNM, pathological tumour, node, metastasis; RCC, renal cell carcinoma; TNM, tumour, node, metastasis; UICC, Union for International Cancer Control.a The pT and pN categories correspond to the T and N categories. Open table in a new tab pTNM, pathological tumour, node, metastasis; RCC, renal cell carcinoma; TNM, tumour, node, metastasis; UICC, Union for International Cancer Control. The natural clinical course varies in RCC, which has led to the development of different prognostic models for the assessment of the patient’s individual risk. Extent of disease, histology, grading and clinical factors have been recognised as having prognostic value in RCC and may be used in localised or in metastatic disease [6.Moch H. Cubilla A.L. Humphrey P.A. et al.The 2016 WHO classification of tumours of the urinary system and male genital organs-part A: renal, penile, and testicular tumours.Eur Urol. 2016; 70: 93-105Abstract Full Text Full Text PDF PubMed Scopus (1573) Google Scholar]. Different pre- or post-operative scores have been developed to assess prognosis in RCC, and are used for risk-adapted follow-up strategies. Integrated prognostic scores offer some predictive advantages over single tumour characteristics and are used preferentially. These models are composed of histological and clinical factors. The most recent modifications of the stage, size, grade and necrosis (SSIGN) score [19.Leibovich B.C. Blute M.L. Cheville J.C. et al.Prediction of progression after radical nephrectomy for patients with clear cell renal cell carcinoma: a stratification tool for prospective clinical trials.Cancer. 2003; 97: 1663-1671Crossref PubMed Scopus (587) Google Scholar] (Table 3) and the University of California Los Angeles Integrated Staging System (UISS) (Table 4) [20.Patard J.J. Kim H.L. Lam J.S. et al.Use of the University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: an international multicenter study.J Clin Oncol. 2004; 22: 3316-3322Crossref PubMed Scopus (302) Google Scholar] score are frequently used.Table 3SSIGN score for localised RCC [19.Leibovich B.C. Blute M.L. Cheville J.C. et al.Prediction of progression after radical nephrectomy for patients with clear cell renal cell carcinoma: a stratification tool for prospective clinical trials.Cancer. 2003; 97: 1663-1671Crossref PubMed Scopus (587) Google Scholar]Adapted from [19.Leibovich B.C. Blute M.L. Cheville J.C. et al.Prediction of progression after radical nephrectomy for patients with clear cell renal cell carcinoma: a stratification tool for prospective clinical trials.Cancer. 2003; 97: 1663-1671Crossref PubMed Scopus (587) Google Scholar], with permission from John Wiley & Sons, Inc.FeatureScorePathological T category of primary tumour (as per 2002 TNM staging)pT1a0pT1b2pT23pT3a-pT3c4pT44Regional lymph node status (as per 2002 TNM staging)pNx or pN00pN1 or pN22Tumour size<10 cm010 cm or more1Nuclear grade1 or 203143Histological tumour necrosisNo0Yes1ScoresGroup5-year metastasis- free survival rate (%)0–2Low risk97.13–5Intermediate risk73.86 or moreHigh risk31.2RCC, renal cell carcinoma; SSIGN, size, stage, grade and necrosis; TNM, tumour, node, metastasis. Open table in a new tab Table 4UISS risk groups and 5-year disease-specific survival [20.Patard J.J. Kim H.L. Lam J.S. et al.Use of the University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: an international multicenter study.J Clin Oncol. 2004; 22: 3316-3322Crossref PubMed Scopus (302) Google Scholar]Reprinted from [20.Patard J.J. Kim H.L. Lam J.S. et al.Use of the University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: an international multicenter study.J Clin Oncol. 2004; 22: 3316-3322Crossref PubMed Scopus (302) Google Scholar] with permission. © 2004 American Society of Clinical Oncology. All rights reserved.Patient groupPrognostic groupT stageFuhrman gradeECOG status5-year disease-specific survival (%)Localised disease (N0, M0)Low risk11–2091.1Intermediate risk11–21 or more80.413–4Any2AnyAny31Any32–4AnyHigh32–41 or more54.74AnyAnyMetastatic diseaseLow riskN1M0AnyAny32N2M0/M11–20Intermediate riskN2M0/M11–21 or more19.530, 1 or more40HighN2M0/M141 or more0ECOG, Eastern Cooperative Oncology Group; UISS, University of California Los Angeles Integrated Staging System. Open table in a new tab RCC, renal cell carcinoma; SSIGN, size, stage, grade and necrosis; TNM, tumour, node, metastasis. ECOG, Eastern Cooperative Oncology Group; UISS, University of California Los Angeles Integrated Staging System. However, among different prognostic scores, a concordance of 0.68–0.89 for cancer-specific survival (CSS) and 0.74–0.82 for recurrence-free survival (RFS) was reported [21.Sun M. Shariat S.F. Cheng C. et al.Prognostic factors and predictive models in renal cell carcinoma: a contemporary review.Eur Urol. 2011; 60: 644-661Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar], indicating that a plateau has been reached for prognostication with available models. Hence, no clear preference for a specific prognostic model may be given. The Memorial Sloan Kettering Cancer Center (MSKCC) system was the gold standard for the risk assessment during cytokine treatment in metastatic RCC (mRCC) [22.Motzer R.J. Mazumdar M. Bacik J. et al.Survival and prognostic stratification of 670 patients with advanced renal cell carcinoma.J Clin Oncol. 1999; 17: 2530-2540Crossref PubMed Google Scholar], and it is still commonly used. Further refinement was introduced with the International Metastatic RCC Database Consortium (IMDC) score, which extended the previous factors to a total number of six in order to increase concordance [23.Ko J.J. Xie W. Kroeger N. et al.The International Metastatic Renal Cell Carcinoma Database Consortium model as a prognostic tool in patients with metastatic renal cell carcinoma previously treated with first-line targeted therapy: a population-based study.Lancet Oncol. 2015; 16: 293-300Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar]:•Karnofsky performance status (PS) <80%;•haemoglobin level below the lower limit of normal;•time from diagnosis to treatment <1 year;•corrected calcium above the upper limit of normal;•platelets greater than the upper limit of normal and•neutrophils greater than the upper limit of normal. A recent evaluation of this model in second-line treatment underscored its predictive value in previously treated mRCC [24.Heng D.Y. Xie W. Regan M.M. et al.Prognostic factors for overall survival in patients with metastatic renal cell carcinoma treated with vascular endothelial growth factor-targeted agents: results from a large, multicenter study.J Clin Oncol. 2009; 27: 5794-5799Crossref PubMed Scopus (1532) Google Scholar] (Table 5). Interestingly, this model is also applicable in further lines of therapy as well as in non-clear cell histology.Table 5Median OS estimates in first- and second-line RCC according to IMDC risk groupsNumber of risk factorsRisk categoryMedian OS (months)First line [24.Heng D.Y. Xie W. Regan M.M. et al.Prognostic factors for overall survival in patients with metastatic renal cell carcinoma treated with vascular endothelial growth factor-targeted agents: results from a large, multicenter study.J Clin Oncol. 2009; 27: 5794-5799Crossref PubMed Scopus (1532) Google Scholar]Second line [23.Ko J.J. Xie W. Kroeger N. et al.The International Metastatic Renal Cell Carcinoma Database Consortium model as a prognostic tool in patients with metastatic renal cell carcinoma previously treated with first-line targeted therapy: a population-based study.Lancet Oncol. 2015; 16: 293-300Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar]0Favourable43.235.31–2Intermediate22.516.63–6Unfavourable7.85.4IMDC, International Metastatic RCC Database Consortium; OS, overall survival; RCC, renal cell carcinoma. Open table in a new tab IMDC, International Metastatic RCC Database Consortium; OS, overall survival; RCC, renal cell carcinoma. Gene signatures can be used to detect different risk groups in RCC. ClearCode34 is a 34-gene expression panel that proved able to classify ccRCC into two subtypes, clear cell A (ccA) and clear cell B (ccB), significantly associated with relapse-free survival and CSS, as well as overall survival (OS) [25.Brooks S.A. Brannon A.R. Parker J.S. et al.ClearCode34: a prognostic risk predictor for localized clear cell renal cell carcinoma.Eur Urol. 2014; 66: 77-84Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar]. Another gene signature, based on a 16-gene assay, was shown to improve prediction of RFS in localised RCC when compared with the SSIGN score according to the Leibovich score (concordance: 0.81 versus 0.74) [26.Rini B. Goddard A. Knezevic D. et al.A 16-gene assay to predict recurrence after surgery in localised renal cell carcinoma: development and validation studies.Lancet Oncol. 2015; 16: 676-685Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar]. Some gene mutations have also been reported as prognostic. The University of Texas Southwestern group identified distinct clinical outcomes in mutation-defined subtypes of ccRCC: a high-risk BAP1-mutant group and a favourable PBRM1-mutant group [27.Kapur P. Peña-Llopis S. Christie A. et al.Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation.Lancet Oncol. 2013; 14: 159-167Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar]. Notably, 80% of patients from both the development and the validation cohorts had localised (or locoregional) disease. In the metastatic setting, the immunohistochemical expression of programmed death ligand 1 (PD-L1) is presently under the spotlight, although the results available so far are still controversial. In 2016, a systematic review and meta-analysis of 6 studies and 1323 cases clearly demonstrated a negative prognostic role of elevated level of PD-L1 tumour expression in RCC [28.Iacovelli R. Nolè F. Verri E. et al.Prognostic role of PD-L1 expression in renal cell carcinoma. A systematic review and meta-analysis.Target Oncol. 2016; 11: 143-148Crossref PubMed Scopus (136) Google Scholar], although discrepancies between PD-L1 expression between the primary tumour and the metastases have been reported. A possible predictive value of PD-L1 expression remains controversial, although recently, PD-L1 tumour expression was shown to be able to identify patients benefiting from a combination of two immune-checkpoint inhibitors [29.Motzer R.J. Tannir N.M. McDermott D.F. et al.Nivolumab plus ipilimumab versus sunitinib in advanced renal-cell carcinoma.N Engl J Med. 2018; 378: 1277-1290Crossref PubMed Scopus (2494) Google Scholar]. In addition, angiogenesis, T-effector/interferon (IFN)-γ response and myeloid inflammatory gene expression signatures have been suggested to predict response to vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI) and immunotherapy [30.McDermott D.F. Huseni M.A. Atkins M.B. et al.Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma.Nat Med. 2018; 24: 749-757Crossref PubMed Scopus (690) Google Scholar]. As a whole, PD-L1 expression should not be routinely used but is a putative biomarker for future treatment selection because of remaining unanswered issues related to the different tests and cut-offs used, to the cells where PD-L1 expression should be checked and to the role of PD-L1 expression heterogeneity (e.g. between different primary tumour sites, or between primary tumour and its metastases). Other putative markers such as circulating DNA, microRNA or DNA methylation status were shown to have prognostic relevance in RCC and warrant future investigation. Overall, these data indicate that molecular analysis may provide additional benefit to already established clinical and histo-anatomical parameters, which may lead to an individual risk assessment in the future. Currently, no specific molecular marker can be recommended for clinical use. Partial nephrectomy (PN) is recommended as the preferred option in organ-confined tumours measuring up to 7 cm (elective indication). This is based on a systematic review including multiple retrospective studies and a prospective, randomised controlled trial (RCT) which compared radical nephrectomy (RN) with PN in solitary T1a-b N0M0 renal tumours <5 cm with normal contralateral kidney function [I, A] [31.MacLennan S. Imamura M. Lapitan M.C. et al.Systematic review of oncological outcomes following surgical management of localised renal cancer.Eur Urol. 2012; 61: 972-993Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar]. PN can be carried out via open, laparoscopic or robot-assisted laparoscopic approaches. Laparoscopic RN is recommended if PN is not technically feasible. In patients with compromised renal function, solitary kidney or bilateral tumours, PN is also the standard of care, with no tumour size limitation (imperative indication). Systematic reviews comparing surgical management of localised RCC (T1-2N0M0) were unable to identify prospective comparative studies reporting on oncological outcomes for minimally invasiv

310 - Differential expression of PD-L1 between sporadic and VHL-associated hereditary clear cell renal cell carcinoma and its correlation with clinicopathological features

310 - Differential expression of PD-L1 between sporadic and VHL-associated hereditary clear cell renal cell carcinoma and its correlation with clinicopathological features

Specific Localization of Missense Mutations in the VHL Gene in Clear Cell Renal Cell Carcinoma.

Missense mutations in the VHL gene during sporadic clear cell renal cell carcinoma were studied to evaluate their localization in relation to functionally important motifs of the VHL protein. Somatic mutations were identified in 124 of 307 samples. All missense mutations in the α-domain were localized in the binding site for elongin C. Substitutions in the β-domain (77%) were found in the HIF-binding site. Five missense mutations were absent in these sites, which illustrates their role in VHL protein formation or suppressor function of other protein cofactors. Mutation c.392A→T (p.N131I) was identified for the first time. Our results hold much promise to estimate the boundaries of functionally important sites in the VHL suppressor gene and contribute to the interpretation of a pathogenic role of mutations in direct DNA diagnostics.

Quantitative proteomics to study a small molecule targeting the loss of von Hippel-Lindau in renal cell carcinomas.

Inactivation of the tumor suppressor gene, von Hippel-Lindau (VHL), is known to play an important role in the development of sporadic clear cell renal cell carcinomas (ccRCCs). Even if available targeted therapies for metastatic RCCs (mRCCs) have helped to improve progression-free survival rates, they have no durable clinical response. We have previously shown the feasibility of specifically targeting the loss of VHL with the identification of a small molecule, STF-62247. Understanding its functionality is crucial for developing durable personalized therapeutic agents differing from those available targeting hypoxia inducible factor (HIF-) pathways. By using SILAC proteomics, we identified 755 deregulated proteins in response to STF-62247 that were further analyzed by ingenuity pathway analysis (IPA). Bioinformatics analyses predicted alterations in 37 signaling pathways in VHL-null cells in response to treatment. Validation of some altered pathways shows that STF-62247's selectivity is linked to an important inhibition of mTORC1 activation in VHL-null cells leading to protein synthesis arrest, a mechanism differing from two allosteric inhibitors Rapamycin and Everolimus. Altogether, our study identified signaling cascades driving STF-62247 response and brings further knowledge for this molecule that shows selectivity for the loss of VHL. The use of a global SILAC approach was successful in identifying novel affected signaling pathways that could be exploited for the development of new personalized therapeutic strategies to target VHL-inactivated RCCs.

Loss of chromosome Y leads to down regulation of KDM5D and KDM6C epigenetic modifiers in clear cell renal cell carcinoma

Recent genomic studies of sporadic clear cell renal cell carcinoma (ccRCC) have uncovered novel driver genes and pathways. Given the unequal incidence rates among men and women (male:female incidence ratio approaches 2:1), we compared the genome-wide distribution of the chromosomal abnormalities in both sexes. We observed a higher frequency for the somatic recurrent chromosomal copy number variations (CNVs) of autosomes in male subjects, whereas somatic loss of chromosome X was detected exclusively in female patients (17.1%). Furthermore, somatic loss of chromosome Y (LOY) was detected in about 40% of male subjects, while mosaic LOY was detected in DNA isolated from peripheral blood in 9.6% of them, and was the only recurrent CNV in constitutional DNA samples. LOY in constitutional DNA, but not in tumor DNA was associated with older age. Amongst Y-linked genes that were downregulated due to LOY, KDM5D and KDM6C epigenetic modifiers have functionally-similar X-linked homologs whose deficiency is involved in ccRCC progression. Our findings establish somatic LOY as a highly recurrent genetic defect in ccRCC that leads to downregulation of hitherto unsuspected epigenetic factors, and suggest that different mechanisms may underlie the somatic and mosaic LOY observed in tumors and peripheral blood, respectively.

Loss of SETD2, but not H3K36me3, correlates with aggressive clinicopathological features of clear cell renal cell carcinoma patients.

Recent studies facilitated by DNA sequencing identified the histone modifying gene SETD2 as the second most frequent mutant gene in sporadic clear cell renal cell carcinoma (ccRCC) patients. SETD2 functions as a tumor suppressor in ccRCC. However, its clinical association and biological functions are not fully delineated. The aim of this study is to evaluate the clinical significance of SETD2 in ccRCC patients. SETD2 and its canonical histone modification product H3K36me3 were analyzed by immunohistochemistry (IHC) in 155 ccRCC patients from two independent cohorts retrospectively. Both SETD2 and H3K36me3 were heterogeneously stained and down-regulated in ccRCC tissues, compared with normal controls. The SETD2 protein deficiency rate was 34.07%, which is much higher than the reported SETD2 gene inactive mutation rate. Furthermore, low SETD2 protein expression, but not H3K36me3 expression, was associated with the aggressive phenotype of ccRCC patients. In addition, cox multivariate analysis identified low SETD2 protein expression as an independent prognostic factor for overall survival of ccRCC patients. Consistently, using RNA-Seq data of ccRCC patients from The Cancer Genome Atlas, we validated our findings that low SETD2 mRNA expression is significantly associated with the aggressive phenotypes, and predicted a worse outcome for ccRCC patients. In conclusion, our study demonstrated a massive down-regulation of SETD2 protein in ccRCC, and identified SETD2 protein, but not H3K36me3, as an independent good prognostic marker, which warrants further study focusing on the non-methyltransferase role of SETD2 in kidney tumor biology.

Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma

Alterations in von Hippel-Lindau gene (VHL) do not determine deregulation of hypoxia-inducible factors (HIFs) in clear-cell renal carcinoma (ccRCC). Their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expressions in sporadic ccRCC are unknown. Therefore, we analyze the impact of VHL alterations and HIF-α production on the expression of TSC proteins and Hsp90 in these tumors.Alterations in VHL gene region exhibited 37/47 (78.7%) tumors. Monoallelic inactivation (intragenic mutation or LOH) was found in 10 (21.3%) and biallelic inactivation (intragenic mutation plus LOH) in 27 (57.4%) ccRCCs. Tumorous expression of HIF-α mRNAs, HIF-α, Hsp90 and TSC2 were VHL independent; TSC2 was underexpressed in all tumors by immunostaining (P<0.001). Immunoblotting revealed that TSC1 production was lower in tumors with monoallelic VHL inactivation than in control (P=0.01) and tissues with biallelic VHL inactivation (P=0.019), while tumors lacking HIF-1α (16/47) concurrently overexpressed HIF-2α and underexpressed TSC1 in comparison to controls (P=0.01 for both) and HIF-1α positive tumors (P=0.015 and P=0.050). Significant portion of variability (56.4%) in tumor diameter was explained by oscillations in nuclear grade, and TSC1 and HIF-2α expression in VHL altered tumors.In conclusion, while TSC2 is broadly downregulated in sporadic ccRCC, TSC1 expression is reduced in two subsets of these tumors, those with monoallelic VHL gene inactivation and those with concurrent low HIF-1α and high HIF-2α expression. Hence, the involvement of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumors, implies the interplay between pVHL and TSC1.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal one-hit cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the Warburg effect. Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NF?B and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

Characterization of VHL missense mutations in sporadic clear cell renal cell carcinoma: hotspots, affected binding domains, functional impact on pVHL and therapeutic relevance.

BackgroundThe VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners. The affected pathways may offer predictive clues to therapy and response to treatment. In this study we focused on the VHL missense mutation pattern in ccRCC, and studied their potential effects on pVHL protein stability and binding partners and discussed treatment options.MethodsWe sequenced VHL in 360 sporadic ccRCC FFPE samples and compared observed and expected frequency of missense mutations in 32 different binding domains. The prediction of the impact of those mutations on protein stability and function was assessed in silico. The response to HIF-related, anti-angiogenic treatment of 30 patients with known VHL mutation status was also investigated.ResultsWe identified 254 VHL mutations (68.3 % of the cases) including 89 missense mutations (35 %). Codons Ser65, Asn78, Ser80, Trp117 and Leu184 represented hotspots and missense mutations in Trp117 and Leu 184 were predicted to highly destabilize pVHL. About 40 % of VHL missense mutations were predicted to cause severe protein malfunction. The pVHL binding domains for HIF1AN, BCL2L11, HIF1/2α, RPB1, PRKCZ, aPKC-λ/ι, EEF1A1, CCT-ζ-2, and Cullin2 were preferentially affected. These binding partners are mainly acting in transcriptional regulation, apoptosis and ubiquitin ligation. There was no correlation between VHL mutation status and response to treatment.ConclusionsVHL missense mutations may exert mild, moderate or strong impact on pVHL stability. Besides the HIF binding domain, other pVHL binding sites seem to be non-randomly altered by missense mutations. In contrast to LOF mutations that affect all the different pathways normally controlled by pVHL, missense mutations may be rather appropriate for designing tailor-made treatment strategies for ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2688-0) contains supplementary material, which is available to authorized users.

Nestin expression on tumour vessels and tumour-infiltrating macrophages define a poor prognosis subgroup of pt1 clear cell renal cell carcinoma.

The behaviour of clear cell renal cell carcinoma (CCRCC) is highly unpredictable. Despite adequate initial surgery, 20 to 30% of patients will develop local recurrence or metastasis during follow-up. Usual clinical and pathology parameters tend to classify most patients in an intermediate prognosis group, and molecular markers to determine prognosis more accurately are needed. A key feature of CCRCC is its abundant vascularization. Factors that upregulate angiogenesis, such as hypoxia and the presence of immune cells including macrophages, also modulate tumour proliferation and metastasis. We studied angiogenesis, as defined by nestin-positive capillaries, and tumour infiltration by macrophages especially in the good prognosis pT1 subgroup of CCRCC. We assessed whether these parameters are associated with metastatic extension and survival in CCRCC. The expression of HIF1α, CAIX, nestin, CD68 and CD163 was assessed by immunohistochemistry on a tissue microarray (TMA) containing tissue samples from 257 consecutive patients with sporadic CCRCC. Factors associated with progression-free (PFS) and overall survival (OS) were analysed. The presence of nestin-positive tumour vessels was independently associated with shorter PFS in the whole cohort and in the pT1 subgroup. The presence of tumour-infiltrating macrophages was independently associated with shorter OS in the whole cohort and in the pT1 subgroup. The presence of nestin-positive endothelial cells is associated with early relapse, especially in the pT1 subgroup and may help to select patients for antiangiogenic treatment. The presence of tumour-infiltrating M2-type macrophages is a strong predictor of short survival and may be used to adapt treatment strategy.

Abstract A19: A novel cell line model for chromophobe renal cell carcinoma, UOK276, derived from an aggressive sarcomatoid differentiated tumor

Abstract Background: Renal cell carcinoma (RCC) represents a heterogeneous group of cancers that arise from the nephron and are subtyped by histopathological features. The most common subtypes are clear cell RCC (~75%) and papillary RCC (~15%); whereas chromophobe renal cell carcinoma (ChRCC) represents only 5% of RCC cases. ChRCC typically demonstrate a well-known karyotype of multiple chromosomal losses and a relatively indolent pattern of local growth, but can present with aggressive features and demonstrate resistance to treatment in a metastatic setting. Some ChRCC cases demonstrate regions of sarcomatoid RCC and the exact cause of this differentiation has yet to be elucidated. Cell line models are an important tool for both the investigation of tumor biology and therapeutic drug efficacy. Currently, numerous cell lines models exist that have been derived from sporadic clear cell or papillary RCCs, but there are few cell lines derived from chromophobe RCCs and none are well characterized. This study produced a novel ChRCC-derived cell line model and provides an initial genetic and metabolic characterization. Materials and Methods: A patient presented with a 20 cm ChRCC with regions of sarcomatoid differentiation that was surgically excised and a section of this tumor was used to establish a spontaneously immortal cell line model, UOK276. This line was grown for over 20 passages and cytogenetically assessed by spectral karyotyping (SKY). Mutation analysis was performed using a cancer gene specific chip, OncoVar V3, which analyses 232 genes. Identified mutations were confirmed in both UOK276 and the original tumor tissue and further investigated for their effects of mRNA and protein expression. UOK276 cells were injected into nude mice to assess the production of xenograph tumors. The metabolic, bioenergetic profile was assessed using a Seahorse XF96 Extracellular Flux Analyzer. Results: The chromosomal SKY analysis did not demonstrate the classic pattern of chromophobe chromosomal losses, but demonstrated hyper-aneuploidy, with a modal number of 49 chromosomes per cell, and identified a balanced translocation t(X;8)(q10;q24). The break on chromosome 8q occurred near the MYC gene, but break-apart FISH analysis demonstrated no alterations to MYC although amplification of this derivative chromosome was observed and increased MYC mRNA expression was demonstrated. Mutation analysis identified a missense mutation (p.H193Y) of TP53, commonly mutated in ChRCC, which was only present in the sarcomatoid region of the tumor. Mutation of TP53 has previously been associated with sarcomatoid differentiation. Protein expression analysis demonstrated the presence of the mutant TP53 protein in UOK276. A heterozygous germline mutation in TRAF7 was identified resulting in an in-frame loss of 4 amino acids (del T22-P25) that was homozygous in the sarcomatoid tumor region and UOK276. Xenograph tumors were successfully grown in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The recent TCGA study of ChRCC demonstrated increased expression of the electron transport chain (ETC) genes suggesting increased oxidative phosphorylation within these tumors. Metabolic analysis of UOK276 demonstrated a relatively low level of oxygen consumption (OCR) in comparison to a normal kidney cell line and this was supported by mRNA expression data showing normal or reduced levels of expression for several ETC-related genes. Conclusions: Our study has produced a novel ChRCC cell line model that exhibits a TP53 mutation, commonly seen in ChRCC, and represents a sarcomatoid differentiated region of the tumor. UOK276 should provide a unique in vitro and in vivo preclinical model system for studying the deregulated pathways and testing therapeutic strategies in sarcomatoid differentiated ChRCC. Citation Format: Youfeng Yang, Cathy D. Vocke, Christopher J. Ricketts, Darmood Wei, Hesed M. Padilla-Nash, Shawna L. Boyle, Robert Worrell, Thomas Ried, Maria J. Merino, W. Marston Linehan. A novel cell line model for chromophobe renal cell carcinoma, UOK276, derived from an aggressive sarcomatoid differentiated tumor. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A19.