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Loss of heterozygosity reveals non-VHL allelic loss in hemangioblastomas at 22q13
Loss of Heterozygosity Reveals Non-VHL Allelic Loss in Hemangioblastomas at 22q13 MARIE E. BECKNER, MD, EIZABURO SASATOMI, MD, PHD, PATRICIA A. SWALSKY, BS, RONALD L. HAMILTON, MD, IAN F. POLLACK, MD, AND SYDNEY D. FINKELSTEIN, MD Hemangioblastomas (HBs) are low-grade (World Health Organization grade I/IV) central nervous system (CNS) tumors that frequently contain VHL (3p26) mutations. They occur sporadically and in von Hippel Lindau (VHL) disease. Encoded pVHL aids degradation of hypoxia-inducible factors (HIFs) in the presence of normal oxygen levels. HBs provide an in vivo view of HIF effects within a CNS tumor. Typically, HBs are cystic tumors containing a mural nodule formed by noninvasive, vacuolated stromal cells that are embedded in a network of capillaries. Nine HBs, consecutively resected from 8 patients at our institution during a recent 2-year time span, were evaluated for additional losses of tumor suppressor genes. Non-VHL microsatellites studied for loss of heterozygosity (LOH) are near tumor suppressor genes lost in gliomas, pituitary adenomas, several CNS tumors on 22q, neurofibromatosis 1, and colon carcinomas (13, 2, 2, 1, and 2 markers for each, respectively). LOH in the region of 3p21.3-3p26.3 occurred in 3 of 8 HBs informative for at least 1 marker (D3S1539, D3S2303, or D3S2373). By using 2 markers
(D22S417 and D22S532) for 22q13.2, LOH was found in 5 of 8 informative HBs. All 3 HBs with allelic losses near VHL also showed LOH at 22q13.2. No consistent losses were found with markers for 1p34, LMYC, 5q21, 5q32, 9p21, 10q23, 17p13, and 19q13. LOH for the 22q13.2 region in HBs suggests that the loss of another tumor suppressor gene is involved in the pathogenesis of HBs in addition to VHL. Absence of LOH for glioma markers is consistent with the low-grade behavior of HBs. HUM PATHOL 35:1105-1111. © 2004 Elsevier Inc. All rights reserved. Key words: hemangioblastoma, von Hippel Lindau, tumor suppressor genes, loss of heterozygosity. Abbreviations: HBs, hemangioblastomas; VHL, von Hippel Lindau; HIFs, hypoxia-inducible factors; LOH, loss of heterozygosity; VEGF, vascular endothelial growth factor; APC, adenomatous polyposis coli; NF1, neurofibromatosis type 1, NF2, neurofibromatosis type 2; CNS, central nervous system; PCR, polymerase chain reaction.
Hemangioblastomas (HBs) are slow-growing central nervous system tumors (World Health Organization grade I/IV) that occur either sporadically or as part of the von Hippel Lindau (VHL) syndrome. HBs are most frequently found in the cerebellum. However, patients with VHL disease can have multiple HBs that sometimes involve the retina, brainstem, spinal cord, and cerebral cortex. HBs occur as well-circumscribed nodules that are often found in the wall of an intracranial (usually posterior fossa) cyst or are associated with a syrinx in the spinal cord. The microscopic appearance of HBs is characterized by a proliferation of capillaries, forming a rich vascular network supporting neoplastic, vacuolated stromal cells. Although the natural history of HBs includes both growth and quiescent phases, the mitotic rates of HBs are usually low, and these tumors characteristically do not behave aggressively.1 Mutational loss of the tumor suppressor gene, VHL, chromosome 3p26.11, often occurs in HBs. A
second allele is lost either after germline mutations in patients with VHL disease or after somatic mutations in sporadic HBs.2-7 Also, somatic mosaicism may occur in VHL disease.8 Mutations of the VHL gene are frequently indicated by loss of heterozygosity (LOH) for nearby microsatellites.7,9-11 The encoded protein, pVHL, regulates several transcriptional cellular responses to hypoxia. Normally, ubiquitination of the ␣ subunit of the hypoxiainducible factor (HIF) transcription factor is mediated by pVHL and leads to HIF degradation in the presence of oxygen.12 Accordingly, the loss of pVHL promotes increased production of proteins encoded by the genes transcriptionally regulated by HIF, including vascular endothelial growth factor (VEGF) and erythropoietin.13-18 Although the loss of VHL leading to increased VEGF expression explains the proliferation of nonneoplastic blood vessels in HBs and increased permeability allowing fluid accumulation,19-22 stromal cell proliferation and histogenesis remain controversial. The loss of VHL alone with activation of HIF has not promoted tumorigenesis in an animal model.23 Mutations of the VHL gene also occur in retinal angiomas, renal carcinomas, pheochromocytomas, islet cell tumors, and endolymphatic sac tumors of the inner ear,9,24-28 suggesting that the potential for tumorigenicity in neuroectodermal and neuroendocrine cells is sensitive to the loss of VHL, possibly promoted by the loss of 1 or more other tumor suppressor genes. A neuroectodermal origin has been proposed for HBs,29,30 as well as a vascular origin.18,31,32 Although stromal cells in HBs are antigenically polymorphous, several studies have shown positive immunohistochem-
From the Department of Pathology and Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, and RedPath Integrated Pathology, Pittsburgh, PA. Accepted for publication May 13, 2004. Supported by The Nick Eric Wichman Foundation, Ellicott City, MD. Address correspondence and reprint requests to Marie E. Beckner, MD, 200 Lothrop Street, PUH, Rm. A-515, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.05.014
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ical staining for antigens, consistent with a neuroectodermal or neuroendocrine origin. Positive results have been obtained with antibodies reactive for S100, glial fibrillary acidic protein, neuron-specific enolase, neuronal cell adhesion molecule (NCAM/CD56), inhibin ␣, and other antigens.29,30,32-37 Expression of VEGF, erythropoietin, and their receptors seen in HB stromal cells also has been found in astrocytes.38-44 In this study, LOH methodology with a variety of chromosomal markers was used to further study HBs. We searched for allelic losses associated with potential tumor suppressor genes by using a consecutive series of 9 HBs. In addition to 3 microsatellite markers near VHL, 20 other markers were also tested for LOH, with an emphasis on those associated with CNS tumors. The markers included those for malignant astrocytic tumors or gliomas (13 markers), pituitary adenomas (2 markers), neurofibromatosis (NF) type 1 (1 marker), 22q13.2 (2 markers) near unidentified tumor suppressor genes and NF2, and colon carcinomas (2 markers) near adenomatous polyposis coli (APC). Although the majority of the non-VHL markers in this survey did not show a significant (P ⬍ 0.05) frequency of LOH, loss of microsatellite markers at 22q13.2 was significant, indicating that another tumor suppressor gene is lost in many HBs in addition to VHL. MATERIALS AND METHODS Specimens from a consecutive series of 9 HBs, resected from 8 patients during a recent 2-year time span, were retrieved from the University of Pittsburgh Medical Center files based on the diagnosis of HB. The morphology and clinical features of all tumors were reviewed. Additional sections of paraffin-embedded tissue blocks containing tumor were obtained for further study. Routine hematoxylin and eosin– stained sections were used to identify regions of tumor and normal (if available) tissues suitable for genotyping. Although mixtures of stromal and vascular cells were allowed for dissections, regions of necrosis, inflammatory infiltrates, and reactive glial cells were avoided in all samples. Each tumor sample and a nearby normal sample (when available) were microdissected from corresponding unstained, deparaffinized histological sections. Microdissection was performed manually with a moistened scalpel blade with the aid of a Zeiss SZ-40 stereomicroscope (Goettingen, Germany). No attempt was made to separately dissect tumor stromal cells from the vasculature. A 0.5- to 1-cm diameter portion of a 4-m histological section, obtained from each sample, was sufficient to form a cloudy solution in 50-L of water in a 1.5-mL conical tube. Dissected tissues were stored for polymerase chain reaction (PCR) amplifications. Loss of microsatellite markers, short tandem tetranucleotide sequence repeat polymorphisms, was used to identify probable losses of nearby alleles for specific tumor suppressor genes. These polymorphic markers normally vary slightly in the number of repeats at a gene locus between paired chromosomes and are identified by flanking sequences. Fluorescent or radioactive tags (P32) on primers (flanking sequences) were incorporated into PCR products that were approximately 120 nucleotides in length. The labeled tumor and normal tissue samples were then evaluated either manually on DNA electrophoretic gels or in an automated ABI
GENESCAN 310 apparatus (Applied Biosystems, Foster City, CA). Results of ABI scans were visualized as peaks reflecting fluorescent signals from the PCR products. They were quantified by calculating ratios of the 2 peaks representing each allele of paired chromosomes. When both DNA polymorphisms were present in the normal tissue (informative sample), 2 PCR products of slightly different lengths (representing different numbers of tandem repeats from each chromosome of a pair) were produced in equal amounts. The ABI readout showed a pair of peaks that were approximately equal in height (ratios ranging from 0.51 to 1.49), representing equal amounts. When LOH was not present in an informative tumor sample, 2 PCR products of slightly different lengths were also produced. When an allele for a gene was missing (LOH), the tumor sample yielded a single peak that was comparable in height to the peaks seen in the normal tissue sample or a pair of peaks with marked differences in height ratios, either less than 0.51 or greater than 1.49. When no heterozygosity was present (noninformative) near a gene, the normal tissue sample showed a single, tall peak representing the combined products derived from both chromosomes. Limiting the interpretation of LOH to ratios either less than 0.51 or greater than 1.49 maintained a high probability for LOH being present in the HBs. Samples that failed to yield peaks for interpretation were considered to be unsatisfactory, presumably because of either insufficient material or interference by blood in the specimen during PCR amplification. When samples yielded unsatisfactory results with the automated ABI method, DNA gels of the PCR products, isotopically labeled with P32, were also performed. Two gel bands with slightly different migration lengths (representing both DNA polymorphisms), when present in the normal tissue, indicated that the sample was informative. If both bands were also present in the tumor tissue, LOH was not present, whereas if only 1 band was present in the tumor sample, then LOH was present. When no heterozygosity was present (noninformative), 2 bands of equal migration lengths were seen in the normal tissue, and the tumor sample was not interpreted. One tumor (Patient 5) that failed to yield results for 22 of 23 markers with either the automated or manual method was not studied further. Three tetranucleotide markers (D3S2373, D3S2303, and D3S1539 at 3p21.3, 3p25.3, and 3p26.3, respectively) near the VHL gene (3p26.11) were tested. Twenty additional markers for other suppressor genes were also tested. The markers listed characteristically show LOH in gliomas (D1S407 and D1S1193 at 1p34; LMYC; D9S254 and D9S251 at 9p21; D10S520 and D10S1173 at 10q23; p53I1, D17S974, D17S1289, and D17S1303 at 17p13; D19S400 and D19S559 at 19q13), pituitary adenomas (D5S1392 and D5S1403 at 5q32), several types of tumors with and without mutations in nearby neurofibromatosis type 2 (NF2; D22S532 and D22S417 at 22q13.2), neurofibromatosis type 1 (NF1) (I27), and colon carcinomas with mutations in APC (D5S592 and D5S615 at 5q21). Markers were selected on the basis of their chromosomal locations in relationship to nearby tumor suppressor gene locations described at http://www.cedar. genetics.soton.ac.uk. The Fisher’s exact test was used to detect significant (P ⬍ 0.05) frequencies of non-VHL LOH in informative specimens, using all markers for each site tested.
RESULTS Nine specimens from 8 patients, 4 of each sex, were tested for LOH. Two spinal cord tumors resected
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TABLE 1. Clinicopathologic Findings in Consecutive Cases of HBs Patient no.*
Age
1
21
VHL disease (2 HBs)
Pain
2
64
Polycythemia, SCC & HTN
Headaches, ataxia
3
69
4
36
Headaches, dizziness, unstable gait Headaches, imbalance
6
30
7
52
Retinal lesion, possibly a hemangioma Previous HB resected from same site Recent immigration to USA, history not available Recent 15-lb weight loss
8
28
History
VHL disease (previous HBs resected from other sites)
Tumor Locations
Symptoms
Spinal cord (two tumors) Cerebellum
Peripheral RBCs 1012/L†
Gross Cyst or Syrinx
5.24 (4.3-5.9)
Yes Not mentioned
Cerebellum
Recent phlebotomy 5.26 (4.2-5.2)
Posterior fossa
5.34 (4.2-5.2)
Probably, fluid obtained Yes
Headaches, imbalance
Cerebellum
4.92 (4.13-5.57)
Yes
Headaches, diplopia, fatigue, weakness Nausea & vomiting, dizziness, diplopia
Fourth ventricle Cerebellum
5.46 (4.2-5.4)
Yes
No labs
Yes
Abbreviations: SCC, squamous cell carcinoma; HTN, hypertension; HB, hemangioblastoma; RBC, red blood cell; VHL, von Hippel Lindau. *Patient 5 was excluded because of unsatisfactory PCR results. †Normal ranges in parentheses.
from the same patient were studied separately. The 2 youngest patients were known to have VHL disease. Another patient had a retinal tumor that clinically suggested a diagnosis of VHL disease (Table 1). Most of the HBs were associated with an intracranial cyst (Fig 1A) or syrinx. Gross dimensions of the tissue specimens resected and submitted for pathological evaluation ranged from 0.5 cm (1 spinal tumor) to 4.5 ⫻ 3 ⫻ 2 cm in largest dimensions. On routine sections, the microscopic features of all specimens were typical for HBs, as shown in the tumor resected from Patient 6 (Fig 1). Reticulin stains or anti-CD34 immunoreactivity revealed a vascular network encompassing negatively stained stromal cells (Fig 1C) in 4 of 4 cases. Mitoses were not seen, and proliferation indices (percentage of stromal cells staining for Ki67), obtained in 4 tumors, were ⬍3% in 2 specimens. The rates were moderate (8.9%) and high (19.2%) in tumors resected from patients 4 and 6, respectively. Immunoreactivity for epithelial membrane antigen was negative in all 3 HBs stained. No gross or microscopic features differentiated the tumors demonstrating loss of markers near 22q13.2 from the other HBs. All 8 HBs satisfactory for PCR were informative for at least 1 marker near VHL. LOH for VHL occurred in 3 of 8 HBs with 3 markers (D3S1539, D3S2303, or D3S2373). Three tumors from separate patients showed losses among the 3p markers (Table 2). These 3 tumors included 1 of 2 spinal cord tumors resected from the patient with known VHL disease. Another tumor with LOH near VHL represented either a second or recurrent HB. LOH was present in 75% of 4 tumors informative for all three 3p markers. Most (79%) of the tumor foci sampled in the positive HBs showed LOH for the markers, as shown in Table 3. The studies showed LOH for 2 markers (D22S417 or D22S532) at 22q13.2 in 5 of 8 informative HBs. This frequency of LOH (62.5%) was statistically significant (P ⫽ 0.0256) when compared with the expected fre-
quency of zero. These tumors included both spinal tumors from 1 patient with known VHL disease and from another patient with probable VHL disease (clinical impression of a concurrent retinal angioma). The losses of markers near VHL occurred only in tumors from patients with losses at 22q13. The frequency of finding LOH for 22q13 markers in the 3 HBs demonstrating LOH near VHL, when no association was expected, had a P value of 0.1, representing a trend. One tumor also had a loss of the marker for NF1 (I27; Table 2). An example of graphical ABI results showing LOH in an informative patient’s specimen is shown (Fig 2). The microsatellite regions on 3p and 22q were the only ones that showed LOH at rates higher than 30% in all informative specimens with the selected markers. No non-VHL microsatellite regions other than 22q showed a significant (P ⬍ 0.05) frequency of LOH for markers at the gene loci selected for this study. Among the negative group, informative markers for LMYC were lost most frequently (LOH in 2 of 7 informative assays; P ⫽ 0.4615).
DISCUSSION This study suggests that there is loss of a non-VHL tumor suppressor gene in many HBs at 22q13.2. Although the NF2 gene may be involved, there are several reasons to consider other unidentified tumor suppressor genes that have been proposed for this chromosomal region. Patients with NF2 are at risk for other types of CNS tumors, including schwannomas, meningiomas, ependymomas, and neurofibromas, but HBs have not been noted, and vice versa, patients with VHL disease have not been reported to be at risk for NF2. Although the loss of NF2 has been noted in sporadic meningiomas, ependymomas, perineurial cell tumors, and mesotheliomas,45-49 NF2 mutations have not been
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FIGURE 1. Patient 6’s hemangioblastoma is illustrated. (A) On magnetic resonance imaging, an axial T1-weighted image shows a cystic cerebellar lesion containing a dense mural nodule, enhanced by gadolinium administration. (B) Vacuolated stromal tumor cells are distributed among a network of capillaries. Hematoxylin and eosin staining. (C) Immunostaining of the arborizing tumor vasculature is shown with antiCD34, leaving the stromal cells unstained. Original magnification, ⫻430 (B) and ⫻114 (C).
reported in sporadic HBs in our review of the literature. In another LOH study using a different chromosomal marker (S22S268) near NF2, retention of heterozygosity was found in 4 HBs that were informative.9 Mutations of NF2 commonly lead to truncation50 and loss of immunoreactivity for merlin or schwannomin.45,51,52 Therefore the positive immunoreactivity for merlin found in HBs when tested by other investigators53 indicated that NF2 remains intact in HBs. Gliomas, some meningiomas, and several malignancies from non-CNS sites have shown either LOH for markers or cytogenetic abnormalities near NF2, but the gene was intact when thoroughly tested.49,54-56 Multiple abnormalities in subregions of chromosome 22 near NF2 have been found in human brain tumors57,58 and in cancers of the head and neck.59 Additional studies report losses involving chromosome 22 that may or may not have been associated with NF2 mutations. A pleomorphic xanthoastrocytoma showed LOH for 22q, cytogenetic studies on pituitary adenomas showed losses of chromosome 22, and LOH for 22q12 has been reported for a neurofibrosarcoma occurring in NF1.60-62 Other tumor suppressor genes that play a role in the pathogenesis of CNS tumors have been proposed to exist on 22q near NF2.54,55,63-65 LOH for 22q has been reported for informative sporadic and VHL pheochromocytomas at 53% and 21% occurrence rates, respectively.64 Identifying another tumor suppressor gene in addition to VHL in HBs may help to clarify the pathogenesis of HBs and the possible consequences of increased HIFs in normoxia within these tumors. Another well-known tumor suppressor gene, hSNF5/INI1, at 22q.11 is lost in some aggressive, primitive CNS tumors that occur in children, such as atypical rhabdoid tumor–malignant rhabdoid tumor.66,67 However, mutations of hSN5F/INI1 have been ruled out in studies showing LOH for nearby markers in gliomas, insulinomas, ependymomas, neurinomas, etc.68-71 Loss of hSNF5/INI1 would be unexpected in low-grade CNS tumors, such as HBs. A previous study that examined 7 HBs for nonVHL genetic defects found retention of heterozygosity for single markers near tumor suppressor genes at 5q21 (APC), 11p15.5 (WT2), 13q (RB), 17p13.1-p13.2 (p53), and 17p21 (BRCA1). Three to 4 of the HBs were informative for each of the markers used.9 We also failed to find significant frequency of LOH for microsatellites near 5q21 (APC) and 17p13 (p53). Only 2 of 11 and 3 of 11 informative assays using multiple markers near 5q21 and 17p13, respectively, showed significant ABI peak ratios in specimens from patients 1, 4, 7, and 8. Comparative genomic hybridization has shown nonVHL genetic defects in 10 sporadic HBs, including losses of chromosomes 6, 9, and 18q and a gain of chromosome 19 in 50%, 30%, 30%, and 30%, respectively.72 We found retention of heterozygosity for 9p21 in 7 HBs that were informative for a single marker. We did not find significant frequency of LOH near 19q13 using 2 markers, D19S400 and D19S555, that were informative in 8 and 7 HBs, respectively. Only 1 of 2
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TABLE 2. Study results in each patient (Pt)’s tumor(s) Pt 1 Associated Gene/Tumor VHL VHL VHL NF2 & other at 22q13 NF1
Microsatellite Markers
1 Tumor
2nd Tumor
Pt 2
Pt 3
Pt 4
Pt 6
Pt 7
Pt 8
D3S1539 D3S2303 D3S2373 D22S417
e e LOH LOH
e e e LOH
e NI NI e
U NI e LOH
LOH LOH LOH LOH
e NI e NI
LOH e LOH NI
e e NI e
D22S532 I27
NI NI
NI NI
e e
NI e
NI NI
e NI
LOH LOH
e e
st
NOTE. Samples from patient 5 were excluded because of unsatisfactory PCR results. Abbreviations: LOH, loss of heterozygosity; e, absence of LOH; NI, noninformative specimens; U, unsatisfactory.
tumor foci showed a significant ABI peak ratio in the specimen from patient 4 using D19S400. This study helps to establish the extent of microdissection needed to detect loss of VHL in HBs. Although our dissection with a handheld scalpel blade included both stromal and endothelial cells, the 50% loss of VHL in HBs of patients informative for two 3p markers is comparable to what has been achieved elsewhere with laser capture microdissection that excluded blood vessels from the tumor tissue. In sporadic HBs, Lee et al11 found LOH with D3S1038, D3S1110, and 104/105 at or near VHL in 52.6% of 19 informative HBs by using laser capture to specifically analyze the stromal cell component. Another study of VHL patients showed LOH with DS1317 and DS1110 out of multiple markers in only 1 of 7 cerebellar hemangioblastomas informative for at least 1 marker near VHL.9 Also, when 3p allelic losses were present in our study, they usually occurred in multiple foci, suggesting that the loss of VHL occurred early in tumor development and that the losses were widespread when present. In regard to tumor angiogenesis, HBs and malignant astrocytomas both show large numbers of supporting blood vessels. Tumor angiogenesis in high-grade gliomas is largely associated with regional tumor hypoxia stimulating VEGF production and indirectly with loss of p53 that allows VEGF production. In HBs, tumor angiogenesis is attributed to the loss of pVHL, resulting in stabilized HIFs that elevate VEGF transcription. However, despite the shared ability of these 2 tumor types to produce increased VEGF, there are striking differences in their behavior and vasculatures. Information ob-
tained regarding the genetic defects of brain tumors may help to explain variations in vascular responses within different types of tumors. HBs provide an in vivo model to study exuberant tumor angiogenesis in the CNS under normoxic conditions that does not lead to malignant biologic behavior. Genes transcribed by HIF include VEGF, which encodes the most powerful of all known promoters of tumor angiogenesis, and transforming growth factor ␣, which encodes another promoter of angiogenesis.12 These are among a large group of known promoters of tumor angiogenesis.73 Tumor suppressor genes whose losses may promote abnormal angiogenesis within malignant astrocytomas but appeared to be retained in HBs included p16, p15 at 9p21, PTEN at 10q23, and p53 at 17p13. The retention of heterozygosity in HBs for markers commonly lost in gliomas illustrates differences in the 2 tumor types and supports the clinical usefulness of using LOH to categorize CNS tumors for prognostic purposes. In regard to HB histogenesis, although LOH cannot be used to indicate derivation from astrocytes or other neuroectodermal cells, allelic losses in the 22q13.2 region have occurred in other CNS tumors of neuroectodermal origin. In summary, LOH near the VHL gene was detected in 3 of 8 informative HBs without selective harvesting of the stromal cells. The significant frequency of LOH for an additional putative tumor suppressor gene at 22q13.2 indicates that the loss of this unidentified gene in addition to VHL most likely plays a role in the pathogenesis of HBs.
TABLE 3. Correlation Between VHL and 22q13 Markers Showing LOH in Hemangioblastomas Patient no. 1
Tumor Locations
3 4
Spinal cord Spinal cord Cerebellum Posterior fossa
7
Fourth ventricle
VHL Markers Showing LOH
VHL LOH in Tumor Foci
22q13 Markers Showing LOH
22q13 LOH in Tumor Foci
D3S2373 None None D3S1539 D3S2303 D3S2373 DS1539 D3S2373
1/2 None None 2/2 2/2 2/2 1/3 3/3
D22S417 D22S417 D22S417 D22S417
1/2 1/2 1/1 1/2
D22S532
2/3
Abbreviations: VHL, von Hippel Lindau gene; LOH, loss of heterozygosity.
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FIGURE 2. A representative example of ABI GENESCAN 310 apparatus (Applied Biosystems, Foster City, CA) results used in the analysis for loss of heterozygosity (LOH) is shown. Polymerase chain reaction (PCR) products were generated with flanking sequences of a microsatellite gene marker, allowing incorporation of fluorescent labels during amplification. Results of automated analysis of a patient’s specimens are shown, with the fluorescent PCR products represented as solid black peaks that migrated according to size (base pairs [bp]). Specimens from informative patients yielded 2 peaks with different migration distances caused by normal variation in the number of tetranucleotide repeats in the microsatellite alleles. Six standards of varying sizes, represented by open peaks greater than 600 fluorescent signal units, were included. (A) Normal tissue with heterozygosity for the microsatellite marker (informative) yielded fluorescent PCR products that migrated as black peaks at 164 and 176 bp, with heights of 567 and 473 signal units, respectively. (B) Tumor tissue from the same patient showed significant loss in height of the black peak migrating at 164 bp compared with normal tissue, indicating LOH. The ratio of peak heights (fluorescence), reflecting relative amounts of the 164-bp and 176-bp PCR products, greatly exceeded the normal range (0.51-1.49) of ratios expected in informative specimens.
Acknowledgment. The authors thank Leslie Viramontes, HT(ASCP), and colleagues, University of Pittsburgh Medical Center Immunohistochemistry Laboratory, for performing special stains; Dr Douglas E. Moul, Department of Psychiatry, University of Pittsburgh for statistical assistance; and Dee Ann M. Cleary, Department of Pathology, University of Pittsburgh, for administrative support. The authors also thank The Nick Eric Wichman Foundation, Ellicott City, MD, for financial support and encouragement.
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profile of the VHLgene in von Hippel-Lindau disease and in sporadic hemangioblastoma. Hum Mutat 12:424-430, 1998 7. Tse JY, Wong JH, Lo KW, et al: Molecular genetic analysis of the von Hippel-Lindau disease tumor suppressor gene in familial and sporadic cerebellar hemangioblastomas. Am J Clin Pathol 107:459466, 1997 8. Murgia A, Martella M, Vinanzi C, et al: Somatic mosaicism in von Hippel-Lindau disease. Hum Mutat 15:114, 2000 9. Crossey PA, Foster K, Richards FM, et al: Molecular genetic investigations of the mechanism of tumourigenesis in von HippelLindau disease: Analysis of allele loss in VHL tumours. Hum Genet 93:53-58, 1994 10. Vortmeyer AO, Gnarra JR, Emmert-Buck MR, et al: von Hippel-Lindau gene deletion detected in the stromal cell component of a cerebellar hemangioblastoma associated with von Hippel-Lindau disease. Hum Pathol 28:540-543, 1997 11. Lee JY, Dong SM, Park WS, et al: Loss of heterozygosity and somatic mutations of the VHL tumor suppressor gene in sporadic cerebellar hemangioblastomas. Cancer Res 58:504-508, 1998 12. Kondo K, Kaelin WG Jr: The von Hippel-Lindau tumor suppressor gene. Exp Cell Res 264:117-125, 2001 13. Krieg M, Marti HH, Plate KH: Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel-Lindau tumor suppressor gene loss of function. Blood 92:3388-3393, 1998 14. Maxwell PH, Wiesener MS, Chang GW, et al: The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygendependent proteolysis. Nature 399:271-275, 1999 15. Pastore Y, Jedlickova K, Guan Y, et al: Mutations of von Hippel-Lindau tumor-suppressor gene and congenital polycythemia. Am J Hum Genet 73:412-419, 2003 16. Hatva E, Bohling T, Jaaskelainen J, et al: Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas. Am J Pathol 148:763-775, 1996 17. Wizigmann-Voos S, Breier G, Risau W, et al: Up-regulation of vascular endothelial growth factor and its receptors in von HippelLindau disease-associated and sporadic hemangioblastomas. Cancer Res 55:1358-1364, 1995 18. Vortmeyer AO, Frank S, Jeong SY, et al: Developmental arrest of angioblastic lineage initiates tumorigenesis in von HippelLindau disease. Cancer Res 63:7051-7055, 2003 19. Wizigmann-Voos S, Plate KH: Pathology, genetics and cell biology of hemangioblastomas. Histol Histopathol 11:1049-1061, 1996 20. Flamme I, Krieg M, Plate KH: Up-regulation of vascular endothelial growth factor in stromal cells of hemangioblastomas is correlated with up-regulation of the transcription factor HRF/HIF2alpha. Am J Pathol 153:25-29, 1998 21. Krieg M, Haas R, Brauch H, et al: Up-regulation of hypoxiainducible factors HIF-1alpha and HIF-2alpha under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 19:5435-5443, 2000 22. Machein MR, Plate KH: VEGF in brain tumors. J Neurooncol 50:109-120, 2000 23. Mack FA, Rathmell WK, Arsham AM, et al: Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth. Cancer Cell 3:75-88, 2003 24. Chan CC, Vortmeyer AO, Chew EY, et al: VHL gene deletion and enhanced VEGF gene expression detected in the stromal cells of retinal angioma. Arch Ophthalmol 117:625-630, 1999 25. Koch CA, Vortmeyer AO, Zhuang Z, et al: New insights into the genetics of familial chromaffin cell tumors. Ann N Y Acad Sci 970:11-28, 2002 26. Curley SA, Lott ST, Luca JW, et al: Surgical decision-making affected by clinical and genetic screening of a novel kindred with von Hippel-Lindau disease and pancreatic islet cell tumors. Ann Surg 227:229-235, 1998 27. Aubert-Petit G, Baudin E, Cailleux AF, et al: [Neuro-endocrine tumors and von Hippel-Lindau disease. 3 cases]. Presse Med 28:1231-1234, 1999 28. Vortmeyer AO, Huang SC, Koch CA, et al: Somatic von Hippel-Lindau gene mutations detected in sporadic endolymphatic sac tumors. Cancer Res 60:5963-5965, 2000 29. Becker I, Paulus W, Roggendorf W: Histogenesis of stromal
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LOH REVEALS NON-VHL ALLELIC LOSS AT 22q13 (Beckner et al) cells in cerebellar hemangioblastomas. An immunohistochemical study. Am J Pathol 134:271-275, 1989 30. Bohling T, Maenpaa A, Timonen T, et al: Different expression of adhesion molecules in stromal cells and endothelial cells of capillary hemangioblastoma. Acta Neuropathol (Berl) 92:461-466, 1996 31. Chaudhry AP, Montes M, Cohn GA: Ultrastructure of cerebellar hemangioblastoma. Cancer 42:1834-1850, 1978 32. Lach B, Gregor A, Rippstein P, et al: Angiogenic histogenesis of stromal cells in hemangioblastoma: Ultrastructural and immunohistochemical study. Ultrastruct Pathol 23:299-310, 1999 33. Tanimura A, Nakamura Y, Hachisuka H, et al: Hemangioblastoma of the central nervous system: Nature of the stromal cells as studied by the immunoperoxidase technique. Hum Pathol 15:866869, 1984 34. Feldenzer JA, McKeever PE: Selective localization of gammaenolase in stromal cells of cerebellar hemangioblastomas. Acta Neuropathol (Berl) 72:281-285, 1987 35. Grant JW, Gallagher PJ, Hedinger C: Haemangioblastoma. An immunohistochemical study of ten cases. Acta Neuropathol (Berl) 76:82-86, 1988 36. Hufnagel TJ, Kim JH, True LD, et al: Immunohistochemistry of capillary hemangioblastoma. Immunoperoxidase-labeled antibody staining resolves the differential diagnosis with metastatic renal cell carcinoma, but does not explain the histogenesis of the capillary hemangioblastoma. Am J Surg Pathol 13:207-216, 1989 37. Hoang MP, Amirkhan RH: Inhibin alpha distinguishes hemangioblastoma from clear cell renal cell carcinoma. Am J Surg Pathol 27:1152-1156, 2003 38. Masuda S, Chikuma M, Sasaki R: Insulin-like growth factors and insulin stimulate erythropoietin production in primary cultured astrocytes. Brain Res 746:63-70, 1997 39. Marti HH, Wenger RH, Rivas LA, et al: Erythropoietin gene expression in human, monkey and murine brain. Eur J Neurosci 8:666-676, 1996 40. Masuda S, Okano M, Yamagishi K, et al: A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J Biol Chem 269:19488-19493, 1994 41. Siren AL, Knerlich F, Poser W, et al: Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain. Acta Neuropathol (Berl) 101:271-276, 2001 42. Issa R, Krupinski J, Bujny T, et al: Vascular endothelial growth factor and its receptor, KDR, in human brain tissue after ischemic stroke. Lab Invest 79:417-425, 1999 43. Krum JM, Rosenstein JM: VEGF mRNA and its receptor flt-1 are expressed in reactive astrocytes following neural grafting and tumor cell implantation in the adult CNS. Exp Neurol 154:57-65, 1998 44. Nagai A, Nakagawa E, Choi HB, et al: Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture. J Neuropathol Exp Neurol 60:386-392, 2001 45. Harwalkar JA, Lee JH, Hughes G, et al: Immunoblotting analysis of schwannomin/merlin in human schwannomas. Am J Otol 19:654-659, 1998 46. De Vitis LR, Tedde A, Vitelli F, et al: Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas. Hum Genet 97:632-637, 1996 47. Birch BD, Johnson JP, Parsa A, et al: Frequent type 2 neurofibromatosis gene transcript mutations in sporadic intramedullary spinal cord ependymomas. Neurosurgery 39:135-140, 1996 48. Lasota J, Fetsch JF, Wozniak A, et al: The neurofibromatosis type 2 gene is mutated in perineurial cell tumors: A molecular genetic study of eight cases. Am J Pathol 158:1223-1229, 2001 49. Sekido Y, Pass HI, Bader S, et al: Neurofibromatosis type 2 (NF2) gene is somatically mutated in mesothelioma but not in lung cancer. Cancer Res 55:1227-1231, 1995 50. Jacoby LB, MacCollin M, Louis DN, et al: Exon scanning for mutation of the NF2 gene in schwannomas. Hum Mol Genet 3:413419, 1994 51. Huynh DP, Mautner V, Baser ME, et al: Immunohistochem-
ical detection of schwannomin and neurofibromin in vestibular schwannomas, ependymomas and meningiomas. J Neuropathol Exp Neurol 56:382-390, 1997 52. Den Bakker MA, van Tilborg AA, Kros JM, et al: Truncated NF2 proteins are not detected in meningiomas and schwannomas. Neuropathology 21:168-173, 2001 53. Hitotsumatsu T, Iwaki T, Kitamoto T, et al: Expression of neurofibromatosis 2 protein in human brain tumors: An immunohistochemical study. Acta Neuropathol (Berl) 93:225-232, 1997 54. Arakawa H, Hayashi N, Nagase H, et al: Alternative splicing of the NF2 gene and its mutation analysis of breast and colorectal cancers. Hum Mol Genet 3:565-568, 1994 55. Ng HK, Lau KM, Tse JY, et al: Combined molecular genetic studies of chromosome 22q and the neurofibromatosis type 2 gene in central nervous system tumors. Neurosurgery 37:764-773, 1995 56. Lomas J, Bello MJ, Alonso ME, et al: Loss of chromosome 22 and absence of NF2 gene mutation in a case of multiple meningiomas. Hum Pathol 33:375-378, 2002 57. Rey JA, Bello MJ, de Campos JM, et al: Abnormalities of chromosome 22 in human brain tumors determined by combined cytogenetic and molecular genetic approaches. Cancer Genet Cytogenet 66:1-10, 1993 58. Huang B, Starostik P, Kuhl J, et al: Loss of heterozygosity on chromosome 22 in human ependymomas. Acta Neuropathol (Berl) 103:415-420, 2002 59. dos Reis PP, Poli-Frederico RC, dos Santos RM, et al: Distinct regions of loss of heterozygosity on 22q in different sites of head and neck squamous cell carcinomas. Med Sci Monitor 8:BR89-BR94, 2002 60. Rey JA, Bello MJ, Kusak ME, et al: Involvement of 22q12 in a neurofibrosarcoma in neurofibromatosis type 1. Cancer Genet Cytogenet 66:28-32, 1993 61. Kaulich K, Blaschke B, Numann A, et al: Genetic alterations commonly found in diffusely infiltrating cerebral gliomas are rare or absent in pleomorphic xanthoastrocytomas. J Neuropathol Exp Neurol 61:1092-1099, 2002 62. Bello MJ, de Campos JM, Kusak ME, et al: Chromosomal abnormalities in pituitary adenomas. Cancer Genet Cytogenet 124: 76-79, 2001 63. Rey JA, Pestana A, Bello MJ: Cytogenetics and molecular genetics of nervous system tumors. Oncol Res 4:321-331, 1992 64. Bender BU, Gutsche M, Glasker S, et al: Differential genetic alterations in von Hippel-Lindau syndrome-associated and sporadic pheochromocytomas. J Clin Endocrinol Metab 85:4568-4574, 2000 65. Oskam NT, Bijleveld EH, Hulsebos TJ: A region of common deletion in 22q13.3 in human glioma associated with astrocytoma progression. Int J Cancer 85:336-339, 2000 66. Versteege I, Sevenet N, Lange J, et al: Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394:203-206, 1998 67. Packer RJ, Biegel JA, Blaney S, et al: Atypical teratoid/ rhabdoid tumor of the central nervous system: Report on workshop. J Pediatr Hematol Oncol 24:337-342, 2002 68. Weber M, Stockhammer F, Schmitz U, et al: Mutational analysis of INI1 in sporadic human brain tumors. Acta Neuropathol (Berl) 101:479-482, 2001 69. Wild A, Langer P, Ramaswamy A, et al: A novel insulinoma tumor suppressor gene locus on chromosome 22q with potential prognostic implications. J Clin Endocrinol Metab 86:5782-5787, 2001 70. Kraus JA, de Millas W, Sorensen N, et al: Indications for a tumor suppressor gene at 22q11 involved in the pathogenesis of ependymal tumors and distinct from hSNF5/INI1. Acta Neuropathol (Berl) 102:69-74, 2001 71. Sevenet N, Lellouch-Tubiana A, Schofield D, et al: Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotypephenotype correlations. Hum Mol Genet 8:2359-2368, 1999 72. Sprenger SH, Gijtenbeek JM, Wesseling P, et al: Characteristic chromosomal aberrations in sporadic cerebellar hemangioblastomas revealed by comparative genomic hybridization. J Neurooncol 52:241-247, 2001 73. Beckner M: Factors promoting tumor angiogenesis. Cancer Invest 17:594-623, 1999
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(D22S417 and D22S532) for 22q13.2, LOH was found in 5 of 8 informative HBs. All 3 HBs with allelic losses near VHL also showed LOH at 22q13.2. No consistent losses were found with markers for 1p34, LMYC, 5q21, 5q32, 9p21, 10q23, 17p13, and 19q13. LOH for the 22q13.2 region in HBs suggests that the loss of another tumor suppressor gene is involved in the pathogenesis of HBs in addition to VHL. Absence of LOH for glioma markers is consistent with the low-grade behavior of HBs. HUM PATHOL 35:1105-1111. © 2004 Elsevier Inc. All rights reserved. Key words: hemangioblastoma, von Hippel Lindau, tumor suppressor genes, loss of heterozygosity. Abbreviations: HBs, hemangioblastomas; VHL, von Hippel Lindau; HIFs, hypoxia-inducible factors; LOH, loss of heterozygosity; VEGF, vascular endothelial growth factor; APC, adenomatous polyposis coli; NF1, neurofibromatosis type 1, NF2, neurofibromatosis type 2; CNS, central nervous system; PCR, polymerase chain reaction.
Hemangioblastomas (HBs) are slow-growing central nervous system tumors (World Health Organization grade I/IV) that occur either sporadically or as part of the von Hippel Lindau (VHL) syndrome. HBs are most frequently found in the cerebellum. However, patients with VHL disease can have multiple HBs that sometimes involve the retina, brainstem, spinal cord, and cerebral cortex. HBs occur as well-circumscribed nodules that are often found in the wall of an intracranial (usually posterior fossa) cyst or are associated with a syrinx in the spinal cord. The microscopic appearance of HBs is characterized by a proliferation of capillaries, forming a rich vascular network supporting neoplastic, vacuolated stromal cells. Although the natural history of HBs includes both growth and quiescent phases, the mitotic rates of HBs are usually low, and these tumors characteristically do not behave aggressively.1 Mutational loss of the tumor suppressor gene, VHL, chromosome 3p26.11, often occurs in HBs. A
second allele is lost either after germline mutations in patients with VHL disease or after somatic mutations in sporadic HBs.2-7 Also, somatic mosaicism may occur in VHL disease.8 Mutations of the VHL gene are frequently indicated by loss of heterozygosity (LOH) for nearby microsatellites.7,9-11 The encoded protein, pVHL, regulates several transcriptional cellular responses to hypoxia. Normally, ubiquitination of the ␣ subunit of the hypoxiainducible factor (HIF) transcription factor is mediated by pVHL and leads to HIF degradation in the presence of oxygen.12 Accordingly, the loss of pVHL promotes increased production of proteins encoded by the genes transcriptionally regulated by HIF, including vascular endothelial growth factor (VEGF) and erythropoietin.13-18 Although the loss of VHL leading to increased VEGF expression explains the proliferation of nonneoplastic blood vessels in HBs and increased permeability allowing fluid accumulation,19-22 stromal cell proliferation and histogenesis remain controversial. The loss of VHL alone with activation of HIF has not promoted tumorigenesis in an animal model.23 Mutations of the VHL gene also occur in retinal angiomas, renal carcinomas, pheochromocytomas, islet cell tumors, and endolymphatic sac tumors of the inner ear,9,24-28 suggesting that the potential for tumorigenicity in neuroectodermal and neuroendocrine cells is sensitive to the loss of VHL, possibly promoted by the loss of 1 or more other tumor suppressor genes. A neuroectodermal origin has been proposed for HBs,29,30 as well as a vascular origin.18,31,32 Although stromal cells in HBs are antigenically polymorphous, several studies have shown positive immunohistochem-
From the Department of Pathology and Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, and RedPath Integrated Pathology, Pittsburgh, PA. Accepted for publication May 13, 2004. Supported by The Nick Eric Wichman Foundation, Ellicott City, MD. Address correspondence and reprint requests to Marie E. Beckner, MD, 200 Lothrop Street, PUH, Rm. A-515, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.05.014
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ical staining for antigens, consistent with a neuroectodermal or neuroendocrine origin. Positive results have been obtained with antibodies reactive for S100, glial fibrillary acidic protein, neuron-specific enolase, neuronal cell adhesion molecule (NCAM/CD56), inhibin ␣, and other antigens.29,30,32-37 Expression of VEGF, erythropoietin, and their receptors seen in HB stromal cells also has been found in astrocytes.38-44 In this study, LOH methodology with a variety of chromosomal markers was used to further study HBs. We searched for allelic losses associated with potential tumor suppressor genes by using a consecutive series of 9 HBs. In addition to 3 microsatellite markers near VHL, 20 other markers were also tested for LOH, with an emphasis on those associated with CNS tumors. The markers included those for malignant astrocytic tumors or gliomas (13 markers), pituitary adenomas (2 markers), neurofibromatosis (NF) type 1 (1 marker), 22q13.2 (2 markers) near unidentified tumor suppressor genes and NF2, and colon carcinomas (2 markers) near adenomatous polyposis coli (APC). Although the majority of the non-VHL markers in this survey did not show a significant (P ⬍ 0.05) frequency of LOH, loss of microsatellite markers at 22q13.2 was significant, indicating that another tumor suppressor gene is lost in many HBs in addition to VHL. MATERIALS AND METHODS Specimens from a consecutive series of 9 HBs, resected from 8 patients during a recent 2-year time span, were retrieved from the University of Pittsburgh Medical Center files based on the diagnosis of HB. The morphology and clinical features of all tumors were reviewed. Additional sections of paraffin-embedded tissue blocks containing tumor were obtained for further study. Routine hematoxylin and eosin– stained sections were used to identify regions of tumor and normal (if available) tissues suitable for genotyping. Although mixtures of stromal and vascular cells were allowed for dissections, regions of necrosis, inflammatory infiltrates, and reactive glial cells were avoided in all samples. Each tumor sample and a nearby normal sample (when available) were microdissected from corresponding unstained, deparaffinized histological sections. Microdissection was performed manually with a moistened scalpel blade with the aid of a Zeiss SZ-40 stereomicroscope (Goettingen, Germany). No attempt was made to separately dissect tumor stromal cells from the vasculature. A 0.5- to 1-cm diameter portion of a 4-m histological section, obtained from each sample, was sufficient to form a cloudy solution in 50-L of water in a 1.5-mL conical tube. Dissected tissues were stored for polymerase chain reaction (PCR) amplifications. Loss of microsatellite markers, short tandem tetranucleotide sequence repeat polymorphisms, was used to identify probable losses of nearby alleles for specific tumor suppressor genes. These polymorphic markers normally vary slightly in the number of repeats at a gene locus between paired chromosomes and are identified by flanking sequences. Fluorescent or radioactive tags (P32) on primers (flanking sequences) were incorporated into PCR products that were approximately 120 nucleotides in length. The labeled tumor and normal tissue samples were then evaluated either manually on DNA electrophoretic gels or in an automated ABI
GENESCAN 310 apparatus (Applied Biosystems, Foster City, CA). Results of ABI scans were visualized as peaks reflecting fluorescent signals from the PCR products. They were quantified by calculating ratios of the 2 peaks representing each allele of paired chromosomes. When both DNA polymorphisms were present in the normal tissue (informative sample), 2 PCR products of slightly different lengths (representing different numbers of tandem repeats from each chromosome of a pair) were produced in equal amounts. The ABI readout showed a pair of peaks that were approximately equal in height (ratios ranging from 0.51 to 1.49), representing equal amounts. When LOH was not present in an informative tumor sample, 2 PCR products of slightly different lengths were also produced. When an allele for a gene was missing (LOH), the tumor sample yielded a single peak that was comparable in height to the peaks seen in the normal tissue sample or a pair of peaks with marked differences in height ratios, either less than 0.51 or greater than 1.49. When no heterozygosity was present (noninformative) near a gene, the normal tissue sample showed a single, tall peak representing the combined products derived from both chromosomes. Limiting the interpretation of LOH to ratios either less than 0.51 or greater than 1.49 maintained a high probability for LOH being present in the HBs. Samples that failed to yield peaks for interpretation were considered to be unsatisfactory, presumably because of either insufficient material or interference by blood in the specimen during PCR amplification. When samples yielded unsatisfactory results with the automated ABI method, DNA gels of the PCR products, isotopically labeled with P32, were also performed. Two gel bands with slightly different migration lengths (representing both DNA polymorphisms), when present in the normal tissue, indicated that the sample was informative. If both bands were also present in the tumor tissue, LOH was not present, whereas if only 1 band was present in the tumor sample, then LOH was present. When no heterozygosity was present (noninformative), 2 bands of equal migration lengths were seen in the normal tissue, and the tumor sample was not interpreted. One tumor (Patient 5) that failed to yield results for 22 of 23 markers with either the automated or manual method was not studied further. Three tetranucleotide markers (D3S2373, D3S2303, and D3S1539 at 3p21.3, 3p25.3, and 3p26.3, respectively) near the VHL gene (3p26.11) were tested. Twenty additional markers for other suppressor genes were also tested. The markers listed characteristically show LOH in gliomas (D1S407 and D1S1193 at 1p34; LMYC; D9S254 and D9S251 at 9p21; D10S520 and D10S1173 at 10q23; p53I1, D17S974, D17S1289, and D17S1303 at 17p13; D19S400 and D19S559 at 19q13), pituitary adenomas (D5S1392 and D5S1403 at 5q32), several types of tumors with and without mutations in nearby neurofibromatosis type 2 (NF2; D22S532 and D22S417 at 22q13.2), neurofibromatosis type 1 (NF1) (I27), and colon carcinomas with mutations in APC (D5S592 and D5S615 at 5q21). Markers were selected on the basis of their chromosomal locations in relationship to nearby tumor suppressor gene locations described at http://www.cedar. genetics.soton.ac.uk. The Fisher’s exact test was used to detect significant (P ⬍ 0.05) frequencies of non-VHL LOH in informative specimens, using all markers for each site tested.
RESULTS Nine specimens from 8 patients, 4 of each sex, were tested for LOH. Two spinal cord tumors resected
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TABLE 1. Clinicopathologic Findings in Consecutive Cases of HBs Patient no.*
Age
1
21
VHL disease (2 HBs)
Pain
2
64
Polycythemia, SCC & HTN
Headaches, ataxia
3
69
4
36
Headaches, dizziness, unstable gait Headaches, imbalance
6
30
7
52
Retinal lesion, possibly a hemangioma Previous HB resected from same site Recent immigration to USA, history not available Recent 15-lb weight loss
8
28
History
VHL disease (previous HBs resected from other sites)
Tumor Locations
Symptoms
Spinal cord (two tumors) Cerebellum
Peripheral RBCs 1012/L†
Gross Cyst or Syrinx
5.24 (4.3-5.9)
Yes Not mentioned
Cerebellum
Recent phlebotomy 5.26 (4.2-5.2)
Posterior fossa
5.34 (4.2-5.2)
Probably, fluid obtained Yes
Headaches, imbalance
Cerebellum
4.92 (4.13-5.57)
Yes
Headaches, diplopia, fatigue, weakness Nausea & vomiting, dizziness, diplopia
Fourth ventricle Cerebellum
5.46 (4.2-5.4)
Yes
No labs
Yes
Abbreviations: SCC, squamous cell carcinoma; HTN, hypertension; HB, hemangioblastoma; RBC, red blood cell; VHL, von Hippel Lindau. *Patient 5 was excluded because of unsatisfactory PCR results. †Normal ranges in parentheses.
from the same patient were studied separately. The 2 youngest patients were known to have VHL disease. Another patient had a retinal tumor that clinically suggested a diagnosis of VHL disease (Table 1). Most of the HBs were associated with an intracranial cyst (Fig 1A) or syrinx. Gross dimensions of the tissue specimens resected and submitted for pathological evaluation ranged from 0.5 cm (1 spinal tumor) to 4.5 ⫻ 3 ⫻ 2 cm in largest dimensions. On routine sections, the microscopic features of all specimens were typical for HBs, as shown in the tumor resected from Patient 6 (Fig 1). Reticulin stains or anti-CD34 immunoreactivity revealed a vascular network encompassing negatively stained stromal cells (Fig 1C) in 4 of 4 cases. Mitoses were not seen, and proliferation indices (percentage of stromal cells staining for Ki67), obtained in 4 tumors, were ⬍3% in 2 specimens. The rates were moderate (8.9%) and high (19.2%) in tumors resected from patients 4 and 6, respectively. Immunoreactivity for epithelial membrane antigen was negative in all 3 HBs stained. No gross or microscopic features differentiated the tumors demonstrating loss of markers near 22q13.2 from the other HBs. All 8 HBs satisfactory for PCR were informative for at least 1 marker near VHL. LOH for VHL occurred in 3 of 8 HBs with 3 markers (D3S1539, D3S2303, or D3S2373). Three tumors from separate patients showed losses among the 3p markers (Table 2). These 3 tumors included 1 of 2 spinal cord tumors resected from the patient with known VHL disease. Another tumor with LOH near VHL represented either a second or recurrent HB. LOH was present in 75% of 4 tumors informative for all three 3p markers. Most (79%) of the tumor foci sampled in the positive HBs showed LOH for the markers, as shown in Table 3. The studies showed LOH for 2 markers (D22S417 or D22S532) at 22q13.2 in 5 of 8 informative HBs. This frequency of LOH (62.5%) was statistically significant (P ⫽ 0.0256) when compared with the expected fre-
quency of zero. These tumors included both spinal tumors from 1 patient with known VHL disease and from another patient with probable VHL disease (clinical impression of a concurrent retinal angioma). The losses of markers near VHL occurred only in tumors from patients with losses at 22q13. The frequency of finding LOH for 22q13 markers in the 3 HBs demonstrating LOH near VHL, when no association was expected, had a P value of 0.1, representing a trend. One tumor also had a loss of the marker for NF1 (I27; Table 2). An example of graphical ABI results showing LOH in an informative patient’s specimen is shown (Fig 2). The microsatellite regions on 3p and 22q were the only ones that showed LOH at rates higher than 30% in all informative specimens with the selected markers. No non-VHL microsatellite regions other than 22q showed a significant (P ⬍ 0.05) frequency of LOH for markers at the gene loci selected for this study. Among the negative group, informative markers for LMYC were lost most frequently (LOH in 2 of 7 informative assays; P ⫽ 0.4615).
DISCUSSION This study suggests that there is loss of a non-VHL tumor suppressor gene in many HBs at 22q13.2. Although the NF2 gene may be involved, there are several reasons to consider other unidentified tumor suppressor genes that have been proposed for this chromosomal region. Patients with NF2 are at risk for other types of CNS tumors, including schwannomas, meningiomas, ependymomas, and neurofibromas, but HBs have not been noted, and vice versa, patients with VHL disease have not been reported to be at risk for NF2. Although the loss of NF2 has been noted in sporadic meningiomas, ependymomas, perineurial cell tumors, and mesotheliomas,45-49 NF2 mutations have not been
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FIGURE 1. Patient 6’s hemangioblastoma is illustrated. (A) On magnetic resonance imaging, an axial T1-weighted image shows a cystic cerebellar lesion containing a dense mural nodule, enhanced by gadolinium administration. (B) Vacuolated stromal tumor cells are distributed among a network of capillaries. Hematoxylin and eosin staining. (C) Immunostaining of the arborizing tumor vasculature is shown with antiCD34, leaving the stromal cells unstained. Original magnification, ⫻430 (B) and ⫻114 (C).
reported in sporadic HBs in our review of the literature. In another LOH study using a different chromosomal marker (S22S268) near NF2, retention of heterozygosity was found in 4 HBs that were informative.9 Mutations of NF2 commonly lead to truncation50 and loss of immunoreactivity for merlin or schwannomin.45,51,52 Therefore the positive immunoreactivity for merlin found in HBs when tested by other investigators53 indicated that NF2 remains intact in HBs. Gliomas, some meningiomas, and several malignancies from non-CNS sites have shown either LOH for markers or cytogenetic abnormalities near NF2, but the gene was intact when thoroughly tested.49,54-56 Multiple abnormalities in subregions of chromosome 22 near NF2 have been found in human brain tumors57,58 and in cancers of the head and neck.59 Additional studies report losses involving chromosome 22 that may or may not have been associated with NF2 mutations. A pleomorphic xanthoastrocytoma showed LOH for 22q, cytogenetic studies on pituitary adenomas showed losses of chromosome 22, and LOH for 22q12 has been reported for a neurofibrosarcoma occurring in NF1.60-62 Other tumor suppressor genes that play a role in the pathogenesis of CNS tumors have been proposed to exist on 22q near NF2.54,55,63-65 LOH for 22q has been reported for informative sporadic and VHL pheochromocytomas at 53% and 21% occurrence rates, respectively.64 Identifying another tumor suppressor gene in addition to VHL in HBs may help to clarify the pathogenesis of HBs and the possible consequences of increased HIFs in normoxia within these tumors. Another well-known tumor suppressor gene, hSNF5/INI1, at 22q.11 is lost in some aggressive, primitive CNS tumors that occur in children, such as atypical rhabdoid tumor–malignant rhabdoid tumor.66,67 However, mutations of hSN5F/INI1 have been ruled out in studies showing LOH for nearby markers in gliomas, insulinomas, ependymomas, neurinomas, etc.68-71 Loss of hSNF5/INI1 would be unexpected in low-grade CNS tumors, such as HBs. A previous study that examined 7 HBs for nonVHL genetic defects found retention of heterozygosity for single markers near tumor suppressor genes at 5q21 (APC), 11p15.5 (WT2), 13q (RB), 17p13.1-p13.2 (p53), and 17p21 (BRCA1). Three to 4 of the HBs were informative for each of the markers used.9 We also failed to find significant frequency of LOH for microsatellites near 5q21 (APC) and 17p13 (p53). Only 2 of 11 and 3 of 11 informative assays using multiple markers near 5q21 and 17p13, respectively, showed significant ABI peak ratios in specimens from patients 1, 4, 7, and 8. Comparative genomic hybridization has shown nonVHL genetic defects in 10 sporadic HBs, including losses of chromosomes 6, 9, and 18q and a gain of chromosome 19 in 50%, 30%, 30%, and 30%, respectively.72 We found retention of heterozygosity for 9p21 in 7 HBs that were informative for a single marker. We did not find significant frequency of LOH near 19q13 using 2 markers, D19S400 and D19S555, that were informative in 8 and 7 HBs, respectively. Only 1 of 2
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TABLE 2. Study results in each patient (Pt)’s tumor(s) Pt 1 Associated Gene/Tumor VHL VHL VHL NF2 & other at 22q13 NF1
Microsatellite Markers
1 Tumor
2nd Tumor
Pt 2
Pt 3
Pt 4
Pt 6
Pt 7
Pt 8
D3S1539 D3S2303 D3S2373 D22S417
e e LOH LOH
e e e LOH
e NI NI e
U NI e LOH
LOH LOH LOH LOH
e NI e NI
LOH e LOH NI
e e NI e
D22S532 I27
NI NI
NI NI
e e
NI e
NI NI
e NI
LOH LOH
e e
st
NOTE. Samples from patient 5 were excluded because of unsatisfactory PCR results. Abbreviations: LOH, loss of heterozygosity; e, absence of LOH; NI, noninformative specimens; U, unsatisfactory.
tumor foci showed a significant ABI peak ratio in the specimen from patient 4 using D19S400. This study helps to establish the extent of microdissection needed to detect loss of VHL in HBs. Although our dissection with a handheld scalpel blade included both stromal and endothelial cells, the 50% loss of VHL in HBs of patients informative for two 3p markers is comparable to what has been achieved elsewhere with laser capture microdissection that excluded blood vessels from the tumor tissue. In sporadic HBs, Lee et al11 found LOH with D3S1038, D3S1110, and 104/105 at or near VHL in 52.6% of 19 informative HBs by using laser capture to specifically analyze the stromal cell component. Another study of VHL patients showed LOH with DS1317 and DS1110 out of multiple markers in only 1 of 7 cerebellar hemangioblastomas informative for at least 1 marker near VHL.9 Also, when 3p allelic losses were present in our study, they usually occurred in multiple foci, suggesting that the loss of VHL occurred early in tumor development and that the losses were widespread when present. In regard to tumor angiogenesis, HBs and malignant astrocytomas both show large numbers of supporting blood vessels. Tumor angiogenesis in high-grade gliomas is largely associated with regional tumor hypoxia stimulating VEGF production and indirectly with loss of p53 that allows VEGF production. In HBs, tumor angiogenesis is attributed to the loss of pVHL, resulting in stabilized HIFs that elevate VEGF transcription. However, despite the shared ability of these 2 tumor types to produce increased VEGF, there are striking differences in their behavior and vasculatures. Information ob-
tained regarding the genetic defects of brain tumors may help to explain variations in vascular responses within different types of tumors. HBs provide an in vivo model to study exuberant tumor angiogenesis in the CNS under normoxic conditions that does not lead to malignant biologic behavior. Genes transcribed by HIF include VEGF, which encodes the most powerful of all known promoters of tumor angiogenesis, and transforming growth factor ␣, which encodes another promoter of angiogenesis.12 These are among a large group of known promoters of tumor angiogenesis.73 Tumor suppressor genes whose losses may promote abnormal angiogenesis within malignant astrocytomas but appeared to be retained in HBs included p16, p15 at 9p21, PTEN at 10q23, and p53 at 17p13. The retention of heterozygosity in HBs for markers commonly lost in gliomas illustrates differences in the 2 tumor types and supports the clinical usefulness of using LOH to categorize CNS tumors for prognostic purposes. In regard to HB histogenesis, although LOH cannot be used to indicate derivation from astrocytes or other neuroectodermal cells, allelic losses in the 22q13.2 region have occurred in other CNS tumors of neuroectodermal origin. In summary, LOH near the VHL gene was detected in 3 of 8 informative HBs without selective harvesting of the stromal cells. The significant frequency of LOH for an additional putative tumor suppressor gene at 22q13.2 indicates that the loss of this unidentified gene in addition to VHL most likely plays a role in the pathogenesis of HBs.
TABLE 3. Correlation Between VHL and 22q13 Markers Showing LOH in Hemangioblastomas Patient no. 1
Tumor Locations
3 4
Spinal cord Spinal cord Cerebellum Posterior fossa
7
Fourth ventricle
VHL Markers Showing LOH
VHL LOH in Tumor Foci
22q13 Markers Showing LOH
22q13 LOH in Tumor Foci
D3S2373 None None D3S1539 D3S2303 D3S2373 DS1539 D3S2373
1/2 None None 2/2 2/2 2/2 1/3 3/3
D22S417 D22S417 D22S417 D22S417
1/2 1/2 1/1 1/2
D22S532
2/3
Abbreviations: VHL, von Hippel Lindau gene; LOH, loss of heterozygosity.
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FIGURE 2. A representative example of ABI GENESCAN 310 apparatus (Applied Biosystems, Foster City, CA) results used in the analysis for loss of heterozygosity (LOH) is shown. Polymerase chain reaction (PCR) products were generated with flanking sequences of a microsatellite gene marker, allowing incorporation of fluorescent labels during amplification. Results of automated analysis of a patient’s specimens are shown, with the fluorescent PCR products represented as solid black peaks that migrated according to size (base pairs [bp]). Specimens from informative patients yielded 2 peaks with different migration distances caused by normal variation in the number of tetranucleotide repeats in the microsatellite alleles. Six standards of varying sizes, represented by open peaks greater than 600 fluorescent signal units, were included. (A) Normal tissue with heterozygosity for the microsatellite marker (informative) yielded fluorescent PCR products that migrated as black peaks at 164 and 176 bp, with heights of 567 and 473 signal units, respectively. (B) Tumor tissue from the same patient showed significant loss in height of the black peak migrating at 164 bp compared with normal tissue, indicating LOH. The ratio of peak heights (fluorescence), reflecting relative amounts of the 164-bp and 176-bp PCR products, greatly exceeded the normal range (0.51-1.49) of ratios expected in informative specimens.
Acknowledgment. The authors thank Leslie Viramontes, HT(ASCP), and colleagues, University of Pittsburgh Medical Center Immunohistochemistry Laboratory, for performing special stains; Dr Douglas E. Moul, Department of Psychiatry, University of Pittsburgh for statistical assistance; and Dee Ann M. Cleary, Department of Pathology, University of Pittsburgh, for administrative support. The authors also thank The Nick Eric Wichman Foundation, Ellicott City, MD, for financial support and encouragement.
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