Molecular genetic analysis of von Hippel-Lindau disease

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

Molecular genetic diagnosis of von Hippel-Lindau disease in familial phaeochromocytoma

Inherited predisposition to phaeochromocytoma is seen in multiple endocrine neoplasia type 2 syndromes, von Hippel-Lindau (VHL) disease, and neuro-fibromatosis type 1. In addition familial phaeochromocytoma alone has been reported. To investigate the genetic basis for familial phaeochromocytoma alone, we screened three affected kindreds for mutations in the RET proto-oncogene and the VHL tumour suppressor gene. We did not detect MEN 2 associated RET mutations in any family, but missense VHL gene mutations (V155L and R238W) were identified in two kindreds with no clinical evidence of VHL disease. Patients with familial, multiple, or early onset phaeochromocytoma should be investigated for germline VHL and RET gene mutations as the molecular diagnosis of multisystem familial cancer syndromes enables appropriate counselling and screening to be provided.     

Utility of a nitric oxide electrode for monitoring the administration of nitric oxide in biologic systems

Von Hippel-Lindau (VHL) disease is an inherited autosomal dominant neoplastic disorder causing central nervous system haemangioblastomas. The VHL gene (3p25-3p26) is known to be a tumour suppressor gene, with its inactivation being responsible for a predisposition to tumour development. As far as we know, the present report of VHL disease manifestation in identical twins is unique. Genetic inquiry into the family background did not reveal this disease among their progenitors. For presymptomatic diagnosis of 17 presently unaffected family members, constitutional DNA of the twins was screened for VHL germline mutations, using loss of heterozygosity studies and exon-specific DNA sequencing. To determine the influence of somatic mutations of the VHL gene in tumourigenesis, DNA of five surgically removed intracerebral haemangioblastomas of the identical twins was analyzed in comparison with their constitutional DNA by DNA sequencing of the complete VHL coding region. However, no allelic losses were found for the VHL gene or for various other tumour suppressor genes (p53, BRCA1, BRCA2, DCC, and MCC). Furthermore, no mutations were found in the constitutional DNA of either twin sister or in the DNA of all five tumour lesions. Based on our observations, we conclude that in certain VHL families, presymptomatic molecular diagnosis of the disease is not feasible and requires close clinical surveillance of all individuals at risk.     

Two Li-Fraumeni syndrome families with novel germline p53 mutations: loss of the wild-type p53 allele in only 50% of tumours

We describe two Li-Fraumeni syndrome families. Family A was remarkable for two early childhood cases of adrenocortical tumours, family B for a high incidence of many characteristic cancers, including a childhood case of choroid plexus tumour. Using direct sequencing, we analysed exons 5-9 of the p53 gene in constitutional DNA of individuals from both families and found two novel germline mutations in exon 5. In family A, we detected a point substitution in codon 138 (GCC to CCC), which resulted in the replacement of the alanine by a proline residue. Family B harboured a single-base pair deletion in codon 178 (CAC to -AC), resulting in a frameshift and premature chain termination. Three out of six tumours examined from both families, a renal cell carcinoma, a rhabdomyosarcoma and a breast cancer, showed loss of heterozygosity and contained only the mutant p53 allele. The remaining three neoplasms, both adrenocortical tumours and the choroid plexus tumour retained heterozygosity. Immunohistochemistry with anti-p53 antibody confirmed accumulation of p53 protein in tumours with loss of heterozygosity, while the remaining tumours were p53 negative. These results support the view that complete loss of activity of the wild-type p53 need not be the initial event in the formation of all tumours in Li-Fraumeni individuals.     

Explaining the unexplained in clinical infectious diseases: looking forward

BACKGROUND: Abdominal lymphangioma is a rare tumour usually classified with mesenteric and retroperitoneal cysts. This experience of abdominal lymphangiomas contrasts the differences between tumours in children and adults. METHODS: Between 1970 and 1996, six patients had surgical resection of an abdominal lymphangioma. RESULTS: There were three children aged 4 years or less and three adults aged 36-76 years. Two children presented with an acute abdomen and one with a rapidly enlarging abdominal girth. Lymphangiomas were located in the mesentery and gastrointestinal tract. In adults, symptoms lasted from months to years and lymphangiomas were found in the pancreas, spleen and retroperitoneum. CONCLUSION: In this series, abdominal lymphangioma presented more acutely in children and usually involved the mesentery, whereas in adults the history was longer and the tumour was found in the retroperitoneum.     

Microsatellite instability in early onset and familial colorectal cancer

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is often considered to be the most common form of inherited colorectal cancer, although its precise incidence is unknown. The clinical diagnosis of HNPCC relies on a combination of family history and young age of onset of colorectal cancer, but as many familial aggregations of colorectal cancer do not fulfil the strict diagnostic criteria, HNPCC might be underdiagnosed. The majority of HNPCC families have germline mutations in mismatch repair (MMR) genes, such as MSH2 or MLH1, so that HNPCC cancers characteristically exhibit DNA replication errors (RERs) at microsatellite loci. Although an RER positive phenotype in tumours can also result from somatic mutations in an MMR gene, the prevalence of RER + tumours should provide a maximum estimate of the incidence of germline MMR gene mutations in patients with early onset and familial colorectal cancer. We investigated colorectal cancers for RERs from (1) a population based study of 33 patients with colorectal cancer aged 45 years or less, (2) 65 kindreds with familial colorectal cancer which only partially fulfilled the criteria for the diagnosis of HNPCC, and (3) 18 cancers from 12 HNPCC kindreds. Seven of 33 patients (21%) with colorectal cancer aged 45 years or less had an RER + cancer, with only two of these having a clear family history of HNPCC. A greater proportion of RER + tumours (5/7) occurred proximal to the splenic flexure than RER - tumours (4/26; chi2 = 6.14, p < 0.025). RERs were detected in all 18 cancers from HNPCC patients but in only six of 65 non-HNPCC familial colorectal cancer kindreds (9%; chi2 = 52.2, p < 0.0005). These findings suggest that most cancers in patients diagnosed at 45 years of age or less and familial aggregations of colorectal cancer which do not fulfil HNPCC diagnostic criteria do not have germline mutations in MSH2 and MLH1. Hence population screening for germline mutations in these genes is unlikely to be an efficient strategy for identifying people at high risk of developing colorectal cancer.     

Sequence analysis suggests the presence of an IG-like domain in the N-terminal region of alpha-dystroglycan which was crystallized after mutation of a protease susceptible site (Arg168-->His)

PURPOSE: Retinal detachment after Nd: YAG laser vitreolysis is rarely reported. The pathogenic role of Nd: YAG laser is analyzed from 3 cases of severe retinal detachment. METHODS: Three men aged (40 to 59 years old) had one or more risk factors for retinal detachment: myopia, complicated cataract surgery, personal or family history of retinal detachment. Nd: YAG laser treatment onto vitreous strand was performed because of: retinal traction, repeated vitreous hemorrhage, anterior vitreous strand in a aphakic patient with cystoid macular edema. Within six weeks following Nd: YAG laser vitreolysis, severe retinal detachment with several tears occurred, complicated with proliferative vitreoretinopathy in two patients, and with choroidal hemorrhage in one. In two cases, several surgical procedures with silicon oil were required and visual acuity remained under 20/1000. In the last case retinal reattachment was obtained with scleral buckling and cryopexy, the patient recovered a visual acuity of 20/100 with a macular pucker. DISCUSSION: Retinal detachment occurred shortly after laser YAG vitreolysis. We assumed Nd: YAG laser was possibly responsible and investigated the possible mechanisms: direct retinal tear, vitreous strand traction, side effect of intraocular shock wave. CONCLUSION: Nd: YAG laser vitreolysis seems to be able to cause retinal detachment. Its use is therefore not recommended, particularly in patients at risk, especially since the efficacy of this treatment has not been proved.     

Immunohistochemical expression of the mucin-type glycoprotein A-80 and prognosis in human breast cancer

Immunohistochemical expression of the tumour associated mucin-type glycoprotein A-80 was investigated in a series of 173 breast cancer patients with a clinical follow-up between 13 and 19 years. A routine immunoperoxidase technique was used in formalin-fixed, paraffin-embedded surgical tumour specimens. One hundred and fifty of 173 tumours (87%) immunostained with MAb A-80. The degree of A-80 immunoreactivity was related to the tumour grade but not to lymph node status, tumour size, or nuclear DNA distribution pattern. In univariate analysis the degree of A-80 expression was found to be of significant prognostic value both in node negative and in node positive breast cancer patients (P = 0.03). Patients with non-A-80 immunoreactive tumours had significant longer distant metastases-free survival times and fewer relapses than women with carcinomas composed of A-80 immunoreactive tumour cells. This prognostic value was reduced in a multivariate analysis, including lymph node status, tumour size, and nuclear DNA distribution pattern, but retained borderline significance (P = 0.08). In conclusion, the findings of this study indicate that expression of the mucin-type glycoprotein A-80 as determined by immunohistochemistry seems to be related to clinical outcome in breast cancer patients.     

T helper-2 type of interleukins with de novo germinal center formation in the spleen during murine pregnancy

von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing those afflicted to hemangioblastomas of the central nervous system and the retina, renal cell carcinomas, pheochromocytomas, and pancreatic tumors. The disease has been associated with mutations of the VHL gene. The screening of 92 unrelated patients with VHL disease for point mutations in this gene revealed 61 DNA variants. In addition, a search for EcoR1 rearrangements revealed germline anomalies in 5 patients. The 61 variants could be subdivided in 20 mutations predicted to alter the open reading frame (8 nonsense mutations, 8 frame shift mutations, and 4 mutations in consensus splicing sites) and 43 DNA sequence variants of a priori unknown biological consequence (4 in-frame insertions or deletions, 36 missense mutations, and 3 apparently silent variations). The 3' end of the coding sequence of the VHL gene, which encodes the Elongin binding domain was the site of 5 of 20 truncating mutations (25%) and of 18 of 41 DNA variants (44%) causing uncertain functional impairment. A similar screening in 18 patients with sporadic hemangioblastoma revealed 2 missense DNA variants. In order to corroborate this latter observation, a systematic screening for germline alteration of the VHL gene might be performed in a larger series of sporadic hemangioblastoma. If this preliminary result is confirmed, more than 10% of sporadic hemangioblastoma might be related to a mild VHL disease, thus a follow-up program similar to that recommended in cases of VHL disease should probably be discussed in the corresponding families.     

Survey of subfertility patients attending a community clinic over a two year span, before and after national folate campaigns

TNF (tumour necrosis factor) is a new immunological anti-cancer agent which is too toxic for systemic use and relatively ineffective when used alone, but remarkably effective when used in closed circuit perfusion techniques together with other anti-cancer agents. Several studies have shown that when TNF is used with melphalan in closed circuit perfusion treatment for multiple melanoma metastases confined to a limb, a response rate of 80% can be achieved compared to a best response rate of 40% with melphalan alone. These findings confirm the difference in tumour responses which can be achieved with the appropriate use of regional chemotherapy in treatment of locally advanced tumours and the importance of surgical oncologists and vascular radiologists in maintaining and developing skills in integrated regional cancer treatment.     

Exposure of macrophage-like cells to titanium particles does not affect bone resorption, but inhibits bone formation

Endocrine tumours of the pancreas, even in case of liver involvement, are generally characterized by a slower evolution and a better prognosis, if compared with ductal carcinoma. This fact gives reason to a radical surgical approach, whenever possible, and to the research of any effective adjuvant treatment. For this purpose, hepatic transarterial chemoembolization (TACE) has been proposed in recent years for the treatment of metastatic endocrine tumours. Out of 80 patients suffering from endocrine tumours of the pancreas, observed between January 1985 and December 1996, 28 (35%) presented liver metastases at the time of diagnosis. Twelve of these patients were submitted to palliative resection of pancreatic tumour and one or more cycles of TACE. Overall survival was 50% (6/12); median survival was 35.4 months (range 4-75). These results suggest that chemoembolization, combined with surgical resection of primary malignancy, appears to be able to control the disease for a certain time and to increase the survival rate.     

CEA-immunoscintigraphy with 99m-technetium correlates with tumour cell differentiation in colorectal cancer

The clinical usefulness of immunoscintigraphy with the monoclonal anti-CEA (carcinoembryonic antigen) antibody BW431/26, directly labelled with 99m-Technetium in targeting colorectal carcinomas was investigated in 43 patients. In addition, tumour cell grading and CEA-expression were examined immunohistochemically. Best imaging results were obtained in pelvic tumour lesions (sensitivity 80%). Tumour grading correlated with radioimmunoimaging, well differentiated tumours being detectable at a higher rate (P = 0.09). Immunoscintigraphy preceded the findings of conventional diagnostic methods in 3 patients. In 4 cases immunoscintigraphy was decisive for patients management. Therefore, immunoscintigraphy with 99m-Technetium is valuable in directing patients management if conventional diagnostic methods remain undecisive.     

Variations in tumour oxygen tension (pO2) during accelerated radiotherapy of head and neck carcinoma

The study was performed to assess the effect of accelerated radiotherapy on oxygenation of primary tumours and metastatic nodes in patients with advanced head and neck tumours. In 14 patients with head and neck tumour, oxygen tension (pO2) was evaluated in normal tissues and tumours (primary tumour or metastatic neck node) before (0 Gy) and after 2 weeks (32 Gy) of accelerated radiotherapy (70 Gy in 3.5 weeks, with three daily fractions). Radiotherapy was combined with carbogen breathing in 5 patients. pO2 was measured using a polarographic technique. For pooled normal tissues, median pO2 was 38 mmHg before treatment and 46 mmHg after 2 weeks. For tumours, very low values (< 2 mmHg) represented 20% of the recorded values before treatment and 10% after 2 weeks. The relative increase in tumour oxygenation was more pronounced for primary tumours (median pO2 12 mmHg before treatment versus 26 mmHg after 2 weeks, P < 0.05) than for metastatic nodes (respectively, 20 and 27 mmHg P = 0.1). For the 5 patients who breathed carbogen during accelerated radiotherapy, the median pO2 was 44 mmHg at 2 weeks, compared with 13.5 mmHg before treatment (P = 0.05). Very low pO2 values, corresponding to tumour hypoxia, were found in the tumours (primary and metastatic neck nodes) prior to accelerated treatment. During the first 2 weeks of accelerated treatment, an increase in median pO2 was found in nine of the 14 tumours, together with a decrease in the frequency of very low values.     

Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite

Decreased retinal blood flow has been measured in streptozotocin (STZ)-induced diabetes of 1 week's duration, and primary insulin intervention was effective in maintaining normal retinal blood flow in diabetic rats. Retinal blood-flow abnormalities precede clinical diabetic retinopathy in both diabetic animals and patients. An important characteristic of diabetic retinopathy is the difficulty of reversibility once it has been established. Because altered retinal hemodynamics is a possible marker of early diabetic retinopathy, we investigated in this study whether retinal blood-flow changes in rats can be normalized by secondary insulin intervention following short and chronic periods of untreated STZ-induced diabetes. Subcutaneous insulin pumps were placed into diabetic rats for 1 week after 1 week of diabetes (2-week group) and after 3 weeks of diabetes (4-week group). Retinal circulatory parameters were determined using image analysis of video fluorescein angiogram recordings. For the 2-week group, retinal blood flow was significantly (P < 0.05) reduced in the untreated diabetic rats compared with nondiabetic and insulin-treated diabetic rats (80.6+/-29.2, 131.9+/-50.1, and 151.3+/-54.0 pixels2/s respectively). Retinal blood flow was also significantly (P < 0.05) reduced in the 4-week untreated diabetic rats compared with nondiabetic rats (95.7+/-22.2 vs. 125.7+/-29.5 pixels2/s). In contrast to the shorter-duration group, insulin treatment for 1 week after 3 weeks of diabetes did not totally normalize retinal blood flow (117.5+/-32.4 pixels2/s). These results suggest that vascular abnormalities could become more resistant to normalization following short-term (1 week) insulin treatment after longer periods of untreated diabetes.     

Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes

OBJECTIVE: von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia type 2 (MEN2) are autosomal dominant cancer syndromes. In both conditions, phaeochromocytoma is a prominent feature. It has recently been suggested that phaeochromocytoma can be the presenting and sole clinical manifestation of these multi-organ syndromes. The aim of this study was to ascertain the incidence of VHL and MEN2 among patients with sporadic phaeochromocytoma by mutational analysis. PATIENTS: Twenty-seven unrelated patients with biochemically and/or anatomically proven sporadic phaeochromocytoma were evaluated. DESIGN AND MEASUREMENTS: Constitutional DNA obtained from the patients was analysed by single stranded conformational analysis (SSCP) for mutations within the VHL gene coding sequence and by denaturing gradient gel electrophoresis (DGGE) for predominant mutations in exons 10, 11 and 16 of the RET proto-oncogene. The incidence of patients positive for either VHL or RET germline mutations was assessed. RESULTS: Twenty-six of 27 patients had normal SSCP patterns in all three VHL gene exon segments and only one patient, with an atypical clinical presentation, had an aberrant pattern in exon 3 which upon DNA sequencing was shown to harbor a G to A transversion mutation at nucleotide 695. All patients had normal RET exon 10, 11 and 16 DGGE migration patterns. CONCLUSION: Most, if not all, patients with typical unilateral sporadic phaeochromocytoma do not have von Hippel-Lindau disease or MEN2. Thus, clinical and/or molecular investigation for von Hippel-Lindau disease and MEN2 in this patient population does not appear to be indicated.     

BRCA1 mutations found in archived early onset breast tumours

Inherited mutations in the BRCA1 gene are thought to account for approximately 5% of breast cancers in women under the age of 45 years. In order to determine whether mutations could be found at the expected frequency, 60% of the protein coding region of BRCA1 was screened in 75 archived early-onset breast tumours, taken from women under 45 years of age. Two of the 75 tumours (2.7%) had detectable mutations, in close agreement to that predicted. Since BRCA1 mutations found in breast tumours are invariably germline, two immediate consequences are apparent. Firstly, family members of affected patients are likely to carry mutations as well, and should be considered for BRCA1 screening; and secondly, persons harbouring a germline BRCA1 mutation should be examined frequently and indefinitely for new primary tumours in remaining breast tissue.     

Screening for tumour suppressor p16(CDKN2A) germline mutations in Israeli melanoma families

Approximately ten percent of patients with malignant melanoma have family histories of the disease, suggesting a genetic predisposition. Germline mutations in tumour suppressor p16 gene have been implicated as disease causing mutations in some of the melanoma families. The frequency of families with p16 germline mutations among melanoma prone families varies from eight to fifty percent. The range of the variability is influenced apparently by the number of melanoma affected individuals within the family, as well as by other, yet unidentified factors. Ethnic background is known to determine both the frequency and the nature of germline alterations. Recently, specific mutations in tumour suppressor genes involved in breast cancer and in colon cancer were found at elevated frequency among Ashkenazi Jews. This report describes results of a screening for p16 germline alterations in a collection of Israeli melanoma families. We have analyzed genomic DNA from thirty one Ashkenazi and non-Ashkenazi Jewish melanoma families, as well as from thirty melanoma patients without an apparent family history of the disease. The entire coding region of the p16 gene was screened by single strand conformation polymorphism analysis and direct DNA sequencing. We have detected a number of carriers with the Ala148 Thr polymorphism at the end of the second exon and several instances of 500(G=>C) substitution at the 3' untranslated portion of the gene.     

Reflection measurement during retinal laser coagulation in patients. Development of an automatically controlled dosimeter

PURPOSE: Retinal laser coagulation has limited reproducibility, because every laser exposition is unpredictably affected by pigmentation and media opacities. This can lead to complications. A feedback-controlled dosimeter would make retinal laser treatment a safer, more reproducible and faster procedure. METHOD: A reflectometer was developed that allows monitoring of the reflection of laser light during standard photocoagulation. Hundreds of coagulations in rabbit eyes and about 12,000 coagulations in patients undergoing routine laser treatment were recorded. RESULTS: The results of the first reflectance measurements in humans are presented along with extensive animal studies. A typical reflectance history was found for different lesion intensities. The degree of retinal whitening correlates with reflectance characteristics. These characteristics allow an early prediction of lesion intensity. Motion during laser exposure affects each coagulation in a fashion similar to spot size and exposure time. CONCLUSION: Reflectometry is the currently most effective means of monitoring lesion development. An automatic dosimeter for retinal photocoagulation seems feasible.     

Metabolism and acid-base status during hypoxic ventilatory depression

In the present study, leukocyte DNA from 143 patients with familial clustering of breast and/or ovarian cancer and tumour DNA from 96 breast carcinomas were screened for base mutations in the estrogen receptor gene (ESR). Three patients with a family history of cancer were carrying a Gly160Cys germline substitution. This alteration was also detected in eight (four females and four males) of 729 controls (366 female, 363 males), indicating that the substitution probably represents a polymorphism. However, in the 229 female controls in whom family history of cancer was known, one of two who had a sister with breast cancer was carrying the variant allele. Hence, a possible clinical significance of the glycine into cysteine cannot be completely ruled out and should be further investigated. Somatic mutations were not detected in any of the tumours studied, and the present data do not provide support for somatic ESR base mutations as an important mechanism for hormonal therapy resistance in estrogen receptor-positive breast carcinomas.     

201Tl single photon emission tomography in the evaluation of residual and recurrent astrocytoma

Twenty-five patients with malignant astrocytoma, either postoperatively (15 cases) or with recurrent tumour versus gliosis (10 cases) were included in this study. 201Tl single photon emission computed tomography (SPECT) was performed with the calculation of early and delayed uptake values and retention index. A high mean value of early and delayed uptake correlated with a low retention index in patients with high-grade astrocytoma in both postoperative residual and recurrent groups, versus a lower mean value of early and delayed uptake with a high retention index in patients with low-grade tumours. All postoperative cases with high-grade astrocytoma had high 201Tl uptake > 1.5, whereas 66.6% of cases with low-grade astrocytoma had low 201Tl uptake < 1.5. There was a correlation between retention index of 201Tl and tumour grade with r = 0.47. Also, recurrent cases showed remarkable differences in early and delayed 201Tl uptake (P < 0.05) and retention index (P < 0.001) compared with postradiation gliosis. There was a higher sensitivity in detection of tumour viability by 201Tl SPECT of 100% versus 80% using computed tomographic scanning and in the differentiation between recurrent tumour and postradiation gliosis.