Jennifer Varley

Are there low-penetrance TP53 Alleles? evidence from childhood adrenocortical tumors.

We have analyzed a panel of 14 cases of childhood adrenocortical tumors unselected for family history and have identified germline TP53 mutations in >80%, making this the highest known incidence of a germline mutation in a tumor-suppressor gene in any cancer. The spectrum of germline TP53 mutations detected is remarkably limited. Analysis of tumor tissue for loss of constitutional heterozygosity, with respect to the germline mutant allele and the occurrence of other somatic TP53 mutations, indicates complex sequences of genetic events in a number of tumors. None of the families had cancer histories that conformed to the criteria for Li-Fraumeni syndrome, but, in some families, we were able to demonstrate that the mutation had been inherited. In these families there were gene carriers unaffected in their 40s and 50s, and there were others with relatively late-onset cancers. These data provide evidence that certain TP53 alleles confer relatively low penetrance for predisposition to the development of cancer, and they imply that deleterious TP53 mutations may be more frequent in the population than has been estimated previously. Our findings have considerable implications for the clinical management of children with andrenocortical tumors and their parents, in terms of both genetic testing and the early detection and treatment of tumors.

Cancer phenotype correlates with constitutional TP53 genotype in families with the Li-Fraumeni syndrome.

The Li–Fraumeni cancer predisposition syndrome is associated with germline TP53 mutations in the majority of families. We have investigated cancer incidence in 34 Li–Fraumeni families, according to their constitutional TP53 mutation status. Families with germline missense mutations in the core DNA binding domain showed a more highly penetrant cancer phenotype than families with other TP53 mutations or no mutation. Cancer phenotype in families carrying such mutations was characterized by a higher cancer incidence and earlier ages at diagnosis, especially of breast cancer and brain tumours, compared with families carrying protein truncating or other inactivating mutations (P=0.03 for all cancers, P=0.006 for breast cancers, P=0.05 for brain tumours). Proband cancers showed significantly younger ages at diagnosis in those with missense mutations in the DNA binding domain than in those with protein inactivating mutations (P=0.031). In individuals with the former type of mutation, there was a significantly lower proportion of tumours which showed loss of the wild-type TP53 allele (P=0.004). These results are consistent with observations in experimental systems which demonstrate that certain mutations exhibit gain of function and/or dominant-negative properties. Our results support an enhanced oncogenic potential for such mutations in human populations.

Identification of a novel fusion gene involving hTAFII68 and CHN from a t(9;17)(q22;q11.2) translocation in an extraskeletal myxoid chondrosarcoma.

A proportion of extraskeletal myxoid chondrosarcomas (EMC) have been shown to have a characteristic translocation t(9;22)(q22;q12) involving the EWS gene at 22q12 and the CHN orphan nuclear receptor gene at 9q22. This translocation appears to be largely specific for EMC, but has not been detected in all such tumours. We report here a case of EMC with a t(9;17)(q22;q11.2) as the sole chromosome abnormality. We have determined that the translocation results in the fusion of the entire coding region of CHN to the N-terminal transactivation domain of RBP56/hTAFII68. This is the first report of a translocation involving RBP56/hTAFII 68, a protein with sequence homology to both EWS and TLS/FUS. The involvement of RBP56/hTAFII68 may explain some unusual features of the tumour.

Comparative genomic hybridisation of ductal carcinoma in situ of the breast: identification of regions of DNA amplification and deletion in common with invasive breast carcinoma.

Comparative genomic hybridisation has been used to map copy number changes in nine cases of ductal carcinoma in situ of the breast obtained from wax-embedded archive material. A wide variety of abnormalities were detected including gain of regions of 1q, 17q, 19q, 20p and 20q and loss on 13q, 14q, 17p, 16q and 22q. Amplification of areas on 10p, 8q and 20q were also observed. Chromosomal alterations were more frequent in higher grade DCIS and closely resemble those previously detected in invasive breast cancer using the same technique. These data provide strong molecular support for the view that DCIS is a precursor lesion of invasive breast carcinoma.

A detailed study of loss of heterozygosity on chromosome 17 in tumours from Li-Fraumeni patients carrying a mutation to the TP53 gene.

We have studied a total of 36 tumours from 28 patients with germline mutations to the TP53 gene for loss of heterozygosity at TP53 using techniques of both direct sequencing and restriction fragment length polymorphism analysis. All patients were from families conforming to the definition of classical Li – Fraumeni syndrome (LFS) or were Li – Fraumeni-like (LFL). The data we have obtained show that loss of the wild-type TP53 gene is observed in under half (44%) of all tumours, and that the pattern of LOH at TP53 may be mutation specific. LOH has been observed in premalignant as well as invasive tumours. Two tumours (6%) show loss of the mutant allele and retention of the wild-type. To confirm that TP53 is indeed the target for LOH events on chromosome 17, we have used additional microsatellite repeats to examine patterns of allelic imbalance along the length of chromosome 17. Data from this analysis indicate that TP53 is the target of loss, but reveal some other interesting patterns of allelic imbalance at other loci on chromosome 17.

Patterns of silver staining of human chromosomes

The chromosomes of twenty individuals with normal karyotypes were studied to determine the patterns of staining with the Ag-AS technique. These patterns were shown to be variable from one individual to another, but characteristic and constant within each individual. In addition, one patient with chronic lymphocytic leukemia was studied, and shown to have an Ag-AS staining pattern that was distinctly different from that of normal subjects.

Absence of mutations in the ATM gene in breast cancer patients with severe responses to radiotherapy

The effectiveness of cancer radiotherapy is compromised by the small proportion (approximately 5%) of patients who sustain severe normal tissue damage after standard radiotherapy treatments. Predictive tests are required to identify these highly radiosensitive cases. Patients with the rare, recessively inherited, cancer-prone syndrome ataxia-telangiectasia (A-T) sustain extremely severe normal tissue necrosis after radiotherapy and their cultured cells are also highly radiosensitive. Clinically normal carriers (heterozygotes) of the A-T gene have an increased risk of breast cancer, account for approximately 4% of all breast cancer cases and show a modest increase in cellular radiosensitivity in vitro. It has been suggested that a substantial proportion of highly radiosensitive (HR) breast cancer patients may be A-T heterozygotes, and that screening for mutations in the A-T gene could be used as a predictive test. We have tested this hypothesis in a group of cancer patients who showed adverse reactions to radiotherapy. Sixteen HR breast cancer patients showing mainly acute reactions (and seven HR patients with other cancers) were tested for ATM mutations using the restriction endonuclease fingerprinting assay. No mutations typical of those found in obligate A-T heterozygotes were detected. If the estimate that 4% of breast cancer cases are A-T gene carriers is correct, then ATM mutations do not confer clinical radiosensitivity. These early results suggest that screening for ATM mutations in cancer patients may not be of value in predicting adverse reactions.

Cytological evidence of transcription of highly repeated DNA sequences during the lampbrush stage in Triturus cristatus carnifex

Highly repeated, or satellite, DNA fractions have been isolated from total Triturus cristatus carnifex DNA by renaturation kinetics, caesium salt centrifugation and restriction endonuclease digestion. We have shown by DNA/DNA in situ hybridisation and autoradiography that all of these probes bind to C-band positive regions on mitotic or lampbrush chromosomes of T.c. carnifex. Under conditions of DNA to RNA-transcript in situ hybridisation labelled satellite DNA binds to nascent RNA transcripts that are still associated with the DNA axes of many lampbrush loops. The majority of the loops that label heavily in these experiments are located on the long arms of chromosome I, a region previously shown to be rich in highly repeated DNA and to have many of the properties of heterochromatin. These satellite DNA probes also label many loops on a comparable chromosome region in T. marmoratus, a species closely related to T. cristatus. However, in DNA/RNA-transcript hybrids to other more distantly related species of Triturus, there are no chromosome regions that have the same concentration of labelled loop pairs as the long arms of T.c.carnifex and T. marmoratus, although some loop pairs do label. We have cloned two satellite sequences in pBR322, and have obtained the same results using these pure probes as we obtained using satellite probes isolated by other techniques. These results demonstrate unequivocally that satellite DNA is transcribed on lampbrush chromosomes during oogenesis in crested newts.

Extraskeletal Myxoid Chondrosarcoma With Neuroendocrine Differentiation: A Pathologic, Cytogenetic, and Molecular Study of a Case With a Novel Translocation t(9;17)(q22;q11.2)

A case of extraskeletal myxoid chondrosarcoma (EMC) in which there was histochemical, immunohistochemical, and ultrastructural evidence of neuroendocrine differentiation is reported. Genetic investigations showed the recently described novel translocation t(9;17)(q22;q11.2) and associated fusion of the CHN and RBP56 genes, contrasting with the translocation t(9;22)(q22;q12) and EWS/CHN gene fusion found in the majority of EMCs.

Genetic and functional studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family.

We report an extensive Li–Fraumeni-like family in which there is an unusual spectrum of tumours at relatively late onset. A germline TP53 splice donor mutation in exon 4 is present in all affected family members available for testing. The mutation abolishes correct splicing of intron 4 and techniques of RT–PCR have identified three different aberrant transcripts from the mutant TP53 allele. Using the yeast functional assay to analyse transcripts in cells from a number of family members with the mutant allele, TP53 appears wild-type. Functional studies have been carried out on cells from patients with and without cancer who carry the germline mutation, and on cells from unaffected individuals from the same family who do not carry the mutation. Using a number of functional endpoints known to distinguish between cells carrying mutant or wild-type TP53 alleles, we were unable to discriminate normal (wt/wt) from heterozygous (wt/mut) cells by lymphocyte apoptosis and fibroblast survival following low dose rate ionising radiation exposure. However germline mutation carriers show increased sensitivity to radiation-induced chromosome damage in the G2 phase of the cell cycle, and decreased transient and permanent G1 arrest. These studies demonstrate the importance of fully characterising the effects of TP53 germline mutations, and may explain some of the phenotypic features of this family.