The spectrum of RB1 germ-line mutations in hereditary retinoblastoma.

We have searched for germ-line RB1 mutations in 119 patients with hereditary retinoblastoma. Previous investigations by Southern blot hybridization and PCR fragment-length analysis had revealed mutations in 48 patients. Here we report on the analysis of the remaining 71 patients. By applying heteroduplex analysis, nonisotopic SSCP, and direct sequencing, we detected germ-line mutations resulting in premature termination codons or disruption of splice signals in 51 (72%) of the 71 patients. Four patients also showed rare sequence variants. No region of the RB1 gene was preferentially involved in single base substitutions. Recurrent transitions were observed at most of the 14 codons within the RB1. No mutation was observed in exons 25-27, although this region contains two CGA codons. This suggests that mutations within the 3'-terminal region of the RB1 gene may not be oncogenic. When these data were combined with the results of our previous investigations, mutations were identified in a total of 99 (83%) of 119 patients. The spectrum comprises 15% large deletions, 26% small length alterations, and 42 % base substitutions. No correlation between the location of frameshift or nonsense mutations and phenotypic features, including age at diagnosis, the number of tumor foci, and manifestation of nonocular tumors was observed.

Molecular analysis and predictive testing in retinoblastoma.

Predictive testing using molecular analysis is an integral part of contemporary retinoblastoma management. We have made extensive use of segregation analysis for risk assessment in both familial and sporadic disease. Investigation of loss of heterozygosity in tumor samples proved to be invaluable for the identification of linkage phase. In many families, however, accurate carrier risk assessment depends on direct identification of the causative R B I mutation. Consequently, we have developed methods for rapid mutation screening. Using these techniques, mutation analysis can now be offered to an increasing number of individuals.

Spectrum of small length germline mutations in the RB1 gene.

A screening method based on multiplexed automated fragment length analysis of polymerase chain reaction products was used to identify germline mutations in the RB1 gene. By screening 106 unrelated patients with hereditary retinoblastoma, 20 small deletions (1-18 bp) and seven insertions (1-5 bp) were identified. When collating our data with reported mutations, recurrence of small length mutations was observed at nine sites within the RB1 gene. Most of these contained monotonic runs or direct repeats embedded in homocopolymer tracts. While the majority of mutations resulted in premature truncation, two mutations caused an in-frame loss of F755 and G540 to E545, respectively. A genotype-phenotype comparison of patients carrying different small length mutations did not reveal any consistent relation. Particularly, the two patients with in-frame mutations showed a high number of tumours consistent with regular-penetrance retinoblastoma.

Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma.

The retinoblastoma susceptibility (RB1) gene contains an unmethylated CpG-rich island at its 5' end. Using methylation-sensitive restriction enzymes, we have investigated the methylation status of this island in 21 sporadic unilateral retinoblastomas and 30 hereditary retinoblastomas. Three sporadic unilateral tumors were found to have hypermethylated RB1 alleles. In two tumors, the paternal allele was methylated, whereas the maternal allele had been lost. Cultured cells from one of these tumors were studied by the reverse transcription polymerase chain reaction and found to have a reduced level of RB1 mRNA. The third tumor had retained constitutional heterozygosity, and the paternal allele was specifically methylated. The combined data from previously published reports and from this study show that hypermethylation of the RB1 gene occurs in 13% of sporadic unilateral tumors and may reduce gene activity.

Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma.

The interfamilial diversity in penetrance and expressivity of hereditary retinoblastoma was investigated in 29 families. By using a simple parameter for estimating the severity of the disease (diseased-eye-ratio), we were able to identify four families with a discrete low-penetrance phenotype. The underlying genetic defect was identified in three families. One family has a 3-bp deletion in exon 16 that results in the deletion of Asn480. In two further unrelated families, the identical missense mutation at codon 661 in exon 20 (CGG to TGG, Arg to Trp) was identified. These mutations are distinct from the majority of retinoblastoma gene alterations, as they do not result in the disruption of the gene product. We propose that reduced penetrance of retinoblastoma is the result of a residual function of these alleles in retinoblastoma precursor cells.

[Risk of metastasis in retinoblastoma]. / Das Metastasierungsrisiko beim Retinoblastom.

580 patients with retinoblastoma were analysed retrospectively. Median follow up was 5.5 years. 41 patients developed metastases within 5 years. Clinical and histopathological risk factors were analysed by univariate tests with regard to the occurrence of metastases. Significant variables were then reevaluated using Cox proportional hazards method. Four factors correlated independently with the development of metastases: optic nerve invasion with involvement of the resection line, optic nerve invasion without involvement of the resection line, choroidal invasion and enucleation of an affected eye more than 120 days after initial diagnosis. The regression coefficient beta of the Cox-model was used for a score classification with groups of high, medium and low metastatic risk. The 5-year incidence of metastases was 68%, 43% and 4% respectively.

Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma.

DNA samples from 92 unrelated patients with bilateral retinoblastoma were analyzed by Southern blot hybridization with cDNA and genomic clones of the retinoblastoma (RB-1) gene. Qualitative and quantitative evaluation of the Southern blot patterns showed a deletion of all or part of the RB-1 gene in 15 patients. Deletion hot spots were not detected. The study shows that 16% of germ cell mutations are detectable by Southern blot hybridization, but that densitometric analysis is required in most cases.

Risk factors for metastases in patients with retinoblastoma.

The study is based upon a review of data from 583 consecutive patients with retinoblastoma over the years 1956 to 1986. Mean follow-up was 8 years, and median was 5.5 years. In 41 patients, metastases developed within 5 years. The influence of clinical and histopathologic risk factors on the occurrence of metastases was first analyzed by univariate tests. Significant variables were then reevaluated using the Cox proportional hazards method. Four factors were found to be independently associated with the development of metastases: optic nerve invasion with and without involvement of the resection line, choroidal invasion and enucleation of an affected eye more than 120 days after initial diagnosis. The 5-year metastatic risks associated with these factors were 67%, 13%, 8%, and 4%, respectively. The relative risk estimate, calculated from the Cox model, was used for a score classification with groups of low, medium, and high metastatic risk. The 5-year incidence of metastases was 4%, 43%, and 68%, respectively.

Long-term treatment effects in patients with bilateral retinoblastoma: ocular and mid-facial findings.

A total of 99 patients with bilateral retinoblastoma who had been treated between 1965 and 1982 were reexamined in April 1988 to study the late effects of treatment. Their median age at the follow-up visit in 1988 was 16 years (range, 6-27 years), and the median follow-up was 15 years (range, 6-26 years). All patients underwent a full eye examination, morphometric measurements of the mid-face and genetic counselling. Each eye or orbit and the corresponding side of the patient's mid-face were evaluated separately, resulting in 198 data sets from 99 individuals. Subjects were divided into four treatment groups according to whether photo- and cryo-coagulation, enucleation, radiation therapy or various combinations thereof were used. In all, 81 eyes had a visual acuity of greater than 0.4 (in 23 of these, however, only with low-vision aids). Within a dose range of 36-51 Gy, the location of the tumor (36%) or cataract (15%) were the main factors responsible for poor visual acuity, whereas radiation retinopathy and/or optic neuropathy occurred in only three cases. Cataracts were more frequently observed following orthovoltage as compared with megavoltage therapy (P = 0.012). A total of 72 eyes had been enucleated and had not received radiation therapy at any time. Cosmetic results (as measured by several parameters) in these cases were significantly better then those in 28 subjects who underwent combined radiation therapy and enucleation. As defined by various subjective as well as objective findings, mid-facial hypoplasia occurred significantly more often following orthovoltage as compared with megavoltage therapy.

Use of the RB1 cDNA as a diagnostic probe in retinoblastoma families.

Use of an intragenic BamHI restriction fragment length polymorphism within the 5' end of the retinoblastoma gene (RB1) provided improved genetic counselling for five familial and ten non-familial retinoblastoma patients and their relatives. All other polymorphic probes within RB1 were uninformative in three families, and accuracy of diagnosis was improved by use of this polymorphism in two families. In 10/14 informative constitutional DNA-RB tumor DNA pairs, a reduction to homozygosity allowed identification of the RB1 allele at risk to carry a germline RB1 mutation.

New and recurrent tumor foci following local treatment as well as external beam radiation in eyes of patients with hereditary retinoblastoma.

The study is based on a retrospective analysis of data from 200 patients with hereditary retinoblastoma. Apart from four unilateral cases with a positive family history all patients had bilateral disease; 229 appeared suitable for conservative treatment while 167 eyes had to be enucleated. Primary local treatment was performed in 102 eyes, while 127 eyes received primary external beam radiation using a linear accelerator. Follow-up was adjusted by life tables, with 75% of the patients being observed longer than 2 years. Using life-table statistics new and recurrent tumors were found in 41% of all patients and in 95% of these cases they were noted within 26 months after initiation of therapy. Among eyes treated with primary external beam radiation new and recurrent tumors were observed significantly less frequently when 50 Gy (22% compared with 49% after 40 Gy) and a highly accurate beam alignment technique (22% compared with 48% after alignment to the outer bony canthus) were applied. The incidence of recurrent tumors following primary local treatment did not differ significantly with regard to the application of either photo- or cryocoagulation (28% vs 33%), while no recurrence was observed among tumors that were suitable for primary treatment using ruthenium or cobalt plaques.

Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma.

Epigenetic models for tumor formation assume that oncogenic transformation results from changes in the activity of otherwise normal genes. Since gene activity can be inhibited by DNA methylation, and inactivation of tumor suppressor genes is a fundamental process in oncogenesis, we investigated the methylation status of the retinoblastoma suppressor gene (RB gene) on chromosome 13, in blood and tumor cells from 21 retinoblastoma patients. Using methylation-sensitive restriction enzymes and a cloned DNA probe for the unmethylated CpG island at the 5' end of RB gene, we obtained evidence of hypermethylation of this gene in a sporadic unilateral retinoblastoma tumor. The closely linked esterase D gene and a CpG-rich island on chromosome 15 were not affected. We suggest that changes in the methylation pattern of the RB gene play a role in the development and spontaneous regression of some retinoblastoma tumors.

Application of linkage analysis to genetic counselling in families with hereditary retinoblastoma.

Six families with retinoblastoma in more than one member were investigated with DNA markers linked to the retinoblastoma locus because direct analysis had not disclosed the gene defect. In all of the families we could identify the affected chromosome and predict the genetic risk with a high level of confidence (90 to 99%). In one patient the test helped to detect tumour development earlier than usually possible. Several subjects were found not to carry a mutation, thus obviating frequent ophthalmological examinations under anaesthesia as would be necessary otherwise. These results show that linkage analysis can be successfully applied to genetic counselling in families with hereditary retinoblastoma.

[Non-ocular, malignant secondary tumor following spontaneous healing of a retinoblastoma ("retinoma", "retinocytoma")]. / Nicht-okulärer, maligner Zweittumor nach Spontanheilung eines Retinoblastoms ("Retinom", "Retinozytom").

In a 47-year-old patient, the father of a child with bilateral retinoblastoma, three retinomas or retinocytomas were observed in the right eye, which had never been treated, in the course of a routine checkup. The left eye had been enucleated at age three-and-a-half because of a tumor of unknown etiology. At age 55 the patient died of lung cancer. The autopsy revealed a metastasized small-cell bronchial carcinoma. Histopathological examination of the right eye showed, in the region where the tumors had been observed clinically, merely disorganized retinal tissue with proliferations of pigment epithelium in some areas, and well-differentiated retinoblastomas in others. Tumor regression and differentiation appear to be the mechanisms responsible for spontaneous healing of the retinoblastoma. This case also emphasizes the threat posed by non-ocular secondary tumors to carriers of retinoblastoma genes.

Detection of submicroscopic deletions and a DNA polymorphism at the retinoblastoma locus.

DNA samples from 60 unrelated patients with retinoblastoma were screened by Southern blot hybridization using two probes that are closely linked to the retinoblastoma locus within human chromosome band 13q14. Seven of 44 patients with bilateral or multifocal unilateral retinoblastoma and one patient with unifocal unilateral retinoblastoma were found to have a heterozygous deletion for the anonymous DNA sequence H3-8. Three of the eight deletions did not include the esterase D locus and were undetectable by conventional cytogenetic analysis. The findings are compatible with the deletions being the cause of retinoblastoma in these cases and provide a basis for DNA diagnosis in nearly 20% of patients with bilateral and multifocal unilateral retinoblastoma. The H3-8 probe also detects a restriction fragment length polymorphism that is a useful genetic marker in some families.

The prognosis of retinoblastoma in terms of globe saving treatment. A computer assisted study. Part I.

At present there is no satisfactory classification or staging system. In order to compare the Reese classification (Reese & Ellsworth, 1963), the Essen classification (Höpping, 1983) and a new classification, a method of evaluating different parameters was created to get an optimal classification of globe saving treatment. A computer score table for the Essen eye clinic was established and this table could be valuable for all clinics using modern methods of treatment.

The prognosis of retinoblastoma in terms of survival. A computer assisted study. Part II.

At present there is no satisfactory classification or staging system. In order to compare the Reese classification (Reese, 1963), the Essen classification (Höpping, 1983) and a new classification, a method of evaluating different parameters was created in order to get an optimal classification of survival.

[Value of ophthalmoscopy and histology for the prognosis of patients with retinoblastoma]. / Die Wertigkeit von Ophthalmoskopie und Histologie für die Prognose der Patienten mit Retinoblastom.

The predictive value of prognostic factors is of utmost importance for the treatment of patients with retinoblastoma. Functional results, that may be expected following sight saving therapy, can be deducted from ophthalmoscopic findings. Large tumors, tumors located centrally, the presence of extensive retinal detachment or diffuse vitreous seeding are poor prognostic signs. Histopathologic findings of the enucleated eye harbouring retinoblastoma correlate well with the incidence of metastatic disease. Patients in which the tumor has invaded the choroid, optic nerve or sclera have a high risk of developing metastases.