Von Hippel-Lindau Mutations Research Articles

SURG-46. ENDOLYMPHATIC SAC TUMORS: A CASE REPORT AND REVIEW OF THE LITERATURE

Abstract Endolymphatic sac tumors are rare, histologically benign tumors that pose a danger of local bony destruction. Definitive treatment is complete surgical resection but the use of subsequent radiotherapy remains controversial and is typically used for residual or recurrent disease. Additionally, these tumors are associated with von-Hippel-Lindau (VHL) syndrome and are present in 10-30% of patients with this autosomal dominant disorder. We present the case of a 69 year-old woman who presented with cranial nerve 7 and 8 palsies that had started in the 1990s and was found to have a left CPA mass. She underwent pre-operative embolization followed by 2 stage craniotomy and post-operative radiotherapy. Testing for the VHL mutation was negative.

Clear Cell Renal Cell Carcinoma: Characterizing the Phenotype of Von Hippel-Lindau Mutation Using MRI.

The von Hippel-Lindau (VHL) mutation is an important alteration in clear cell renal cell carcinoma (ccRCC); however, its imaging phenotype remains unclear. To investigate whether MRI features can reflect the VHL mutation status. Retrospective. 3 T/fast spin echo T2-weighted, spin-echo echo planar diffusion-weighted, gradient recalled echo T1-weighted, gradient recalled echo chemical-shift T1-weighted, and contrast-enhanced gradient recalled echo T1-weighted sequences. One hundred five patients with ccRCC who underwent preoperative contrast-enhanced MRI and subsequent genomic sequencing: 59 consecutive patients from our institution (38 [64.41%] with VHL mutations) formed a training cohort, and 46 from The Cancer Genome Atlas (TCGA) database (24 [52.17%] with VHL mutations) formed an independent test cohort. Two radiologists, with 23 and 33 years of experience respectively, jointly evaluated the semantic MRI features of the primary lesion in ccRCCs to propose potential features related to VHL mutations in both cohorts. Three additional readers, with 5, 7, and 10 years of experience respectively, independently reviewed all lesions to assess the interobserver agreement of MRI features. A VHL mutational likelihood score (VHL-MULIS) system was constructed using the training cohort and validated using the independent test cohort. Fisher's test or chi-square test, t-test or Mann-Whitney U test, logistic regression, Cohen's kappa (κ), area under the receiver operating characteristic curve (AUC). A two-sided P value <0.05 was considered statistically significant. In both the local and public cohorts, T2-weighted signal intensity and presence of microscopic fat from primary lesions were significantly associated with VHL mutation status. The VHL-MULIS incorporated maximum diameter, T2-weighted signal intensity, and presence of microscopic fat in the training cohort and demonstrated promising diagnostic ability (AUC, 0.82; sensitivity, 0.79; specificity, 0.82) and substantial interobserver agreement (κ, 0.787) in the test cohort. The VHL mutation exhibited a distinct MRI phenotype. Integrating multiple semantic MRI features has potential to reflect the mutation status in patients with ccRCC. 3 TECHNICAL EFFICACY: Stage 2.

Clinicopathological features of BAP1 mutated clear cell renal cell carcinoma

Objective: To investigate the clinicopathological characteristics, immunophenotypes, molecular features, and differential diagnosis of BAP1 mutated clear cell renal cell carcinoma (CCRCC) for better understanding this entity. Methods: Clinical data, histological morphology, immunophenotypes and molecular characteristics of 18 BAP1 mutated CCRCC cases diagnosed at the Department of Pathology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China from January 2020 to December 2022 were analyzed. The patients were followed up. Results: There were 17 males and 1 female patients, aged from 39 to 72 years, with an average age of 56.3 years. Sixteen patients with primary CCRCC were followed up for an average of 24 months, 7 patients had metastases occurred from 4 to 22 months postoperatively. Thirteen of the 16 patients were alive at the time of the last follow-up while 3 patients died 12, 15, and 20 months after the surgery, respectively. One patient underwent retroperitoneal mass resection, but had lung metastasis 32 months after surgery. One case received cervical tumor resection and died at 22 months after the surgery. Characteristic CCRCC regions were identified in 11 of the 18 cases. The tumor cells were arranged in papillary, alveolar, and large nest patterns. Abundant lymphoid tissue, necrosis, and psammoma bodies were seen. Tumor cells showed abundant eosinophilic cytoplasm, and sometimes exhibited rhabdoid differentiation. Round eosinophilic globules were located in the cytoplasm and extracellular matrix. There were 9 cases with WHO/International Society of Urological Pathology grade 3, and 9 cases with grade 4. PAX8 (18/18), carbonic anhydrase 9 (CA9, 16/18), CD10 (18/18), and vimentin (18/18) were positive in the vast majority of tumors.TFE3 was expressed in 5 cases, with strong expression in only 1 case. Eighteen cases were all positive for P504s. Twelve cases harbored a BAP1 mutation combined with von Hippel-Lindau (VHL) mutation, and 2 cases had mutations in BAP1, VHL and PBRM1 simultaneously. SETD2 mutation was not found in any of the cases. Conclusions: BAP1 mutated CCRCC contained papillary, alveolar, and large nest patterns, eosinophilic cytoplasm, high-grade nucleoli, and collagen globules, with P504s positivity. In practical work, when encountering CCRCC containing these features, pathologists should consider the possibility of BAP1 mutations and conduct related molecular tests.

Advancements in Radiogenomics for Clear Cell Renal Cell Carcinoma: Understanding the Impact of BAP1 Mutation.

Recent advancements in understanding clear cell renal cell carcinoma (ccRCC) have underscored the critical role of the BAP1 gene in its pathogenesis and prognosis. While the von Hippel-Lindau (VHL) mutation has been extensively studied, emerging evidence suggests that mutations in BAP1 and other genes significantly impact patient outcomes. Radiogenomics with and without texture analysis based on CT imaging holds promise in predicting BAP1 mutation status and overall survival outcomes. However, prospective studies with larger cohorts and standardized imaging protocols are needed to validate these findings and translate them into clinical practice effectively, paving the way for personalized treatment strategies in ccRCC. This review aims to summarize the current knowledge on the role of BAP1 mutation in ccRCC pathogenesis and prognosis, as well as the potential of radiogenomics in predicting mutation status and clinical outcomes.

VHL mutation as a cause of three generations familial pheochromocytoma

BackgroundPheochromocytoma is a rare disease, and its familial occurrence is quite uncommon. The aim of this paper is to report a three-generation phenotypical expression of a case familial occurrence of pheochromocytoma.Case presentationA 25-year-old female, with a history of adrenalectomy for pheochromocytoma, arrived at the shock room during her third pregnancy with an adrenergic crisis and hypoglycemia. To prevent perinatal complications, the patient was stabilized and the newborn was delivered through a Kerr-type cesarean section. A detailed history revealed that the paternal grandfather of the patient had an unilateral pheochromocytoma, whereas her paternal uncle had a bilateral pheochromocytoma. Additionally, a brother of the patient presented a unilateral pheochromocytoma. Amplicons for PCR assays were designed to span the protein-coding segments of the three Von Hippel–Lindau (VHL) exons, and the PCR products were sequenced using the Sanger method. In the trace of exon 3, we detected in the sample of the proband a heterozygous guanine to adenine transition (NM_000551.4 c. 552G > A) within the protein-coding segment of exon 3 of the VHL gene, which leads to a substitution of the arginine residue at position 161 by a glutamine residue in the encoded peptide (NP_000542.1p.R161Q). This mutation was absent in two unaffected daughters.ConclusionA VHL mutation was suspected and confirmed in this family that was not transmitted to a fourth generation. This case illustrates the importance of molecular genetics methodologies to assist genetic counseling in cases of pheochromocytoma where familial aggregation is presumed.

A Prospective Study of the Diagnostic Performance of Photon-Counting CT Compared With MRI in the Characterization of Renal Masses.

The aim of this study was to assess the interreader reliability and per-RCC sensitivity of high-resolution photon-counting computed tomography (PCCT) in the detection and characterization of renal masses in comparison to MRI. This prospective study included 24 adult patients (mean age, 52 ± 14 years; 14 females) who underwent PCCT (using an investigational whole-body CT scanner) and abdominal MRI within a 3-month time interval and underwent surgical resection (partial or radical nephrectomy) with histopathology (n = 70 lesions). Of the 24 patients, 17 had a germline mutation and the remainder were sporadic cases. Two radiologists (R1 and R2) assessed the PCCT and corresponding MRI studies with a 3-week washout period between reviews. Readers recorded the number of lesions in each patient and graded each targeted lesion's characteristic features, dimensions, and location. Data were analyzed using a 2-sample t test, Fisher exact test, and weighted kappa. In patients with von Hippel-Lindau mutation, R1 identified a similar number of lesions suspicious for neoplasm on both modalities (51 vs 50, P = 0.94), whereas R2 identified more suspicious lesions on PCCT scans as compared with MRI studies (80 vs 56, P = 0.12). R1 and R2 characterized more lesions as predominantly solid in MRIs (R1: 58/70 in MRI vs 52/70 in PCCT, P < 0.001; R2: 60/70 in MRI vs 55/70 in PCCT, P < 0.001). R1 and R2 performed similarly in detecting neoplastic lesions on PCCT and MRI studies (R1: 94% vs 90%, P = 0.5; R2: 73% vs 79%, P = 0.13). The interreader reliability and per-RCC sensitivity of PCCT scans acquired on an investigational whole-body PCCT were comparable to MRI scans in detecting and characterizing renal masses. PCCT scans have comparable performance to MRI studies while allowing for improved characterization of the internal composition of lesions due to material decomposition analysis. Future generations of this imaging modality may reveal additional advantages of PCCT over MRI.

Radiogenomics and Texture Analysis to Detect von Hippel-Lindau (VHL) Mutation in Clear Cell Renal Cell Carcinoma.

Radiogenomics, a burgeoning field in biomedical research, explores the correlation between imaging features and genomic data, aiming to link macroscopic manifestations with molecular characteristics. In this review, we examine existing radiogenomics literature in clear cell renal cell carcinoma (ccRCC), the predominant renal cancer, and von Hippel-Lindau (VHL) gene mutation, the most frequent genetic mutation in ccRCC. A thorough examination of the literature was conducted through searches on the PubMed, Medline, Cochrane Library, Google Scholar, and Web of Science databases. Inclusion criteria encompassed articles published in English between 2014 and 2022, resulting in 10 articles meeting the criteria out of 39 initially retrieved articles. Most of these studies applied computed tomography (CT) images obtained from open source and institutional databases. This literature review investigates the role of radiogenomics, with and without texture analysis, in predicting VHL gene mutation in ccRCC patients. Radiogenomics leverages imaging modalities such as CT and magnetic resonance imaging (MRI), to analyze macroscopic features and establish connections with molecular elements, providing insights into tumor heterogeneity and biological behavior. The investigations explored diverse mutations, with a specific focus on VHL mutation, and applied CT imaging features for radiogenomic analysis. Moreover, radiomics and machine learning techniques were employed to predict VHL gene mutations based on CT features, demonstrating promising results. Additional studies delved into the relationship between VHL mutation and body composition, revealing significant associations with adipose tissue distribution. The review concludes by highlighting the potential role of radiogenomics in guiding targeted and selective therapies.

Novel Approaches with HIF-2α Targeted Therapies in Metastatic Renal Cell Carcinoma.

Germline inactivation of the Von Hippel-Lindau (VHL) tumor suppressor is the defining hallmark in hereditary VHL disease and VHL-associated renal cell carcinoma (RCC). However, somatic VHL mutations are also observed in patients with sporadic RCC. Loss of function VHL mutations result in constitutive activation of hypoxia-inducible factor-2 alpha (HIF-2α), which leads to increased expression of HIF target genes that promote angiogenesis and tumor growth. As of 2023, belzutifan is currently the only approved HIF-2α inhibitor for both VHL-associated and sporadic metastatic RCC (mRCC). However, there is potential for resistance with HIF-2α inhibitors which warrants novel HIF-2α-targeting strategies. In this review, we discuss the potential resistance mechanisms with belzutifan and current clinical trials evaluating novel combinations of belzutifan with other targeted therapies and immune checkpoint inhibitors which may enhance the efficacy of HIF-2α targeting. Lastly, we also discuss newer generation HIF-2α inhibitors that are currently under early investigation and outline future directions and challenges with HIF-2α inhibitors for mRCC.

A nonsense mutation in VHL causing Von Hippel-Lindau syndrome in a large Chinese family—a genetic study of familial neoplastic disease

Von Hippel-Lindau (VHL) syndrome is a multi-organ neoplastic disease characterized by highly vascular and cystic tumors in the central nervous system (CNS), retina, and visceral lesions, which are mainly caused by germline mutations in VHL. We aimed to detect novel mutations in VHL gene in families with VHL. Here, a large consanguineous four-generation family with variant phenotypes of VHL syndrome was recruited, and its molecular genetics were tested via Sanger sequencing. And various tools and databases were used to predict the variant pathogenicity, frequency, and protein function. Genetic investigation detected a c.351G > A nonsense mutation in VHL that altered the downstream reading frame and created a premature TGA stop signal, resulting in severely truncated pVHL (p.Trp117Ter). This mutation is absent from most public databases, and functional prediction bioinformatic tools demonstrated that this residue is conserved and that this variant is highly likely to be deleterious. The c.315G > A nonsense mutation in VHL is the causal mutation of this kindred that may lead to clear familial aggregation of VHL syndrome because of the dysfunction of the truncated pVHL.

Characterizing and predicting ccRCC-causing missense mutations in Von Hippel-Lindau disease.

Mutations within the Von Hippel-Lindau (VHL) tumor suppressor gene are known to cause VHL disease, which is characterized by the formation of cysts and tumors in multiple organs of the body, particularly clear cell renal cell carcinoma (ccRCC). A major challenge in clinical practice is determining tumor risk from a given mutation in the VHL gene. Previous efforts have been hindered by limited available clinical data and technological constraints. To overcome this, we initially manually curated the largest set of clinically validated VHL mutations to date, enabling a robust assessment of existing predictive tools on an independent test set. Additionally, we comprehensively characterized the effects of mutations within VHL using in silico biophysical tools describing changes in protein stability, dynamics and affinity to binding partners to provide insights into the structure-phenotype relationship. These descriptive properties were used as molecular features for the construction of a machine learning model, designed to predict the risk of ccRCC development as a result of a VHL missense mutation. Analysis of our model showed an accuracy of 0.81 in the identification of ccRCC-causing missense mutations, and a Matthew's Correlation Coefficient of 0.44 on a non-redundant blind test, a significant improvement in comparison to the previous available approaches. This work highlights the power of using protein 3D structure to fully explore the range of molecular and functional consequences of genomic variants. We believe this optimized model will better enable its clinical implementation and assist guiding patient risk stratification and management.

THU487 Xenograft of VHL-deficient Pancreatic Neuroendocrine Neoplasm Cells - A Novel Low-grade PNEN In Vivo Model

Abstract Disclosure: A. Telerman: None. Y. Yossef: None. A. Chmelnik: None. A. Tirosh: None. Background: Von Hippel-Lindau (VHL) disease is a familial cancer syndrome caused by a germline mutation in the VHL tumor suppressor gene. Although VHL-related pancreatic neuroendocrine neoplasms (vPNEN) have been studied, their molecular pathogenesis is not fully understood. Aims: To generate a mouse model for studying the mechanisms that promote vPNEN development in vivo. Methods: We induced a frameshift mutation in VHL using the CRISPR/Cas9 technique in the BON1 cell line, originating from high-grade PNEN (FS-BON1). We validated the pseudohypoxic nature of the cells by real-time polymerase chain reaction of VEGF and EPO in FS-BON1 compared to VHL wild-type BON1 (WT-BON1). We compared cell line-derived xenografts (CDXs) growth in athymic Nude-Foxn1nu female mice injected with WT-BON1 (n=9), FS-BON1 (n=9) and media (purified bovine serum, n=5). Tumor diameter and calculated volume and mice weight were recorded weekly. Plasma chromogranin A (CgA) levels were measured in mice sera by ELISA. Results: FS-BON1 showed upregulation of VEGF and EPO compared with WT-BON1, confirming their pseudohypoxic microenvironment. At 14 weeks, 0/9 FS-BON1 CDX reached a volume of 1 cm3 compared with 5/9 of the WT-BON1 group (p=0.03). In time-dependent analysis, FS-BON1-derived xenografts grew significantly slower than WT-BON1 xenografts (p=0.02). No significant differences in CgA serum levels were found between the mice groups. Conclusions: We report a new pseudohypoxic in vivo PNEN model. The pVHL-deficient cells show an unexpected indolent course, suggesting it as a new model for low-grade PNEN. This CDX mouse model can be utilized in further studies to evaluate the mechanism responsible for vPNEN development and for evaluating interventions in low-grade PNEN. Presentation: Thursday, June 15, 2023

VHL-deficiency leads to reductive stress in renal cells

Heritable renal cancer syndromes (RCS) are associated with numerous chromosomal alterations including inactivating mutations in von Hippel-Lindau (VHL) gene. Here we identify a novel aspect of the phenotype in VHL-deficient human renal cells. We call it reductive stress as it is characterised by increased NADH/NAD+ ratio that is associated with impaired cellular respiration, impaired CAC activity, upregulation of reductive carboxylation of glutamine and accumulation of lipid droplets in VHL-deficient cells. Reductive stress was mitigated by glucose depletion and supplementation with pyruvate or resazurin, a redox-reactive agent. This study demonstrates for the first time that reductive stress is a part of the phenotype associated with VHL-deficiency in renal cells and indicates that the reversal of reductive stress can augment respiratory activity and CAC activity, suggesting a strategy for altering the metabolic profile of VHL-deficient tumours.

Selective HIF2A Inhibitors in the Management of Clear Cell Renal Cancer and Von Hippel-Lindau-Disease-Associated Tumors.

Von Hippel-Lindau (VHL) loss is the hallmark event characterizing the clear cell renal cancer subtype (ccRCC). Carriers of germinal VHL mutations have an increased prevalence of kidney cysts and ccRCC as well as hemangioblastoma, pheochromocytoma and pancreatic neuroendocrine tumors. In both sporadic and inherited ccRCC, the primary mechanism of VHL-mediated carcinogenesis is the abnormal stabilization of hypoxia-inducible factors (HIF1A and HIF2A). While HIF1A acts as a tumor suppressor and is frequently lost through inactivating mutations/14q chromosome deletions, HIF2A acts as an oncogene promoting the expression of its target genes (VEGF, PDGF, CAIX Oct4, among others). Selective HIF2a inhibitors block the heterodimerization between HIF2A and ARNT, stopping HIF2A-induced transcription. Several HIF2A inhibitors have entered clinical trials, where they have shown a favorable toxicity profile, characterized by anemia, fatigue and edema and promising activity in heavily pretreated ccRCC patients. Belzutifan, a second-generation HIF2a inhibitor, was the first to receive FDA approval for the treatment of unresectable ccRCC in VHL syndrome. In this review, we recapitulate the rationale for HIF2a blockade in ccRCC, summarize the development of HIF2a inhibitors from preclinical models up to its introduction to the clinic with emphasis on Belzutifan, and discuss their role in VHL disease management.

Elevated erythroferrone distinguishes erythrocytosis with inherited defects in oxygen-sensing pathway from primary familial and congenital polycythaemia.

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.

Biallelic ELOC-Inactivated Renal Cell Carcinoma: Molecular Features Supporting Classification as a Distinct Entity

Approximately 70% of clear cell renal cell carcinoma (ccRCC) is characterized by the biallelic inactivation of von Hippel-Lindau (VHL) on chromosome 3p. ELOC-mutated (Elongin C-mutated) renal cell carcinoma containing biallelic ELOC inactivations with chromosome 8q deletions is considered a novel subtype of renal cancer possessing a morphologic overlap with ccRCC, renal cell carcinoma (RCC) with fibromyomatous stroma exhibiting Tuberous Sclerosis Complex (TSC)/mammalian Target of Rapamycin (mTOR) mutations, and clear cell papillary tumor. However, the frequency and consequences of ELOC alterations in wild-type VHL and mutated VHL RCC are unclear. In this study, we characterize 123 renal tumors with clear cell morphology and known VHL mutation status to assess the morphologic and molecular consequences of ELOC inactivation. Using OncoScan and whole-exome sequencing, we identify 18 ELOC-deleted RCCs, 3 of which contain ELOC mutations resulting in the biallelic inactivation of ELOC. Biallelic ELOC and biallelic VHL aberrations were mutually exclusive; however, 2 ELOC-mutated RCCs showed monoallelic VHL alterations. Furthermore, no mutations in TSC1, TSC2, or mTOR were identified in ELOC-mutated RCC with biallelic ELOC inactivation. Using High Ambiguity Driven biomolecular DOCKing, we report a novel ELOC variant containing a duplication event disrupting ELOC-VHL interaction alongside the frequently seen Y79C alteration. Using hyper reaction monitoring mass spectrometry, we show RCCs with biallelic ELOC alterations have significantly reduced ELOC expression but similar carbonic anhydrase 9 and vascular endothelial growth factor A expression compared with classical ccRCC with biallelic VHL inactivation. The absence of biallelic VHL and TSC1, TSC2, or mTOR inactivation in RCC with biallelic ELOC inactivation (ELOC mutation in combination with ELOC deletions on chromosome 8q) supports the notion of a novel, molecularly defined tumor entity.

Proteostasis Modulation in Germline Missense von Hippel Lindau Disease.

Missense mutated von Hippel Lindau (VHL) protein (pVHL) maintains intrinsic function but undergoes proteasomal degradation and tumor initiation and/or progression in VHL disease. Vorinostat can rescue missense mutated pVHL and arrest tumor growth in preclinical models. We asked whether short-term oral vorinostat could rescue pVHL in central nervous system hemangioblastomas in patients with germline missense VHL. We administered oral vorinostat to 7 subjects (ages 46.0 ± 14.5 years) and then removed symptomatic hemangioblastomas surgically (ClinicalTrials.gov identifier NCT02108002). Vorinostat was tolerated without serious adverse events by all patients. pVHL expression was elevated in neoplastic stromal cells compared with untreated hemangioblastomas from same patients. We found transcriptional suppression of downstream hypoxia-inducible factor (HIF) effectors. Mechanistically, vorinostat prevented Hsp90 recruitment to mutated pVHL in vitro. The effects of vorinostat on the Hsp90-pVHL interaction, pVHL rescue, and transcriptional repression of downstream HIF effectors was independent of the location of the missense mutation on the VHL locus. We confirmed a neoplastic stromal cell-specific effect in suppression of protumorigenic pathways with single-nucleus transcriptomic profiling. We found that oral vorinostat treatment in patients with germline missense VHL mutations has a potent biologic effect that warrants further clinical study. These results provide biologic evidence to support the use of proteostasis modulation for the treatment of syndromic solid tumors involving protein misfolding. Proteostasis modulation with vorinostat rescues missense mutated VHL protein. Further clinical trials are needed to demonstrate tumor growth arrest.

Abstract 133: Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints

Abstract Whether cancer evolution follows a deterministic route prescribed by host factors (germline genetics and/or environment) or proceeds along multiple alternative trajectories due to the stochastic nature of evolution is unknown. Patients with VHL disease have a pathogenic germline mutation in VHL (3p25) and tumorigenesis occurs when the wild-type copy of VHL is lost. Patients develop multiple clonally independent tumors including clear cell renal cell carcinoma (ccRCC), pheochromocytoma or paraganglioma (PPGL) and pancreatic neuroendocrine tumors (pNET), offering an opportunity to test repeatability of evolutionary trajectories between and within patients. We performed deep targeted exome sequencing, in 1520 tumors (993 solid ccRCC; 156 cystic ccRCC; 135 mixed solid/cystic ccRCC; 236 extra-renal) from 130 patients with VHL disease to evaluate somatic variants and copy number alterations, ploidy and chromosomal complexity. Tumor phenotype was evaluated from pathology reports and longitudinal growth kinetics from diagnostic MRI data. Analysis of 889 tumor samples is complete and profiling of the full cohort will be presented at the 2023 AACR meeting. Within clonally independent tumors, we performed two tests to assess for evidence of convergence between tumors from the same patient. First, we tested for parallel events in canonical ccRCC drivers. We found significant enrichment of somatic VHL variants occurring in one patient with a germline whole VHL gene deletion encompassing neighboring BRK1. Contiguous BRK1 loss is associated with less aggressive ccRCC phenotype and our findings suggest a specific constraint in this patient. We next assessed for evidence of convergence by measuring the genetic distance of copy number and mutational profiles between and within patients with resampling and permutation. Similar somatic profiles between tumors were only detected in 1/76 patients whose tumors were characterized by infrequent somatic alterations beyond 3p loss indicating little evidence of convergence. Cystic ccRCCs represent an indolent phenotype not associated with metastasis that may progress to solid tumors over time; we found mixed tumors closely resemble somatic profiles of solid tumors whereas cysts were characterized by infrequent copy number gains and low chromosomal complexity. We also identified cysts with specific losses (chr16, chr17, chr19p) mutually exclusive with 3p LOH suggestive of VHL-loss independent cyst formation. In solid ccRCCs, we found chromosomal complexity and loss of specific chromosomal loci (9p and/or 14q) are associated with diameter potentially reconciling the observation linking risk of metastasis to tumor diameter in VHL-related ccRCC. In summary, within the constrained setting of VHL-related ccRCC, evolution is dominated by chance although specific patient and phenotype specific constraints are observed. Citation Format: Scott T. Shepherd, Alex Coulton, Cathy D. Vocke, Chris J. Ricketts, Mark Ball, Fiona Byrne, Yuliia Dovga, Annika Fendler, Barber Taja, Olga O'Neill, Lina Gerontogianni, Gavin Kelly, Laura S. Schmidt, Daniel R. Crooks, Kirstin Choo, Rabindra Gautam, Zoe Blake, Krista Reynolds, Kevin Litchfield, Maria Merino, Marston W. Linehan, Samra Turajlic. Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 133.

Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma.

Patients with resected localized clear-cell renal cell carcinoma (ccRCC) remain at variable risk of recurrence. Incorporation of biomarkers may refine risk prediction and inform adjuvant treatment decisions. We explored the role of tumor genomics in this setting, leveraging the largest cohort to date of localized ccRCC tissues subjected to targeted gene sequencing. The somatic mutation status of 12 genes was determined in 943 ccRCC cases from a multinational cohort of patients, and associations to outcomes were examined in a Discovery (n = 469) and Validation (n = 474) framework. Tumors containing a von-Hippel Lindau (VHL) mutation alone were associated with significantly improved outcomes in comparison with tumors containing a VHL plus additional mutations. Within the Discovery cohort, those with VHL+0, VHL+1, VHL+2, and VHL+≥3 tumors had disease-free survival (DFS) rates of 90.8%, 80.1%, 68.2%, and 50.7% respectively, at 5 years. This trend was replicated in the Validation cohort. Notably, these genomically defined groups were independent of tumor mutational burden. Amongst patients eligible for adjuvant therapy, those with a VHL+0 tumor (29%) had a 5-year DFS rate of 79.3% and could, therefore, potentially be spared further treatment. Conversely, patients with VHL+2 and VHL+≥3 tumors (32%) had equivalent DFS rates of 45.6% and 35.3%, respectively, and should be prioritized for adjuvant therapy. Genomic characterization of ccRCC identified biologically distinct groups of patients with divergent relapse rates. These groups account for the ∼80% of cases with VHL mutations and could be used to personalize adjuvant treatment discussions with patients as well as inform future adjuvant trial design.

The von Hippel-Lindau Tumor Suppressor Gene Mutations Modulate Lipocalin-2 Expression in Ferroptotic-Inflammatory Pathways.

A previous study of an animal model with tumor suppressor gene von Hippel-Lindau (VHL) conditional knockdown suggested that tissue inflammation and fibrosis play important roles in the development of clear-cell renal cell carcinoma (ccRCC), which is consistent with the epidemiological evidence linking inflammatory kidney disease and renal cancer. Ferroptosis and inflammation have been linked in a recent study, but the exact mechanism remains unclear. This study is aimed at investigating the mechanism of lipocalin-2- (LCN-2-) mediated ferroptosis and inflammation in vhl-mutated HK-2 cells and mouse primary proximal tubule cells (mRTCs) and the polarization of macrophage RAW 264.7 cells. Based on the levels of lipid reactive oxygen species (ROS) and the expression of glutathione peroxidase 4 (GPX4) in HK-2 cells, we observed that a VHL mutation increased ROS production and depressed GPX4 expression, whereas LCN-2 knockdown reversed these effects. Accordingly, VHL appears to affect ferroptosis in an LCN-2-dependent manner. We also revealed that LCN-2 sensitizes HK-2 cells to inflammation and macrophage RAW 264.7 cells to M1-like polarization. This study provides novel insights into the potential therapeutic target and strategy for attenuating the progression of ccRCC by revealing the role of VHL in regulating chronic inflammation within the LCN-2-ferroptosis pathway.

Screening of VHL mutation in different types of kidney cancer in patients of West Bengal (India)

Introduction: The purpose of this study was to screen the patients of West Bengal (India) with different types of renal cancer to see the presence of von Hippel–Lindau (VHL) gene mutations. Materials and Methods: This prospective study included 25 patients with renal cancer operated on between December 2019 and January 2022. Tumor tissue and adjacent normal tissue samples were taken and subjected to genomic DNA isolation, polymerase chain reaction, DNA sequencing, and identification of polymorphism in renal cell carcinoma (RCC) patients by comparing with a database using “ENSEMBL” (genome browser) followed by the role of identified variants in disease-causing using different software. Results: After analysis, we identified six exonic and one intronic variant in the VHL gene. rs34661876 A&gt;G, AG genotype in intron 2–3 has increased the risk of RCC against the odd 6.729 times (P = 0.0041). It is present in 17 out of 25 case samples. rs1642742 G&gt;A, AA genotype in exon 3 has increased the risk of RCC against the odd 22.167 times (P = 0.0001). It is present in 14 out of 25 case samples. The effect of these Single nucleotide polymorphism SNPs/mutations on VHL function were predicted by various bioinformatics software and it was found that rs1399097617 C&gt;T, rs5030830 T&gt;C, and rs1553620326 G&gt;C are disease-causing. Conclusion: If any of the above-mentioned variants are detected in RCC patients, then they will be benefited from the agents that modulate the VHL-hypoxia-inducible factor pathway and will help in developing new strategies for the management of RCC.