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[401] Germline Mutations in the von Hippel-Lindau Disease (VHL) Gene in Japanese VHL. Shuin T1, Yao M2, Kondo K2. 1Dept Urology, Kochi Medical School, 2Dept Urology Yokohama City U. School of Medicine.
To define the molecular basis of VHL patients in Japanese populations and improve their clinical treatment, we analyzed germline mutations of the VHL gene in 45 Japanese patients by single strand conformation poly-morphism (SSCP) and Southern blot analysis. We detected 23 (51.9%) mutations and 3 (6.7%) deletions by SSCP analysis and Southern blot, respectively. The intragenic mutations consisted of 14 missense mutations, 7 micro-deletions or insertions, and 2 splice-site mutations. Interestingly, 9 of 10 mutations in exon 1 are localized in a short region of 37 nucleotides. Five unique sites of mutation were included, which were not seen in previous studies. Unlike Western VHL patients, nonsense mutations were not found in theseJapanese VHL patients. The mutations found in 22 VHL patients without pheochromo-cytoma consisted of 11 missense mutations, 6 microdeletions or insertions, 2 splice-site alterations and 3 deletions. The mutations found in 4 VHL patients with pheochromocytoma consisted of 1 missense mutation at nucleotide 686 (codon 228), 2 missense mutations at nucleotide 712 (codon 238), and a novel 20 base-pair insertion at nucleotide 776 (codon 259). Although the mutations in codon 238 are at the mutational hot spot found in Western VHL patients with pheochromocytomas, a 20 base-pair insertion/duplication of wild-type VHL cDNA sequence is a unique mutation.
Our results suggest that mutations in Japanese VHL patients contain some unique features compared with Western VHL. The VHL gene has a critical role for etiology in Japanese VHL as well as Western VHL. Therefore the present work will improve clinical treatment and follow up of Japanese VHL.
[402] Genotype/Phenotype Correlation in Dutch VHL
Families. Hes FJ1, van Amstel HKP2, Zewald RA2, Hené RJ3,
Feldberg MAM4, van Gils APG4, van Vroonhoven TJMV5, Slootweg PJ6, Elderson
A7, Wittebol-Post D8, Lips CJM1. Dept 1Internal Medicine, 2Human Medical
Genetics, 3Nephrology, 4Radiology, 5Surgery, 6Pathology, 7Neurology, 8Ophthalmology,
U. Hospital Utrecht, The Netherlands.
Objective:
- Three large Dutch families were studied for clinical manifestations of VHL and mutations on chromosome 3p25-26.
Patients and Methods:
- From a total of over 100 family members, 35 patients could be identified by clinical screening, i.e. medical family history, biochemical analysis of blood and urine and radiology. Between 1994 and 1995, germline mutation analysis (by PCR amplification, direct sequencing and Southern blot analysis) was done in 26 persons. Clinical manifestations of VHL were scored by the number of organs affected. To study the natural history of the disease caused by a specific germline mutation, several criteria have to be fulfilled: large families have to be examined with long-term follow-up and with an extensive screening protocol. Limited screening in small families will only reveal symptomatic patients with manifest disease (preselection) and give an incomplete impression of the phenotype for each mutation.
Results:
- A total of 59 affected organs was detected: haemangioblastoma of the central nervous system (23), retinal angioma (7), renal cysts and carcinoma (14), phaeochromocytoma (4), pancreatic lesions (8) and liver lesions (3). DNA analysis revealed that two families had missense mutations (G713A and T722A) resulting in amino acid substitutions (R218Q and V241D, respectively) in the VHL-disease gene. In a third family a deletion was found. The family with a G713A mutation had haemangioblastoma of the central nervous system and renal tumors, but no phaeochromocytoma. In the family with a T722A mutation there was a high incidence of haemangioblastoma of the cerebellum (10/12; 7 cysts, 2 solid, 1 combined tumor) and renal lesions (9/12; 9 cysts, 7 renal cell carcinoma). The family with the deletion was not affected by phaeo-chromocytoma, but with haemangioblastoma in myelium and cerebellum (central and with occlusive hydrocephalus) and renal cysts and carcinoma. Retinal angioma and pancreatic cysts occurred in all three families.
Discussion:
- VHL families with phaeochromocytoma were shown to have missense mutations, whereas families with nonsense mutations or larger deletions did not develop phaeo-chromocytoma. The G713A mutation is found world-wide in a relatively high frequency. So far 4 families (from England, Japan, America and France) were also without phaeochromocytoma. This indicates that for this mutation there is no strong correlation in these studied families between genotype and phenotype (phaeochromocytoma). Renal cell carcinoma was associated in Dutch families with mutations in the 3' end of the VHL-gene. This finding is in good agreement with international publications.
Conclusion:
- Identification of specific germline mutations not only allows presymptomatic diagnosis and genetic counseling in VHL-families, but also can have clinical impact in some cases, by predicting disease phenotype.
[403] von Hippel-Lindau's Disease in Denmark: Past, Present and Future. Kjeldsen E, Rasmusson K, Hørder M. Dept Clinical Biochemistry and Clinical Genetics, KKA, Odense U. Hospital, Denmark.
- In Denmark about 50 families are known to suffer from von Hippel-Lindau disease (VHL) but it is generally believed that the occurrence is underreported. Although VHL is a multisystem disease associated with a diverse spectrum of systemic lesions predisposing individuals to the development of tumors, there have been no previous general attempts for establishing follow-up programs including genetic counseling and screening of healthy individuals at risk in Denmark.
After the VHL gene was cloned in 1993 and a large number of possible disease-causing mutations were identified, we are now in the process of establishing a nation-wide Danish center for molecular genetic diagnosis. Furthermore, we are establishing a database containing information on each patient's symptoms, treatment, time of diagnosis, follow-up and family history.
Due to the multiple organ involvement of VHL, we are also in the process of making thorough data for both patients and physicians in aid of making earlier diagnosis, and to help specialists involved understand the importance of full body screening for all manifestations of this complex disease.
Our initial experience was that it was a difficult task to get involved scientists "talking together" and to gather all the necessary information for proper genetic counseling and establishment of follow-up programs for each patient having VHL-manifestations, but especially for healthy individuals at risk. As an initial attempt to establish strategies for genetic counseling, molecular diagnosis, and how to operate functional follow-up programs, we have included 10 families and preliminary results will be discussed.
[404] Genomic Characterization of Alterations Associated with the VHL Disease In France. Olschwang S1, Richard S2, Boisson C1, Giraud S3, Laurent-Puig P1, Resche F4, Thomas G1. 1INSERM Institut Curie, 2CHU Necker Hôpital, Paris, 3Hôpital. E. Herriot Lyon, 4CHU Nantes, France.
Introduction
The complete cDNA of the gene responsible for VHL disease has been cloned. The VHL gene is divided into three exons, and encodes a 213-amino acid nuclear protein. Germline alterations of the VHL gene have been identified in VHL patients. We present here the results of a systematic screening for germline mutations in a series of 92 unrelated VHL patients referred from different regions of France.
Material and Methods
Blood samples were obtained from a total of 92 apparently unrelated VHL patients and kept frozen at -30° until DNA extraction. The clinical data were collected by S.R. through patient interviews and hospital notes. Patients were classified as type 1 (63 cases) and type 2 (28 cases) according to the absence or presence of phaeochromo-cytoma respectively. Depending on absence or presence of renal and pancreatic manifestations, type 2 was subdivided into type 2A (12 cases) and type 2B (16 cases). Search for the VHL mutations causing the disease was performed using a pre-screening method based on denaturing gradient gel electrophoresis after specific PCR amplification. Search for mutations in exons 2 and 3 required two independent amplifications and enabled the efficient screening of the whole exons. All electrophoretic variants in exon 2 or 3 were sequenced from PCR amplified products. Because of a high GC percentage, exon 1 could not be screened reliably by this method. Direct sequencing was systematically performed on exon 1 PCR products. Search for large alterations was performed using Southern method with the G7 cDNA probe kindly provided by Dr. Zbar.
Results
This procedure identified 52 variants in the series of VHL patients (67%). Mutations were either nonsense (10 cases), missense (35 cases) or silent mutations (3 cases), in-frame insertions or deletions (3 cases), mutations predicted to alter the reading frame (9 cases) or the splicing process (4 cases). Five large alterations could be identified on EcoR1 digested DNA's.
According to the clinical classification, 44 (70%) mutations were identified in type 1 VHL patients; 8 (67%) and 13 (81%) point mutations were identified in type 2A and 2B VHL patients respectively. Combining both approaches, a germline alteration could be identified in 73% of a series of 92 patients.
Also significantly participating in the clinical registration: M Bonnet, A Gaudric, G Lenoir, S Martin, PF Plouin, A Redondo, L Tailandier, R van Effenterre.
[405] Kidney Cancer and Kidney Failure in VHL.Novick AC, Dept of Urology, The Cleveland Clinic Foundation, Cleveland, Ohio.
- Von Hippel-Lindau disease is a rare autosomal dominant disorder characterized by predisposition to tumor development in the retina, cerebellum, spinal cord, pancreas, adrenals and kidneys. Renal cell carcinoma occurs in up to 45% of the patients with von Hippel-Lindau disease. However, in contrast to sporadic renal cell carcinoma, these tumors are often multicentric and bilateral, and occur at a younger patient age. Furthermore, renal cell carcinoma in von Hippel-Lindau disease is often associated with renal cysts that contain either frank carcinoma or a lining of hyperplastic clear cells representing incipient cancer.
Although originally believed to have a less malignant potential than its spontaneous counterpart, renal cell carcinoma in von Hippel-Lindau disease can metastasize and is a major cause of death of these patients. Therefore, surgery is indicated for localized renal cell carcinoma in von Hippel-Lindau disease to prevent tumor progression. The surgical options in patients with bilateral renal cell carcinoma are bilateral nephrectomy or a nephron-sparing operation to avoid end-stage renal failure. While the early results of nephron-sparing surgery in von Hippel-Lindau disease has been promising, recent studies have suggested a high incidence of postoperative tumor recurrence in the remaining portion of the kidney. Less information is available concerning the outcome for patients who require renal replacement therapy following bilateral nephrectomy.
We recently reported the aggregate experience with surgical treatment of renal cell carcinoma in von Hippel-Lindau disease at 8 medical centers in the United States. Our study was done to delineate further the clinical presentation, management, and long-term outcome of these patients. A total of 65 patients with von Hippel-Lindau disease underwent surgery for renal cell carcinoma (54 bilaterally and 11 unilaterally). Only 1 patient presented with metastatic disease. Radical nephrectomy and nephron-sparing surgery were performed in 16 and 49 patients, respectively. Mean post-treatment follow-up was 68 months.
The 5 and 10-year cancer-specific survival rates for all patients were 95% and 77% respectively. The corresponding rates for patients treated with nephron-sparing surgery were 100% and 81%, respectively. Of the latter patients, 25 (51%) had post-operative local tumor recurrence but only 2 had concomitant metastatic disease. Survival free of local recurrence was 71% at 5 years, but only 2 (15%) at 10 years. End-stage renal failure occurred in 15 patients (23%); 6 underwent renal transplantation (5 are alive with satisfactory renal function and no evidence of malignancy) and 9 were treated with chronic dialysis (6 are free of tumor).
Our results indicated that nephron-sparing surgery can provide effective initial treatment for patients with renal cell carcinoma and von Hippel-Lindau disease. These patients must be followed closely, since most will eventually have locally recurrent renal cell carcinoma typically not manifested for several years. The risk for concomitant metastasis appears to be small. When removal of all remaining renal tissue is necessary to achieve control of malignancy, renal transplantation can provide effective replacement therapy for end-stage renal disease.
[406] Lesions of Endocrine Organs in Von Hippel-Lindau Disease. Neumann HPH1, Bender BU1, Brauch H2. 1Dept Nephrology, Albert-Ludwigs-University of Freiburg, 2Institute Pathology, Technical University of Munich, Germany.
- Involvement of endocrine organs in von Hippel-Lindau disease includes adrenal glands, paraganglia, pancreas, testis, ovaries, thyroid, parathyroid and pituitary glands. Reported tumors of endocrine tissue are - frequently - pheochromocytoma, paraganglioma and pancreatic islet cell tumors, and - infrequently - carcinoid, medullary thyroid cancer, pituitary adenoma, and parathyroid adenoma. The risk for such tumors differs in different VHL families. Since germline mutation analysis of the VHL gene is now routine, the risk for developing tumors must be estimated for each type of mutation separately.
The estimated risks need further specification and comments. The risk ratio is derived mainly from the total number of subjects known to be affected; it is not clear how the lesions were ruled out in the remaining subjects. The risk ratio should be separated for symptomatic and asymptomatic subjects, and all characteristics should be estimated for the two groups separately. All predictions should also consider age at presentation, in cumulative distribution diagrams.
For the rare lesions, doubts remain whether tumori-genesis is induced by mutations of the VHL gene. The proof would be to demonstrate loss of heterozygosity at the VHL locus on chromosome 3p for the normal parental allele in tumor tissue from the affected subjects.
The total risk for endocrine tumors in the Freiburg study are currently:
Mutations Tumor Risk (n=)
505 T/C Pheochromocytoma 31/63
775 C/G Pheochromocytoma 10/10
712 C/T Pheochromocytoma 2/5
490 C/A Pheochromocytoma 2/2
505 T/C Islet cell carcinoma 1/63
505 T/C Pituitary adenoma 1/63
746 T/A Pheochromocytoma 2/2
775 C/G Medullary thyroid carcinoma 2/10
505 T/C Carcinoid 1/63
2 kb deletion Carcinoid 1/63
2 kb deletion Papillary ovary carcinoma 1/1
[407] Renal Cell Carcinoma in von Hippel Lindau
Disease: Strategies in Early Detection and Treatment. Lips CJM1,
Hes FJ1, Blijham GH1, Voest EE1, Los M1, Hené RJ2, Vos J2, Feldberg
MAM3, van Gils APG3, Hüppener JWM1,4, van Amstel HKP5, Zewald RA5,
Slootweg PJ4, van Vroonhoven JMV6. Dept 1Internal Medicine, 2Nephrology,
3Radiology, 4Pathology, 5Medical Genetics, 6Surgery, University Hospital,
Utrecht, The Netherlands.
- In three large families with three distinct VHL gene mutations and more than 100 members, radiological investigation revealed 14 patients with renal cell carcinoma. Eight patients were operated on for renal cell carcinoma. Initially, bilateral nephrectomy was performed, since extensive tumor formation was already present, or the risk of additional tumor development was estimated to be high. Although cure was obtained, it appeared that chronic dialysis or renal transplantation was not without complications, the most serious consequence being a decrease in life expectancy as a result of renal failure.
Genotype-phenotype correlations revealed that expres-sion of the disease within a family was very heterogeneous. Some patients developed multiple and aggressive tumors, while in other proven gene-carriers no signs or abnormalities could be detected. Pathological examination revealed that the grossly normal parenchyma that surrounds tumor tissue was scattered with microscopic lesions: benign cysts lined by atypical clear cells and small clear cell carcinomas. Apparently, in most patients, the clinical behavior of these microscopic carcinomas was relatively benign.
The hypothesis was raised that, when progression of growth in a solid tumor is observed, additional mutations have occurred. This results in more aggressive behavior and an increased risk of tumor spread. Immediate removal of the lesion is then indicated. Therefore, since 1992, we performed elective surgery. The procedure of nephron-sparing surgery in patients with VHL was based on a desire to maintain renal function as long as possible, while reducing the risk for metastases. Until now, no local recurrence of tumor formation was observed.
Our provisional conclusion is that nephron-sparing surgery is an effective initial treatment for VHL patients, provided patients are followed closely, because the risk for local recurrence remains.
[408] Renal Involvement in 43 Consecutive Patients
with von Lindau (VHL) Disease. Chauveau D, Chrétien Y, Richard
S, Grünfeld JP. Dept Nephrology and Urology, Hôpital Necker,
Paris, France.
- Renal involvement has emerged as the most prevalent cause of death in VHL disease, so we tested whether and how nephron-sparing surgery (NSS) could be applied in 43 consecutive VHL patients (24 unrelated families) with renal lesions. Renal involvement comprised multiple cysts and bilateral and multifocal carcinomas (RCC) detected by screening in 38 patients, at 30.5 (14-62) years of age. The severity of the renal disease was similar in VHL type 1 (79% of the pedigrees) and 2 (21%). Twenty-nine patients were operated on at a mean age of 33.6 years. 21 patients (28 kidneys or 61% of all operated kidneys) underwent NSS, 4 had complete ablation of involved kidneys and thus required dialysis, 3 had uninephrectomy and one had cyst fenestration. Vascular thrombosis occurred in 4/9 kidneys treated by ex vivo surgery. During a median follow-up of 29 months, local recurrence occurred in 5/21 (24%) patients treated by NSS whereas 2 developed metastasis. Chronic renal failure (creatinine > 120 mmol/l) affected 11 patients; in 9 of them, it was due to sequelae of surgery.
In conclusion, screening of RCC and NSS are of value in VHL patients. However, indications for ex vivo surgery should be drastically restricted and renal sequelae are not uncommon. Renal follow-up is required because of the risk of recurrence.
[409] Nephron-Sparing Operation for Renal Cell Carcinoma in von Hippel-Lindau Disease. Shinohara N, Sazawa A, Demura T, Nonomura K, Koyanagi T. Dept Urology, Hokkaido U. School of Medicine, Sapporo, Japan.
- We present 6 patients with localized renal cell carcinoma in von Hippel-Lindau disease to evaluate the technical feasibility and long-term follow-up outcomes of a nephron-sparing operation. These patients underwent an initial nephron-sparing operation (bilateral 4, unilateral 2), followed by serial CT scans at 3-6 month intervals.
All but one renal lesion could be resected by nephron-sparing operations (10: 6 in situ partial nephrectomy or enucleation, 4 extracorporeal partial nephrectomy with autotransplantation) preserving total renal function within normal limits. In 4 patients with bilateral renal cell carcinoma, postoperative serial CT scan revealed a total of 35 tumorous lesions (34 de novo lesions, 1 unresected lesion), 8 of which showed varying enlargement (mean growth rate: 0.534 ± 0.217 cm/year) for 7-45 months. A secondary renal operation was done in 4 patients with these enlarging renal lesions. At last follow-up, all patients still preserved adequate renal function.
We concluded that the nephron-sparing operation is, even with a high risk of local recurrence, an appropriate approach for localized renal cell carcinoma in von Hippel-Lindau disease.
[410] A Canadian Family With Hereditary Papillary Renal Carcinoma. Schmidt L, Glenn G, Choyke P, Linehan WM, Zbar B. BCDP, SAIC and Cancer Diagnostic Branch, NCI-NIH; Diagnostic Radiology, N.I.H., Bethesda, Maryland.
- Hereditary papillary renal carcinoma (HPRC) has been recognized as a form of inherited kidney cancer characterized by a predisposition to develop multiple, bilateral renal tumors with a papillary histology. Unlike clear cell renal carcinoma, neither genetic linkage to chromosome 3p nor loss of heterozygosity on chromosome 3 is associated with HPRC. Papillary renal tumors have a characteristic karyotype, trisomy of chromosomes 7, 17, often 16 and 20, and loss of Y.
Genetic linkage analysis of families with an inherited disorder provide information for localization of the disease gene to a region of a particular chromosome.
We describe a three generation Canadian family in which 13 of 40 members have papillary renal tumors. The early age of onset, multiple bilateral tumor pattern and papillary histology confirm the diagnosis of HPRC. Simulation studies to determine the power of this pedigree for genetic linkage studies will be presented.
[411] Hemangioblastomas of the Central Nervous System - Why, When and How to Operate on Them Micro-surgically. Scheremet R. Dept Neurosurgery, University of Freiburg, Germany.
- The only proven efficient treatment for hemangio-blastomas is total surgical excision. The operative risk mainly depends on the location of the tumor within the CNS, its relationships to the surrounding tissue and its size. These factors must be weighed against the risk of spontaneous sequelae. These include chronic damage of the surrounding nervous tissue by a growing tumor, an acute life-threatening decompensation especially of cystic tumors in the cranio-cervical region, and acute bleeding with consequent neurological disturbances due to damage to neural tissues.
The risk size of the natural course is as yet unknown. Reliable figures may be expected in the future by serial MRI observations of non-operated tumors.
Analyzing the literature and our own data on operated hemangioblastomas of the CNS within the past six years, we believe that the operative removal of cerebellar hemangio-blastomas is a low risk maneuver. The removal of brainstem and spinal cord tumors carried a slightly higher risk. Outcome correlated more with the preoperative neurological condition than with the size of the tumor.
Due to these observations, we tend to recommend an operation at an early stage. This prophylactic attitude may seem controversial.
In the case of multiple lesions, we try to estimate the potential operative risk for each lesion and to remove those whose natural course seems to be more dangerous.
[412] Renal Cell Carcinoma: Familial and Sporadic Types, Molecular Genetics Glenn G. National Cancer Institute, N.I.H, Bethesda, Maryland
[413] Prospects for New Anti-Cancer Drugs, Based on Tumor Suppressor Genes. Kaelin WG Jr. Dana-Farber Cancer Institute, Boston, Massachusetts.
- Inactivation of multiple tumor suppressor genes is a recurring theme in human cancers. In model systems, restoring the function of even one tumor suppressor gene can dramatically influence the ability of malignant cells to grow in culture and/or to grow as tumors in nude mice. This suggests that 1) tumor suppressor genes cooperate to constrain cell growth and prevent tumor formation and 2) small molecules, which, on one level mimic the activities of tumor suppressor proteins, might be useful in the treatment of cancer.
Considerable progress has been made toward elucidating the functions of human tumor suppressor proteins. For example, the ability of the retinoblastoma protein to regulate cell growth appears to be due, at least in part, to its ability to inhibit a cell-cycle regulatory transcription factor family called E2F. The p53 protein is a sequence-specific DNA-binding protein which, in response to signals such as DNA damage, activates the transcription of certain target genes.
Finally, recent data suggest that the ability of the von Hippel-Lindau protein to suppress tumorigenesis may be related to its ability to inhibit an enzyme called elongin or SIII. Biochemical functions such as these are amenable to high-throughput drug screens. For example, screens are on-going to identify drugs which, for example, will either inhibit E2F or activate genes normally controlled by p53. Likewise, it may, in time, be possible to screen for drugs which will mimic one or more of the activities of the wild-type von Hippel-Lindau protein.
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