Mutational status of K- ras and TP53 genes in primary sarcomas of the heart

We investigated three patients with cardiac angiosarcomas and two with cardiac rhabdomyosarcomas, all for mutations at exons 5, 6, 7 and 8 of the p53 gene and at exon 1 of K- ras. No point mutations were observed in the p53 gene in any of the five cases; however, at exon 1 of K- ras, three patients (60%) presented the same mutation at the first base of codon 13 (G to A transition). Interestingly, this mutation was detected in both rhabdomyosarcoma and angiosarcoma histologic sarcoma types. © 2000 Cancer Research Campaign

The molecular characterization of primary cardiac sarcomas with respect to genetic alterations in p53 and K-ras genes has not been widely studies. However, there is some evidence of the presence of p53 and K-ras gene alterations in sarcomas located in other organs (Muligan et al, 1990). A number of reports dealing with the occurrence of p53 gene mutations in sarcomas show it to be a frequent event, especially in the rhabdomyosarcoma histologic subtype (Felix et al, 1992;Wurl et al, 1996;Kusufuka et al, 1997). Specifically, Naka et al (1997) reported two cases of p53 gene mutations in four patients with primary cardiac angiosarcomas.
Different mutation rates of ras genes have been detected in sarcomas. K-ras mutations in 26% of hepatic angiosarcomas (Przygodzki et al, 1997), point H-ras mutations in 33% of rhabdomyosarcomas and malignant fibrous histiocytomas (Wilke et al, 1993), and N-ras and K-ras mutations in 35% of rhabdomyosarcomas (Stratton et al, 1989). However, genetic studies of heart sarcomas have not been reported.
We investigated the mutational status of p53 and K-ras genes in primary cardiac sarcomas in a small number of patients.

Tumour samples and DNA extraction
We obtained tumour specimens from seven patients in two Madrid hospitals. Five samples corresponded to sarcomas (three angiosarcomas and two rhabdomyosarcomas) with two additional samples corresponding to rhabdomyomas being taken from infants who died of multiple cardiac rhabdomyomas 3 and 7 days after birth. All the histological specimens were fixed in 10% formalin and routinely processed by paraffin-embedding. Histologic confirmation of the diagnosis was performed prior to the molecular study. DNA was extracted from formalin-fixed, paraffin-embedded tissues using chelating resin. Paraffin blocks without samples were used as negative controls for each polymerase chain reaction (PCR) analysis throughout the procedure.

Mutational study of the p53 gene
To establish the presence of point mutations in the conserved exons of TP53, polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis was performed. Exons 5, 6, 7 and 8 of the p53 gene were amplified. PCR was performed under standard conditions in a 25-µl volume containing 2-µl (100 ng) tumour DNA template; 2.5-µl of 10 × PCR buffer and 1.5 U of Ampli Taq Gold (Perkin-Elmer, Roche Molecular Systems Inc., Branchburg, NJ, USA); 200 µM deoxynucleotide triphosphate (dNTP) mix; 0.6 µM of each primer; and various concentrations of magnesium chloride (MgCl 2 ) depending on the primer and distilled water (H 2 O) needed to reach the total volume. For PCR amplification, the samples underwent 40 cycles at 94°C for 1 min, followed by subjection to different annealing temperatures depending on the primer, and 70°C for 1 min. The amplified products were denatured by mixing with 15 µl of denaturing stop solution that contained 98% formamide, 10 ml l -1 edathamil (pH 8.0), 0.02% xylene cyanol and 0.02% bromophenol blue, being heated to 95°C for 5 min and rapidly cooled on ice. Electrophoresis was Mutational status of K-ras and TP53 genes in primary sarcomas of the heart JM Garcia 1 , R Gonzalez 1 , JM Silva 1 , G Dominguez 1 , IS Vegazo 2 , C Gamallo 3 , M Provencio 1 , P España 1 and F Bonilla 1 Departments of 1 Medical Oncology and 2 Pathology, Clinica Puerta de Hierro, 28035-Madrid, Spain; 3 Department of Pathology, Hospital La Paz, 28034-Madrid, Spain Summary We investigated three patients with cardiac angiosarcomas and two with cardiac rhabdomyosarcomas, all for mutations at exons 5, 6, 7 and 8 of the p53 gene and at exon 1 of K-ras. No point mutations were observed in the p53 gene in any of the five cases; however, at exon 1 of K-ras, three patients (60%) presented the same mutation at the first base of codon 13 (G to A transition). Interestingly, this mutation was detected in both rhabdomyosarcoma and angiosarcoma histologic sarcoma types. © 2000 Cancer Research Campaign Keywords: cardiac sarcomas; K-ras mutations; TP53 mutations run on non-denaturing 8-12% polyacrylamide gels for 12-15 h at 250 V. The allelic band intensity on the gels was detected by nonradioisotopic means using a commercially available silver staining method (Oto et al, 1993). All specimens that showed a differential band at SSCP were amplified to obtain templates for DNA sequencing. These amplifications were independent to those used for SSCP analysis. Amplified DNA fragments were purified from 0.9% agarose gels using the Geneclean Kit (Bio-101, Inc., La Jolla, CA, USA) and used for direct DNA sequencing by the ddNTP method with the Sequenase Kit (United States Biochemical Corp. Cleveland, OH, USA).

Mutational analysis of K-ras gene
PCR amplification of K-ras gene, using 100 ng genomic DNA as template, was carried out in a 25-µl reaction volume with a final concentration of 1 × PCR buffer and 1.5 units of Ampli Taq DNA polymerase (Perkin-Elmer, Roche Molecular Systems Inc., Branchburg, NJ, USA); 200 µM dNTP mix; 0.6 µM of each primer; 2.5 mM MgCl 2 and distilled H 2 O to reach the total volumes. For amplification, each sample was denatured at 94°C for 5 min and subjected to 35 cycles (94°C for 30 s, 58°C for 40 s and 72°C for 30 s, followed by a final 7-min extension at 72°C). The amplified products were mixed with 20 µl denaturing stop solution that contained 98% formamide, 10 mml l -1 edathamil (pH 8.0), 0.02% xylene cyanol and 0.02% bromophenol blue, heated to 95°C for 5 min and rapidly cooled on ice. The samples were electrophoresed on non-denaturing 12% polyacrylamide gels at 250 V overnight at room temperature. Products were visualized by nonradioisotopic means using a commercially available silver staining method (Oto et al, 1993). Primers used for amplification of exon 1 of K-ras, which contains codons 12 and 13, were: 5′-GACT-GAATATAAACTTGTGGTAGT and 5′-CTATTGTTGGATCAT-ATTCGTCC. All specimens, with and without differential bands at SSCP, were sequenced following the same method as used in the sequencing process of p53 gene.

RESULTS
The clinicopathologic characteristics of the patients and treatments administered are listed in Table 1.
The five cardiac sarcomas were screened for mutations by PCR-SSCP and direct sequencing at exons 5, 6, 7 and 8 of the p53 gene and exon 1 of the K-ras gene. SSCP analysis of the exons of the p53 gene did not reveal any cases of bands with altered electrophoretic mobility. Considering that SSCP sensitivity is low under certain circumstances (Hayashi, 1991) direct sequence analysis for exons 5, 6, 7 and 8 of the p53 gene in each case were performed and no point mutations were detected. The first exon of K-ras was checked by SSCP in all lesions and three sarcomas showed a mobility shift; however, to avoid false-negatives, direct sequence analysis was performed in all cases. Mutations at codon 13 of K-ras were present in all three cases that displayed changes in mobility. G to A transition at the first base of codon 13, which resulted in one amino acid substitution (Gly-13 to Ser) (Figure 1), was detected in two angiosarcomas and one rhabdomyosarcoma.
Although our series does not permit a statistical study due to its small size, in terms of clinicopathologic features, the three patients with K-ras mutations were under 50 years of age (mean 31 years) and the two patients without K-ras mutations were over 50. Moreover, it is noteworthy that the same K-ras mutation was present in both rhabdomyosarcoma and angiosarcoma.

DISCUSSION
The rare occurrence of primary cardiac sarcomas may explain the almost complete absence of molecular studies involving the molecular status of p53 and K-ras genes in these tumours. To the best of our knowledge, there have been no reports dealing with mutations in the ras gene family, and there is only one report of a study examining p53 gene mutations in cardiac angiosarcomas with point mutations being found in 50% of cases (Naka et al, 1997). No point mutations in the p53 gene were detected in the cases we present here, suggesting that it is difficult to draw conclusions about p53 inactivation in these rare tumours from small series.
However, the current study shows that K-ras gene mutations could be relatively frequent in these rare tumours. Of the five cases studied, we found a mutation at the first position of codon 13 (Gly-13 to Ser) in three sarcomas, two angiosarcomas and one rhabdomyosarcoma, while no mutations were observed in the two cardiac rhabdomyomas assessed. Although the implication of the  Figure 1 Sequencing analysis of patients P2, P3 and P4 shows a nucleotide change (C to T) in the reverse sequence that corresponds to a transition (G to A) of the K-ras gene. This transition at codon 13 causes an amino acid change (Gly-Ser). The change was not observed in the DNA of the control case (C) ras oncogene family in the development of mesenchymal tumours was reported long ago (Stratton et al, 1989) there were no studies available concerning the presence of these point mutations in sarcomas derived from heart tissue. As was mentioned above, the clinicopathologic features of our patients were similar to those reported elsewhere with respect to age, tumour location and histological type. In our series, it is interesting to note that the same mutation was observed in both a heart rhabdomyosarcoma and an angiosarcoma.
In conclusion, we report a small series of heart sarcomas subjected to a specific molecular study that shows the presence of K-ras mutations at codon 13. Nevertheless, there were no differential clinical characteristics except for the patients harbouring these mutations being younger and predilection for the male sex.