Kathrin Radig

MDM2 amplification and loss of heterozygosity at Rb and p53 genes: no simultaneous alterations in the oncogenesis of liposarcomas.

Purpose: The present study aimed to investigate the status of alterations of the MDM2, Rb and p53 genes in a series of 45 liposarcomas. Furthermore, the possible correlation with histological and clinical parameters was studied. Methods:MDM2 amplification was examined by non-radioactive Southern blot hybridization with a human MDM2 cDNA probe. Mutations in the p53 gene were screened by polymerase chain reaction/single-strand conformation polymorphism analysis and direct sequencing. To study loss of heterozygosity (LOH) at the tumor-suppressor genes Rb and p53, we used four polymorphic intragenic Rb markers (introns 1, 17, 20, and 25) and two p53 markers (intron 1 and exon 4). Results:MDM2 amplification was found in 19 of 45 liposarcomas (42.2%). The frequency of LOH in Rb and p53 was nearly identical (22%). In 4 of 9 tumors (44.4%) with LOH, allelic loss was a concurrent event in both genes. Of 45 liposarcomas, 6 (13.3%) showed p53 mutations. Overall, alterations of the p53/MDM2/Rb pathway occurred in 30 of 45 liposarcomas (66.6%). In contrast to myxoid and pleomorphic variants, well-differentiated liposarcomas were characterized by a high frequency of MDM2 amplification, a lack of LOH of Rb and p53, and p53 mutations. Conclusions: Obviously MDM2 amplification and LOH at the Rb and p53 genes do not occur simultaneously in the oncogenesis of liposarcomas, as is the case for MDM2 amplification and p53 gene mutations (with one exception). We suggest that well-differentiated, myxoid and pleomorphic liposarcomas are characterized by a different pattern of molecular alterations.

Clinicopathologiclmplications of MDM2, p53 and K-ras gene alterations in osteosarcomas: MDM2 amplification and p53 mutations found in progressive tumors

It is widely recognized that various oncogenes and tumor suppressor genes contribute to tumorigenesis and progression of osteosarcomas. However, whether genetic alternations enable us to predict the prognosis of patients with osteosarcomas is unclear. Southern blotting and polymerase chain reaction/single strand conformation polymorphism (PCR-SSCP) analyses were performed to search for MDM2, ras family and p53 gene alterations in 17 patients with high-grade osteosarcomas. Amplification of the MDM2 gene was found in three tumors, two of which were obtained from a regional lymph node metastasis and the other from a locally advanced lesion. Point mutations of the p53 gene were found in exons 4 and 5 in two tumors each. One of the four tumors with p53 mutations was obtained from a lymph node metastasis, one from a recurrent tumor and another from the primary tumor of a patient who developed lung metastases. Coexistence of MDM2 amplification with point mutation of the p53 gene was observed in two tumors. A point mutation of the K-ras oncogene was detected at codon 13 in two tumors. MDM2 amplification and p53 mutation may reflect tumor progression, although no correlation between alteration and response to chemotherapy or patient survival was demonstrated.

p53 gene mutations in soft-tissue sarcomas - correlations with p53 immunohistochemistry and DNA ploidy

The significance ofp53 mutations in a group of 67 soft-tissue tumors was examined using single-strand conformation polymorphism and direct sequencing analysis. Molecular findings were correlated with immunohistochemical detection of thep53 protein and DNA ploidy status. Mutations of thep53 gene were detected in 13 (19.5%) out of 67 cases of soft-tissue tumors. Only there were localized outside the conservative regions of thep53 gene. Six mutations were described for the first time in these tumors. Most of the mutations were point mutations in exons 5–8 and, in one case, a deletion at the 3′-splice site of exon 5 could be demonstrated. There was no significant correlation between the occurrence ofp53 mutations and the histological grade, although a high number of mutations were defined in poorly differentiated tumors (grade 3). Molecular finding of ap53 gene mutation and immunohistochemical detection ofp53 expression did not correlate, which may be due to the high percentage of nonsense mutations in our study (50%). We confirm that only DNA sequencing allows a unique identification and differentiation of mutations in thep53 gene. Other factors may be responsible for the detection ofp53 protein in many cases. Histological grade correlated with aneuploidy. The frequency of mutations observed was in accordance with values quoted in the literature. Generally,p53 mutations andp53 overexpression are more likely to represent a late event in the oncogenesis of soft-tissue tumors.

On telomere shortening in soft-tissue tumors.

Abstract Purpose: Specific simple DNA repeats occur at the telomeric ends of mammalian chromosomes. Loss of (G+C)-rich repeats can result in genetic instability, associated with tumorigenesis. So far, data on telomere shortening have not been available for different types of soft-tissue tumors. Methods: Using tumor material and the blood of the corresponding patient, high-molecular-mass DNA was prepared by digestion with proteinase K and extraction with phenol/chloroform. A 10-μg sample of DNA was digested with the restriction enzyme HinfI. DNA fragments were separated in a 0.7% agarose gel, and in-gel hybridization was performed with the telomere-specific repeat probe (TTAGGG)3. Results: Shortening of the telomere repeat was observed in 14/30 soft-tissue tumors; 5 tumors showed elongated telomere repeats, whereas the telomeres appeared unchanged in 11 tumors. Decreased telomere repeat length correlated with advanced age, DNA ploidy, and a higher proliferation index. There was no association between telomere repeat length and tumor grade. Interestingly, in contrast to other entities, all malignant schwannomas and leiomyosarcomas showed significantly reduced telomere lengths. An explanation for the telomere heterogeneity in liposarcomas may include differential telomerase reactivation in well and poorly differentiated tumors. Conclusions: Telomere shortening is frequent but not a uniform phenomenon in different types of soft-tissue tumor. Studies on telomerase activity should be performed in the same cohort of sarcomas.

p53 Gene mutations in osteosarcomas of low-grade malignancy

Alterations in tumor suppressor gene p53, localized on chromosome 17p13, are considered to play a significant role in the initiation and, to some extent, even in the progression of various malignant tumors. In this respect, investigations on conventional highly malignant osteosarcomas have shown a mutation rate of approximately 20%. However, currently, data on the mutation rate in the group of variant histology osteosarcomas of low-grade malignancy do not exist. Therefore, we investigated a panel of low malignant entities (five low malignant intramedullary osteosarcomas grade 1; one intramedullary osteosarcoma grade 2; eight parosteal osteosarcomas, including one local recurrence grades 1 and 2, and five periosteal osteosarcomas grade 2) with polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis focusing on exons 4 to 8 of the p53 gene followed by direct sequencing. Point mutations were found in one low-grade osteoblastoma-like osteosarcoma and in two periosteal osteosarcomas grade 2 (one missense, one silent, and one nonsense mutation). This mutation rate of 15.7% (3 of 19) is comparable to that determined in highly malignant osteosarcomas. Moreover, the analysis of clinical data did not show any difference in the behavior of tumors with p53 mutations compared with those without. Therefore, we suggest that alterations in p53 gene are an early event in the tumorigenesis of malignant osteoblastic tumors without impact on progression of these tumors.

Expression of MDR1/p-glycoprotein and multidrug resistance-associated protein in childhood solid tumours

We evaluated the expression of MDR1/p-glycoprotein in paediatric tumours using reverse transcriptase polymerase chain reaction (RT-PCR), RNA dot blot analysis, and immunohistochemistry on formalin fixed paraffin-embedded material with JSB-1 and C-219 monoclonal antibodies, and compared these three techniques. The expression of multidrug resistance-associated protein (MRP) gene was examined by RT-PCR assay. We studied MDR1/p-glycoprotein and MRP expression in 13 samples from 10 neuroblastoma patients, 11 samples from 10 nephroblastoma patients, 2 rhabdomyosarcomas, 1 adrenocortical carcinoma and 10 benign tumours or tumour-like lesions. Eleven of 13 neuroblastomas, 7 of 11 nephroblastomas, 2 rhabdomyosarcomas, 1 adrenocortical carcinoma, and 7 of 10 benign tumours or tumour-like lesions showed MDR1 PCR products. By RNA dot blot analysis, MDR1 transcripts were detectable in 11 of 34 specimens. Immunohistochemically, we detected positive reaction products for JSB-1 in 26 of 36 samples. There was a significant correlation between the immunoreactivity for JSB-1 and the expression of MDR1 mRNA expression by RTPCR (P=0.0001). However, the presence of p-glycoprotein immunostaining does not correlate with the MDR1 expression shown by RT-PCR in every case. As for MRP mRNA expression, 9 of 13 neuroblastomas and 10 of 11 nephroblastomas revealed PCR products.

Telomeric lengths and telomerase activity in liposarcomas.

To assess the role of telomerase in the development of liposarcomas, we measured telomerase activity in 36 malignant and seven benign lipomatous neoplasias from 34 patients. A sensitive polymerase chain reaction–based telomerase assay (the telomeric repeat amplification protocol) was applied. Shortening or elongation of telomeric repeat fragment lengths, as measured by using hybridization with a telomere‐specific oligonucleotide probe, was correlated with the presence of telomerase activity. The latter was demonstrable in 69% of malignant tumors. Benign tumors can be distinguished from malignant neoplasias on the basis of telomerase activity. However, telomerase expression seems to be characteristic of poorly differentiated liposarcomas. Myxoid/round cell liposarcomas exhibited a higher telomerase activity level than the classical low‐grade variants. Telomerase activity was not correlated with age at the time of diagnosis or with sex. In most cases, telomerase‐positive tumors showed higher proliferation indices than did neoplasias lacking telomerase. All eight recurrences expressed telomerase activity, reflecting a close association of telomerase with the biological behavior of liposarcomas. Our findings suggest that telomerase may play a key role in the establishment and progression of malignant lipomatous tumors. Mol. Carcinog. 24:144–151, 1999.

Genetic instability in osteoblastic tumors of the skeletal system

At the histological level, the differential diagnosis of osteoblastic bone tumors is characterized by several problems that cannot be solved by conventional histological methods including immunohistology. Differentiating aneurysmal bone cyst from telangiectatic osteosarcoma or giant cell tumor from giant cell-containing highly malignant osteosarcoma are only two examples reflecting the complexity of this field. To develop a new approach to these diagnostic problems, we analyzed the genetic instability in a large number of bone-forming tumor-like lesions as well as in benign and malignant osteoblastic tumors. Our research concentrated on genetic alterations in cell cycle regulator genes: mutations in the p53 gene and ras gene, loss of heterozygosity at the p53, p16 and Rb-locus, and amplification of the mdm2-gene and the c-myc-gene. In addition to cell cycle regulators, the telomerase activity has also been analyzed. The results show that the number of genetic alterations increases with the malignancy of the tumors. The highest number of genetic alterations could thus be found in conventional intraosseous osteosarcoma. In tumor-like lesions, genetic alterations have rarely been observed. The results of this study show that analyzing the genetic instability probably contributes to an improvement in the differential diagnosis of osteoblastic tumors.

ALVEOLAR SOFT PART SARCOMA OF THE UTERINE CORPUS: REPORT OF TWO CASES AND REVIEW OF THE LITERATURE

Alveolar soft-part sarcoma (ASPS) is a rare tumor of uncertain histogenesis, mainly localized in the extremities and less frequently found in the head, neck and trunk. The present report describes two cases of ASPS localized in the uterus. In general, this entity is very rarely encountered in the female genital tract. Including the two cases presented here, 27 patients have been described in the literature. Whereas the prognosis for ASPS in soft tissues is usually poor (most of the patients died of lung metastases), those localized in the female genital tract are associated with a much better survival. This could be explained by the fact that their diameter seldom exceeds 5 cm, the size which is regarded as being the critical prognostic limit. Histologically, both tumors showed the same characteristics as known for ASPS in other localizations: organoid or nest-like arrangement of tumor cells; rounded or polygonal cells with cytoplasm of varying density; thin-walled, sinusoidal vascular spaces between tumor cell nests. Immunohistochemically, we found positivity for desmin and, to a lesser extent, for vimentin. Cytokeratin was negative, which is important in differential diagnosis to other rare uterine neoplasias like clear cell (mesonephroid) adenocarcinomas or metastases.

p53 and ras Mutations in Ewing's Sarcoma

The role of tumor suppressor genes and oncogenes in the development of Ewings sarcoma has not yet been fully clarified. In this study, we analyzed the frequency of p53 tumor suppressor gene mutation in exons 4-8 by PCR-SSCP and direct sequencing, and the expression of p53-protein in Ewings sarcoma (ES) by using immunohistochemistry. The overexpression of MDM2, which acts as a functional inactivator of p53, was studied by immunohistochemistry. In addition, a screening for point mutations in the hot spot regions codon 12 and 13 of exon 1 and codon 61 of exon 2 of ras-genes (H-ras, N-ras, K-ras) was performed. In one case, a p53 gene mutation could be confirmed in codon 238 of exon 7 (1/24). Overexpression of MDM2 was found in five cases; in ras-genes, no mutations were detected. Compared with other highly malignant mesenchymal pediatric tumors such as osteosarcomas, mutations of p53 and ras in Ewings sarcomas are an extraordinarily rare event. However, their frequency is comparable to that of PNET, suggesting that the low incidence of these mutations in ES and PNET could be group-specific for tumors of neuroectodermal genesis.