Gastric Neuroendocrine Tumor Presenting Atypically in von Hippel–Lindau Disease: A Case Report

Focus on kidney cancer

Proteostasis Modulators Prolong Missense VHL Protein Activity and Halt Tumor Progression

The Tumor Suppressor von Hippel-Lindau Gene Product and Metastasis: New Thoughts on an Old Molecule

4532 Background: The prevalence of von Hippel-Lindau (VHL) mutations in clear cell renal cell carcinoma (ccRCC) is about 30–50%. The association between VHL mutation and patient prognosis remains controversial, despite extensive in vitro research on its role in hypoxia response. We have previously reported a robust prognostic classification of ccRCC using gene expression profiling. In view of reports of activity of anti-angiogenesis agents, we studied the association of VHL mutation with tumor profiling subtypes, survival and other clinical parameters. Methods: All exons of the VHL gene in 88 ccRCC samples were sequenced. Expression profiling using oligonucleotide arrays (54,675 probe sets) was also performed, and the samples classified using hierarchical clustering. For identification of VHL mutation specific signatures, nearest shrunken centroids with internal validation was used with a 10% misclassification cutoff. Results: 35% of the samples had VHL mutations. Mutation status was not significantly associated with survival on univariate analysis (p = 0.54). Hierarchical clustering yielded two ccRCC subtypes with divergent clinical outcomes (HR = 4.13, p < 0.001). VHL mutations were significantly associated with the good-prognosis profiling subtype of ccRCC tumors (OR 3.3, p = 0.04), but no effect modification between VHL mutation and the prognostic subtypes was found (p=0.31). No standard clinical parameter was associated with VHL mutation. No significant association between VHL mutation and downstream hypoxia response gene expression, including VEGF (p = 0.14), PDGF (p = 0.5), TGF-A (p = 0.24) and GLUT1 (p = 0.45) was found. Conclusion: While VHL mutation is associated with a biologically distinct good-prognosis profiling tumor subtype, its lack of prognostic value on univariate analysis suggests an expanded study to evaluate effect modification. The absence of a specific gene classifier for VHL mutation and the lack of association between VHL gene mutation and known hypoxia response genes suggest that VHL mutations result in heterogenous tissue phenotypes. These results support molecular subtyping of ccRCC in laboratory and clinical studies; in particular, this may be critical for trials involving anti-angiogenesis agents. No significant financial relationships to disclose.

Background: The von Hippel-Lindau (VHL) gene is a tumor suppressor gene. Although VHL gene alterations play a key role in the pathogenesis of renal cell carcinoma (RCC), and several therapies targeting this molecular pathway have been studied, such as sunitinib, pazopanib and axitinib. However, it is not clear whether VHL mutation is a prognostic factor for immunotherapy in renal cell carcinoma. Methods: The impact of VHL mutations on survival outcomes in RCC patients received immunotherapy or non-immunotherapy were verified in the Memorial Sloan Kettering Cancer Center (MSKCC) trials and The Cancer Genome Atlas (TCGA) RCC cohort, respectively. An in-house Chinese RCC cohort (n=950) was used for the analysis of immune-related markers. The relationship between clinical pathologic features and VHL were analyzed with using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics associated with overall survival using Cox regression and the Kaplan-Meier method. Results: In the MSKCC cohort, a total of 143 RCC patients receiving immunotherapy were enrolled, of which 117 (81.8%) were clear cell RCC and 143 (100%) microsatellite-stable RCC. VHL mutation was significantly associated with high TMB score (P =0.017). Kaplan-Meier survival analysis showed that patients with VHL mutation obtained better OS compared to VHL wild-type (50 months vs. 23 months, HR, 0.36; 95% CI 0.21-0.64; P<0.001). In the TCGA cohort, all RCC patients received non- immunotherapy. No association was observed between VHL and OS (HR, 1.00; 95% CI 0.73-1.38; P=0.981). VHL mutations were significantly associated with higher TMB levels (P=0.006) and MSI-H (P=0.004) in the in-house Chinese RCC cohorts. Conclusions: Our results suggest that VHL mutation may be associated with better OS in RCC patients receiving immunotherapy. The exact mechanisms underlying VHL are needed to be further evaluated. Citation Format: Dong Shen, Yufeng Huang, Zhiwei Wu, Junling Zhang, Mengli Huang, Yanan Chen, Xihua Xia, Yuezong Bai. Prognosis of renal cell carcinoma patients with VHL mutations to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5090.

To evaluate the relationship between the mutation of the von Hippel-Lindau (VHL) gene and expression of vascular endothelial growth factor (VEGF) in sporadic clear cell renal cell carcinoma (CCRCC) and angiogenesis. Polymerase chain reaction (PCR) was used to detect the mutation of VHL gene in the specimens of cancerous tissue and normal tissues away from tumor from 77 patients with CCRCC. Immunohistochemistry was used to examine the expression of VEGF. CD34 staining was used to measure the microvascular density (MVD). VHL gene mutations were detected in 40 cases (51.9%). The expression rate of VEGF was 79.2% (61 cases). The positive rate of VEGF in the cases with VHL mutation was 92.5%, significantly higher than that in the cases without VHL mutation (64.9%, P = 0.003). The levels of MVD was higher in the cases with VHL mutation and those with VEGF expression were 760.80/mm2 and 715.95/mm2 respectively, both significantly higher than those in the cases without VHL-mutation and those without VEGF expression (547.03/mm2 and 437.44/mm2 respectively, all P = 0.001). The cases with expression of VEGF were divided into two groups according the presence or absence of VHL gene mutations or not. The MVD of the cases with VEGF expression and VHL mutation was 760.80 mm2, significantly higher than that of the cases with VEGF expression and without VHL mutation (547.03 mm2, P = 0.011). The mutation rate of VHL gene is high among the Chinese with sporadic CCRCC. VHL gene mutation increases significantly the VEGF expression, thus, and perhaps via other mechanism too, promoting the angiogenesis in tumor. The high level of MVD of the cases with VHL gene mutation may be related to the high malignant potential of CCRCC.

PARENCHYMAL SPARING SURGERY IN PATIENTS WITH HEREDITARY RENAL CELL CARCINOMA: 10-YEAR EXPERIENCE

Von Hippel–Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome with a prevalence of 1 in 39,000 live births that results from a germline mutation of the VHL gene with over 90% penetrance by 65 years of age despite variable expression [1, 2]. VHL is a tumor suppressor gene located on the short arm of the chromosome 3 (3p25–26) that is made of three exons and widely expressed in both fetal and adult tissues [3, 4]. A patient with heritable VHL disease inherits a germline mutation of the gene from the affected parent and a normal gene from the unaffected parent (wild type). The Knudson’s two-hit hypothesis of tumorigenesis applies to the initiation of the tumor growth when a somatic mutation affects the wildtype VHL allele [5]. Although germline VHL mutations are present in all the cells of the affected individuals who inherit the genetic trait, only the cells that are somatically mutated in the wild-type gene and are parts of a susceptible organ experience tumor growth [2]. In fact, patients with VHL disease are predisposed to develop specific central nervous system (CNS) and visceral lesions [1, 2, 4]. Affected individuals may develop retinal hemangioblastomas, endolymphatic sac tumors (ELST) of the labyrinth, or craniospinal hemangioblastomas in the cerebellum, brainstem, or spinal cord [2]. Furthermore, visceral involvement in some patients affects the kidney with renal cell carcinoma (RCC) or cysts, the adrenal glands with pheochromocytomas, the pancreas with tumors or cysts, and the epididymus or the broad ligament with cystoadenomas [2]. Clinical criteria or genetic tests help in diagnosing VHL disease [1]. Obviously, patients with a family history of VHL and CNS lesions (hemangioblastomas), RCC, pheochromocytoma, pancreatic cysts or ELST will meet the criteria for diagnosis of VHL [1]. However, 20% of patients lack a family history, but meet the criteria for VHL, if they present two or more CNS hemangioblastomas, or one CNS hemangioblastoma and a visceral VHL-associated tumor [1]. A classification of the VHL disease in type 1 or 2A–C based on the VHL mutation, molecular defect, and clinical manifestations have been proposed [4]. Patients at high risk undergo testing for germline VHL mutations with 100% detection rate if they have a positive family history [1, 6]. On the contrary, patients with negative family history present with tissue mosaicism that may hamper the use of testing their peripheral blood leukocytes lacking the VHL mutation [7]. The somatic VHL mutations are present in approximately 50% of sporadic RCC, whereas 20% of cases present hypermethylated VHL gene in the same group of tumors [4]. Hypermethylation of the VHL gene has only been described in the RCC, while somatic mutations account for 30% of sporadic hemangioblastomas without any description of VHL gene hypermethylation in this group of tumors [4]. In the context of sporadic tumors, the two-hit model occurs exclusively somatically rather than in the germline [4]. The VHL gene is widely expressed in tissues, including the ones not affected by VHL-transformed phenotype. The VHL mRNA encodes a 213 amino acid residue-protein (pVHL) with an apparent molecular weight of ~24 to 30 kDa (VHL30) [1, 4]. A second pVHL isoform of approximately 19 kDa (VHL19) is the by-product of inframe start codon (ATG) at codon 54 [4]. Both isoforms have tumor suppressor activity. pVHL shuttles between the nucleus and the cytoplasm, and the nuclear-cytoplasmic trafficking is required for the function of the VHL tumor suppressor protein [4]. Primarily, pVHL complexes with J Mol Med (2009) 87:591–593 DOI 10.1007/s00109-009-0466-z

Mutations in the von Hippel-Lindau (VHL) gene are frequently detected in human sporadic renal cell carcinoma (RCC). We analysed 102 Swedish RCCs for VHL mutations by PCR-SSCP and sequencing. In 47 patients (46.1%), 70 different mutations were found, and most of them represented novel variations of the VHL gene. Mutations in the VHL gene were found in 54% of clear cell renal cell carcinomas (CCRCC) and in 18% of chromophilic cancers but in no chromophobe cancers or oncocytomas (P=0.016). Three novel hotspot or founder mutations were detected in our study: four CCRCCs carried a missense mutation (glutamic acid to lysine) at codon 160 which is critical in the stabilization of the H1 helix of the alpha domain and the alpha-beta domain interface in the VHL protein. Five CCRCCs and one chromophilic RCC harbored a 15-nucleotide in-frame deletion (codons 41-45) at a duplex tandem repeat sequence site. Moreover, this deletion was in linkage disequilibrium with a C-->T transition in the promoter region. The frequency of linkage was 17 times more common than chance. Five patients with this linked mutation resided in the same hospital district and at least three of them showed the two sequence variants in the tumor-adjacent tissue. In 5/6 patients the wild-type allele was lost in the tumor samples, suggesting a causal role for the mutations in RCC. These linked mutations might be novel polymorphisms maintained in a relative isolated population. Multiple mutations in VHL were found in 17 tumors out of 47 tumors with the VHL mutation. A higher multiple mutation detected rate (33%) was observed in grade 3 CCRCCs than those in grade 1 (22%) and grade 2 (9%) (P=0.04). This is evidence on the association between VHL mutation and extent of nuclear atypia.

Novel compound <scp>VHL</scp> heterozygosity (<i><scp>VHL</scp></i> T124A/L188V) associated with congenital polycythaemia

Kidney cancer: Identification of novel targets for therapy

Oncological Outcomes of Partial Nephrectomy for Multifocal Renal Cell Carcinoma Greater Than 4 cm

5012 Background: The clinical response to VEGF-targeted therapy in metastatic RCC is robust, yet variability in outcome exists. VHL is often mutated in RCC, but the relation to therapeutic outcome is unclear. Identification of tumor molecular characteristics associated with outcome would aid in patient selection and interpretation of clinical trials. Materials and Methods: Patients with metastatic, clear-cell RCC with available baseline frozen or paraffin-embedded tumor samples who received VEGF-targeted monotherapy with sunitinb, sorafenib, axitinib or bevacizumab at Cleveland Clinic or University of California San Francisco, were included. Patient characteristics, VHL gene status (mutated or not mutated) and objective response rate (ORR) were documented. ORR was investigator-assessed per RECIST criteria. Fisher’s exact test and logistic regression models were used to assess ORR in univariate and multivariate analyses, respectively. Results: One-hundred twenty-three patients were evaluable. The ORR was 36.5% for the entire cohort. VHL mutation was found in 48% of patients and was seen across all three exons. Patients with VHL mutation had an ORR of 46% versus 28% for pts without a mutated VHL gene (p=0.06). In multivariate analysis, presence of a VHL mutation remained an independent prognostic factor associated with improved ORR (p=.02) when multiple clinical prognostic factors (ECOG PS, hemoglobin, corrected calcium, LDH, prior radiation, prior therapy and number of metastatic sites) were also considered. Conclusion: This is the first large study testing the impact of VHL mutation on response to VEGF-targeted agents in metastatic RCC. Although objective responses were more likely among patients with mutated VHL gene, lack of VHL mutation did not preclude a response. Additional tissue analysis and identification of biomarkers relevant to response to VEGF-targeted agents in metastatic RCC are warranted. [Table: see text]

Von Hippel-Lindau (VHL) disease is a dominantly inherited multisystem family cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal carcinoma, phaeochromocytoma, pancreatic islet cell tumours and endolymphatic sac tumours. In addition, renal, pancreatic and epididymal cysts occur. Morbidity and mortality from VHL disease can be reduced by the identification and surveillance of affected individuals and at-risk relatives so that complications are diagnosed at an early presymptomatic stage. The detailed mapping and subsequent isolation of the VHL tumour suppressor gene has enabled molecular genetic analysis in families and patients with definite or possible VHL disease. Initially, linked DNA markers were used in informative families to modify individual risks and then to make appropriate alterations in surveillance programs. However, currently most DNA analysis involves the characterisation of germline mutations. World-wide, mutations have been identified in almost 500 families (including 132 in our laboratory). These studies have revealed considerable heterogeneity both in the type and in the location of mutations within the VHL gene. In our experience, most recurrent mutations result from de novo mutations at hypermutable sequences, although a founder effect for the Tyr98His ('Black Forest') mutation has been reported in German and American families. Although many mutations are predicted to impair the ability of pVHL to combine with the elongin regulatory subunits, analysis of genotype-phenotype relationships suggests that the VHL protein has multiple and tissue specific functions. Calculation of tumour risks for different classes of VHL mutations has provided important prognostic information especially with respect to the likelihood of phaeochromocytoma. However, there is evidence that retinal involvement does not correlate with allelic heterogeneity, but that the variability in retinal angiomatosis is influenced by modifier gene effects. VHL gene mutation analysis also provides a basis for investigating the genetic basis of familial phaeochromocytoma and renal cell carcinoma, and apparently isolated retinal angiomas. Results to date suggest that a substantial proportion of patients with familial pheochromocytoma have VHL gene mutations but in contrast, most familial clusters of clear cell renal cell carcinoma (RCC) without evidence of VHL do not have germline VHL mutations.

Inactivation of tumour suppressor gene(s) (TSGs) on 3p appears to be a critical event in the pathogenesis of clear cell renal cell carcinoma (CC-RCC). Analysis of loss of heterozygosity (LOH) in sporadic RCC samples has implicated roles for TSGs in three specific regions of 3p in RCC development: (1) 3p12-p14, which includes the breakpoint of the familial t(3;8) constitutional translocation involved in hereditary RCC development and a recently cloned putative TSG, the FHIT gene: (2) 3p21.2-p21.3, a common region of deletion in many cancers including lung; and (3) 3p25-p26, which contains the von Hippel-Lindau (VHL) disease TSG. We and others have shown that most primary sporadic CC-RCCs contain somatic VHL gene mutations, clearly implicating inactivation of the VHL gene in the pathogenesis of CC-RCC. It is not known if CC-RCC without VHL gene mutations have alternative mechanisms of VHL gene inactivation or result from an alternative non-VHL pathway to RCC, e.g., inactivation of TSGs in 3p12-p21. We and others have reported hypermethylation and silencing of the VHL TSG in RCC from patients with VHL disease and in CC-RCC cell lines. However, the incidence and specificity of VHL methylation in primary sporadic RCC has not been defined. Therefore, we analysed methylation of the VHL, CDKN2, MYC, and H19 genes in primary RCC samples. Hypermethylation of the VHL promoter region was detected in 11% (11/99) of the primary RCCs analysed. In 10 of these tumours, there was no evidence of concomitant VHL gene mutation. VHL methylation was specific to CC-RCC (15%, 7/45) but was not detected in any non-CC tumours (n = 16). None of the 11 RCCs methylated at VHL had evidence of methylation at either CDKN2 or MYC (methylation at CDKN2 was, however, detected in 3%, or 1/33, of RCCs without VHL methylation). A normal methylation pattern at H19 was demonstrated in the three RCCs with methylated VHL analysed. Previous studies have suggested that, in addition to VHL, other 3p TSGs at 3p12-p14 and 3p21 may be involved in CC-RCC tumourigenesis. However, the interpretation of these studies has been difficult because information on VHL gene status has not been available for these data sets. Therefore, we investigated a subset of 55 sporadic RCCs (of known VHL gene methylation and mutation status) for LOH at polymorphic markers close to candidate TSG loci in the 3p14.2 and 3p21.2-p21.3 regions. Among tumours with LOH at one or more 3p markers, the incidence of 3p25 allele loss was higher in tumours with VHL alterations (mutation or methylation) than in those without. For tumours without detectable VHL alterations, the frequency of 3p14-p21 LOH was significantly higher than the frequency of 3p25-p26 LOH (93%, 13/14 vs. 43%, 6/14; P = 0.013), whereas, in RCC samples with VHL methylation or mutation, the frequency of 3p14-p21 LOH did not differ from that of sp25-p26 (72%, 18/25 vs. 59%, 13/22; P = 0.376). None of the 11 RCCs with 3p25 allele loss that were informative at 3p21 and 3p14 showed LOH at 3p25 only. These findings suggest that (1) VHL methylation is a specific and important event in the pathogenesis of CC-RCC; (2) in CC-RCC with 3p LOH but without VHL inactivation, mutations in TSGs at 3p14-p21 appear to have a primary role in tumourigenesis; and (3) inactivation of other 3p TSGs in addition to VHL may also be required for malignant transformation in tumours with VHL gene inactivation.