Sporadic hemangioblastomas are characterized by cryptic VHL inactivation
- Ganesh M Shankar†1, 5,
- Amaro Taylor-Weiner†5,
- Nina Lelic1,
- Robert T Jones6,
- James C Kim2,
- Joshua M Francis5,
- Malak Abedalthagafi6,
- Lawrence F Borges1,
- Jean-Valery Coumans1,
- William T Curry1,
- Brian V Nahed1,
- John H Shin1,
- Sun Ha Paek7,
- Sung-Hye Park8,
- Chip Stewart5,
- Michael S Lawrence5,
- Kristian Cibulskis5,
- Aaron R Thorner9,
- Paul Van Hummelen9,
- Anat O Stemmer-Rachamimov2,
- Tracy T Batchelor3,
- Scott L Carter5,
- Mai P Hoang2,
- Sandro Santagata6,
- David N Louis2,
- Fred G Barker1,
- Matthew Meyerson5, 6, 9, 10,
- Gad Getz2, 5,
- Priscilla K Brastianos4, 5Email author and
- Daniel P Cahill1Email author
© Shankar et al.; licensee BioMed Central. 2014
Received: 19 November 2014
Accepted: 25 November 2014
Published: 24 December 2014
Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.
KeywordsCentral nervous system Hemangioblastoma Deep sequencing Somatic gene alterations Von Hippel-Lindau gene Hypoxia-inducible signaling
Hemangioblastomas comprise 1–2.5% of primary intracranial and 7-10% of spinal cord tumors, with a benign pathology notable for neoplastic “stromal” cells embedded in a dense network of vascular channels . These tumors are largely diploid with few chromosomal abnormalities ,, and have served as the prototypic lesions in the study of genomic drivers of hypoxia-mediated metabolism in cancer . Approximately 25% of hemangioblastomas are a result of Von Hippel-Lindau (VHL) disease, which is inherited in an autosomal dominant manner through a germline inactivating mutation of the tumor suppressor gene VHL on chromosome 3, with subsequent inactivation of the second allele by somatic mutation or gene loss in the neoplastic “stromal” cell. The clinical spectrum of VHL disease includes other neoplastic lesions, including retinal angiomatosis, pheochromocytoma and clear cell renal carcinoma.
Findings from prior studies analyzing sporadic hemangioblastomas for somatic mutations, loss of heterozygosity or deletion of VHL
Loss of heterozygosity
Rate of somatic mutation (%)
Rate of LOH (%)
Rate of deletion (%)
Kanno et al. 
Oberstrass et al. 
Tse et al. 
Olschwang et al. 
Lee et al. 
Glasker et al. 
Gijtenbeek et al. 
Lemeta et al. 
We considered two possibilities for this difference between the reported frequency of mutations in VHL between familial and sporadic hemangioblastomas. The first explanation is that sporadic cases of hemangioblastoma may develop via alterations in genes involved in hypoxia-sensing pathways other than VHL itself that also result in a histologically-identical phenotype of exuberant angiogenesis. A candidate gene for such an alteration is TCEB1, which encodes the required VHL binding partner elongin C and, in the recent genomic sequencing of clear cell renal carcinoma was found to be somatically mutated in the minority of cases that do not harbor VHL mutations . A second explanation is that prior bulk sequencing techniques were technically limited in capturing the full extent of VHL inactivation. Neoplastic “stromal” cells comprise roughly 10-20% of the hemangioblastoma tumor cell mass with the remainder consisting of pericytes, vascular endothelium and other nucleated cells, such as lymphocytes, that are present in the vascular channels . Given the relatively low purity of neoplastic cells, previous studies may not have detected the entire spectrum of alterations in VHL.
The emergence of next generation sequencing technologies has rapidly accelerated the discovery of somatic alterations in cancer genes, by exploiting greater depth-of-coverage to increase the sensitivity of detection within low purity samples ,-. Application of these techniques to hemangioblastomas can comprehensively examine the role of VHL across the spectrum of this disease. In addition, whether hemangioblastomas form from an embryologically arrested “hemangioblast” stem cell that subsequently differentiates into the discrete constituent cell types that support the architecture of this vascular tumor ,- is unclear. While developmental studies have shown a common precursor of the differentiated cell types (endothelial, stromal) found in the hemangioblastoma cell mass, whether neoplastic cells contribute to these different populations was not well-characterized. By analyzing the extent of genomic alterations and their subclonal and clonal status within the tumor mass of sporadic hemangioblastomas, this issue can be addressed. In this study, we characterize sporadic hemangioblastomas of cerebellum and spinal cord by whole exome sequencing and deep-coverage sequence analysis.
Materials and methods
This study was approved by the Institutional Review Board at the hospitals providing specimens. Written informed consent was obtained for all samples undergoing whole exome sequencing. The discovery and validation cohorts of sporadic hemangioblastomas involving the cerebellum and spinal cord were obtained from patients treated at Massachusetts General Hospital (MGH, Boston MA), Brigham and Women’s Hospital (BWH, Boston MA), and Seoul National University (South Korea). Patients were identified as likely sporadic hemangioblastomas based on (1) prior testing that was negative for germline VHL mutations by sequencing of the coding regions (exons 1–3), (2) lack of family history of hemangioblastomas or VHL disease, and/or (3) lack of other stigmata of VHL disease, such as renal cancer, pheochromocytoma, or retinal angiomas. Pathology was verified by the Departments of Pathology at MGH and BWH (DNL, SS and MA) and tumor rich areas were microdissected.
DNA was extracted from FFPE tumor specimens and matching peripheral blood samples using standard techniques (QIAamp, Qiagen) and quantified by PicoGreen dye (Invitrogen). Whole exome sequencing was performed by hybrid capture and next-generation sequencing as described previously . In brief, DNA was fragmented by sonication (Covaris Inc., Woburn, MA) to 150 bp and further purified using Agencourt AMPure XP beads. 50 ng of size-selected DNA was then ligated to specific adaptors during library preparation (Illumina TruSeq, Illumina Inc., San Diego, CA). Each library was made with sample-specific barcodes and quantified by quantitative PCR (Kapa Biosystems, Inc., Woburn, MA), and two libraries were pooled to a total of 500 ng for exome enrichment using the Agilent SureSelect hybrid capture kit (Whole Exome_v1.1; Agilent Technologies, Santa Clara, CA). Several captures were pooled further and sequenced in one or more lanes to a final equivalent of two exomes per lane on a HiSeq 2500 system (Illumina Inc, San Diego, CA).
The validation cohort was sequenced for 560 cancer-associated genes and 39 translocations previously implicated in cancer (Additional file 1: Table S1). Briefly, DNA was sonicated to achieve an average fragment size of 250 bp, size selected and barcoded. Multiplexed pools were hybridized with biotinylated baits (Agilent SureSelect) designed to capture exonic sequences, including those of VHL and ARID1B. The captures were sequenced on the Illumina HiSeq 2500 in Rapid Run Mode.
Separately, Sanger sequencing of the TCEB1 Tyr79Cys hotspot, and the promoter and exon 1 of VHL, was performed on PCR amplified products from tumor genomic DNA. Chromatograms were manually reviewed to evaluate for the presence of somatic mutations, which may be represented as a minor allele.
Somatic variant, germline variant, and small deletions and insertion calling was performed within the Firehose environment at the Broad Institute with the previously published MuTect, MapReduce, and Indelocator algorithms -. Pindel, an algorithm for detecting insertions and deletions (indels) , was used to detect indels in the coding regions of VHL. Because the samples were all obtained from formalin fixed, paraffin embedded (FFPE) pathology specimens, we applied a filtering algorithm to reduce the number of artifactual mutations introduced by FFPE . The FFPE filter consists of two steps. First, the filter estimates the component of total sequencing error rate due to FFPE artifacts in a CpG island by scanning all reference C (or G) sites counting sites sequenced as T (or A) in the two possible read pair orientations. Second, the orientation of each C > T (or G > A) mutation is compared to a model of balanced read pair orientation (binomial with p = 0.5: no artifact) and a biased orientation characteristic of FFPE artifacts (binomial with p = 0.96), as FFPE-induced artifacts affect one of the DNA strands. The filter removes mutations consistent with the FFPE orientation bias to the degree where less than 1% of the surviving mutations in a given sample are consistent with FFPE artifacts. Significance of mutated genes was determined by MutSigCV .
Copy number and rearrangement analysis
Segmented copy data was obtained using copy number ratios. These were calculated as the ratio of tumor read depth to the average read depth observed in a panel of normal samples. Subsequently we converted the copy ratio data to allelic ratio, which allows for detection of copy neutral LOH and gives another constraint on copy number alterations. ABSOLUTE  was used to estimate sample purity and ploidy and then calculate cancer cell fraction (inferred percentage of neoplastic nuclei that harbor a given mutation) and cell copy number of mutations and local DNA copy changes. Rearrangements were determined by the dRanger and BreakPointer algorithms .
Loss of heterozygosity analysis of validation cohort
Where af is the true allele fraction between 0 and 1, and alt is the alternate allele counts, ref is the reference allele count, and m is each single nucleotide variant detected along chromosome 3p. Samples demonstrating allelic fractions with bimodal distributions were called positive for a deletion or copy neutral loss of heterozygosity.
- (1)if the LOH event represents a copy neutral event, then the tumor purity (PT) is given by the following equation:
- (2)if the LOH event was a result of haploidization, then the tumor purity is given by:
Five micron sections of the FFPE specimens from the discovery cohort were prepared and stained for HIF1-α (NB100-131, Novus Biologicals), VEGF (SC-152, Santa Cruz Biotechnology), or PDGFR-β (AB32570, Abcam) by protocols described in the product data sheets. IHC was reviewed and scored by MPH.
Baseline clinical characteristics of discovery and validation cohorts of sporadic hemangioblastomas sequenced in this study
Discovery (n = 10)
Validation (n = 22)
Average Age (IQR)
Prior VHL testing
Additional VHL-related lesions
To identify somatic mutations in the WES data, a filter was first applied to exclude artifact mutations introduced by the processing of formalin-fixed, paraffin-embedded (FFPE) specimens, which are characterized by artificially induced C > T . These artifact mutations are distinct from real CpG > TpG mutations, which dominate in most cancers. The FFPE filter accordingly removed 10 spurious mutations (Additional file 4: Figure S3).
Because exon 1 of VHL was covered at a lower mean depth (13×) than exons 2 (213×) and 3 (51×) (Figure 2b), the promoter region and exon 1 were separately PCR amplified and Sanger sequenced for the four specimens that did not demonstrate evidence for biallelic VHL inactivation. None of these specimens, for which exon 1 was initially covered at a mean of 7.4×, demonstrated somatic mutations in the promoter region or exon 1 of VHL by Sanger sequencing, although this method is not highly sensitive for detection of mutations with low allelic fractions. Of note, two missense mutations in ARID1B were identified in two separate hemangioblastomas, at amino acid positions 647 and 651, with predicted allelic fractions of 2-6% and cancer cell fraction of 8-32%, suggesting subclonality.
VHL mutations have been previously detected in sporadic hemangioblastomas -. Our study is the first to comprehensively set an upper bound on the frequency of other alterations in hemangioblastoma formation, and further confirm that VHL alterations can be detected in the vast majority of hemangioblastomas, establishing VHL inactivation as the central event in formation of this tumor. Based on the implications of prior work, these findings were one of the possible anticipated results in this prototypic tumor type, and were facilitated by next generation sequencing techniques. In addition, utilization of this technology allowed for quantification of allelic frequency, revealing the cell non-autonomous role of VHL-inactivated hemangioblasts/stromal cells driving and sustaining the tumor cell mass via recruitment of intermixed normal endothelial cells.
Biallelic VHL inactivation in hemangioblastoma results in insufficient degradation of HIF-1α, which then results in the inappropriate transcription of effectors normally expressed in hypoxic states, such as vascular endothelial growth factor (VEGF) or platelet derived growth factor B (PDGF B) . Accordingly, the tumor cell mass is composed largely of non-neoplastic endothelial cells and surrounding pericytes, with only a minor component consisting of neoplastic “stromal” cells that actually bear driver mutations. The process of tumorigenesis is thus driven by a subpopulation of neoplastic cells that recruit and interact with diverse cellular types to form the tumor mass ,. Genomic analyses that do not sufficiently account for such cell-type purity issues can incompletely characterize the spectrum of alterations within the neoplastic cellular compartment. Sporadic hemangioblastomas therefore offered an enticing opportunity to use the most current analytics in cancer genomics to uncover cryptic driver alterations, which may be present in only a minor subpopulation of the overall tumor mass, evading detection by prior bulk tissue analyses.
As predicted by the benign clinico-pathologic features, hemangioblastomas were notable for a relatively low frequency of mutations. Within this context of infrequent mutations and copy number alterations, we found a markedly high prevalence of recurrent somatic mutations (50%) and LOH of the gene locus on chromosome 3p (72%). Together, these alterations account for biallelic inactivation of VHL in 47% and an inactivating alteration in at least one allele in 78% of sporadic hemangioblastomas. We detected these alterations in specimens with tumor cell fractions as low as 9%. It is possible that we could have captured an even higher prevalence of VHL inactivation via somatic mutation or LOH with deeper sequencing across VHL in its entirety.
Prior studies have reported the presence of mutations in VHL in 10 to 44% of sporadic hemangioblastoma . The frequency of aneuploidy of chromosome 3 has ranged from 18 to 69% in these sporadic tumors ,. One study identified concurrent somatic mutation and LOH of VHL in only 1 of 13 sporadic hemangioblastomas  and another study identified missense mutations with deletion of VHL by comparative genomic hybridization in only 2 of 16 sporadic hemangioblastomas . This is likely a consequence of lower VHL mutant allele fractions confined to the neoplastic “stromal” cells in sporadic hemangioblastomas, as compared to familial tumors that are characterized by heterozygous germline VHL alterations.
Mutations in ARID1B, which behaves as a tumor suppressor in other cancers -, were detected in this study in 20% of the discovery cohort, a level that did not reach statistical significance by our analysis. Furthermore, the mutations were present with allelic fractions of only 2-6% and these lesions were found to be subclonal. Further analysis is needed to determine whether such subclonal variants are functionally important in supporting the development of sporadic hemangioblastomas ,.
VHL inactivation in the pathogenesis of sporadic hemangioblastoma mirrors the VHL alterations seen in benign renal cysts, which may serve as precursors of clear cell renal cancer . These cysts are notable for biallelic VHL inactivation in renal tubular epithelial cells; whereas, clear cell renal cancer is additionally characterized by mutations in genes involved in the PTEN-mTOR pathway ,. Similar to benign renal cysts, hemangioblastomas rarely demonstrate aggressive malignant features, and we did not detect additional clonal genetic drivers in hemangioblastomas, including other candidate genomic loci on chromosome 3p of BAP1, SETD2, and PBRM1 implicated in clear cell renal carcinoma.
The analysis of tumor purity by ABSOLUTE also demonstrates that the extent of VHL inactivation in these tumors parallels “stromal” cell content estimated by IHC, rather than representing a homogenous genome that would characterize a tumor cell mass entirely derived from an ancestral hemangioblast clone. This important distinction lends further support to the model that stromal cells are genetically unique in VHL deficiency and stimulate reactive angiogenesis by recruiting normal endothelial cells via pseudo-hypoxia mediated paracrine signaling. This model contrasts with the alternate proposed pathway of an embryologically arrested “hemangioblast” ancestral clone that trans-differentiates into the various cell types contained in the tumor ,,.
In summary, we find that inactivation of VHL is highly recurrent and characteristic of the majority of sporadic hemangioblastomas. In addition, we do not detect other common recurrent somatic mutations in these tumors. These data therefore confirm the central role of VHL inactivation in all hemangioblastomas, suggesting that treatment strategies for both familial and sporadic cases could be uniformly considered, as VHL loss in the neoplastic subpopulation likely orchestrates the architecture of these tumors. For example, VEGF inhibitors have been used in cases of multifocal familial hemangioblastomas with treatment responses ,; their use in surgically-challenging sporadic disease should be considered. Advances in deep-coverage sequence analysis have thus resulted in a more complete understanding of the molecular basis of this canonical cancer type, and hold promise for the discovery of similarly cryptic drivers of tumorigenesis in other cancers with complex and diverse cellular composition.
In this study, we performed whole exome sequencing of matched tumor-normal specimens of sporadic hemangioblastoma and found that VHL inactivation is more highly prevalent in this disease process than previously noted and that no other gene alteration was identified at a statistically significant level in these tumors. High depth sequencing was required to unmask the low allelic fraction of VHL mutations and copy number alterations found in the neoplastic “stromal” cells, which are present only in a fraction of cells comprising the overall tumor mass. The central role of VHL in the formation of sporadic hemangioblastomas suggests that the approach to these tumors can be universally considered as a single entity together with familial cases, which arise as a result of VHL disease.
Matthew Meyerson, Gad Getz, Priscilla K Brastianos and Daniel P Cahill are co-senior authors.
This work was supported by the Massachusetts General Hospital Neuro-Oncology Fund for Genomic Discovery. GMS was supported by NIH National Institutes of Neurologic Disorders and Stroke R25NS065743 and American Brain Tumor Association Basic Research Fellowship supported by the Humor to Fight the Tumor Event Committee. DPC was supported by the Burroughs Wellcome Career Award. PKB was supported by the American Brain Tumor Association Basic Research Fellowship and Brain Science Foundation. The authors thank Othon Iliopoulos for helpful discussions and Michelle Toohey and Ariel Warren for clinical assistance.
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