PTEN/MMAC1 gene mutation is a rare event in soft tissue sarcomas without specific balanced translocations
Article first published online: 3 JAN 2003
Copyright © 2003 Wiley-Liss, Inc.
International Journal of Cancer
Volume 104, Issue 2, pages 175–178, 20 March 2003
How to Cite
Saito, T., Oda, Y., Kawaguchi, K.-i., Takahira, T., Yamamoto, H., Tamiya, S., Tanaka, K., Matsuda, S., Sakamoto, A., Iwamoto, Y. and Tsuneyoshi, M. (2003), PTEN/MMAC1 gene mutation is a rare event in soft tissue sarcomas without specific balanced translocations. Int. J. Cancer, 104: 175–178. doi: 10.1002/ijc.10918
- Issue published online: 4 FEB 2003
- Article first published online: 3 JAN 2003
- Manuscript Accepted: 25 OCT 2002
- Manuscript Revised: 26 AUG 2002
- Manuscript Received: 31 MAY 2002
- Ministry of Education, Science, Sports and Culture of Japan. Grant Number: 12670167
- soft tissue sarcoma
The tumor suppressor gene PTEN/MMAC1 was identified on chromosome 10q23.3, which is homozygously deleted in many human malignancies. The loss of chromosome 10q was also frequently reported in some types of soft tissue sarcomas. Our study was designed to investigate the frequency of PTEN/MMAC1 gene mutation and to evaluate the role of the PTEN/MMAC1 gene in the tumorigenesis of soft tissue sarcomas without specific balanced translocations. We analyzed 51 cases of soft tissue sarcomas without specific balanced translocations for PTEN/MMAC1 mutations by polymerase chain reaction-single strand conformation polymorphism and direct sequencing. Mutations in the PTEN/MMAC1 gene were found in only 2 cases (3.9%). Both tumors with PTEN/MMAC1 mutation were leiomyosarcomas arising from the retroperitoneum and inferior vena cava, respectively. Two of 3 leiomyosarcomas arising from the intra-abdominal cavity examined harbored mutations of this tumor suppressor gene. This result suggests that leiomyosarcomas derived from the intra-abdominal cavity might have different tumorigenesis from those of an extremity or the trunk, from the viewpoint of PTEN/MMAC1 mutation, although PTEN/MMAC1 gene mutations are rare event in these soft tissue sarcomas. © 2003 Wiley-Liss, Inc.
The pathogenesis of soft tissue sarcoma (STS) is unclear; however, at least 2 different groups of sarcomas with distinctly different patterns of chromosome aberrations can be distinguished. One group, which includes synovial sarcoma and myxoid liposarcoma, frequently shows specific balanced translocations as the sole cytogenetic anomaly, and their possible contributions to the pathogenesis have been suggested. Tumors of the other group, including leiomyosarcomas (LMS), malignant fibrous histiocytomas (MFH) and malignant peripheral nerve sheath tumors (MPNST), are characterized by complex karyotypic changes and extensive heterogeneity.1, 2, 3, 4, 5, 6 It has also been suggested, based on cytogenetic study or comparative genomic hybridization, that losses of DNA sequences are more frequent than gains in these STS without specific balanced translocations.7 Mapping the regions of loss of heterozygosity (LOH) may help to identify putative tumor suppressor loci that play a role in the tumorigenesis of STS. Therefore, deletions in some chromosome segments have been hypothesized to be important early events in tumorigenesis or the progression of STS.1, 2, 3, 4, 5, 6
The tumor suppressor gene PTEN/MMAC1 was identified on chromosome 10q23.3, which is homozygously deleted in many human malignancies.8 Germ-line PTEN mutations have been detected in patients with autosomal dominant cancer predisposition syndromes.9, 10 Somatic mutations and deletions of this gene have been reported in many types of sporadic tumors,11 including endometrial cancers,12, 13 glioblastomas,14 prostate cancers15 and melanomas.16 The PTEN/MMAC1 gene encodes a phosphatidylinositol (PI) phosphatase, and the product is reported to induce apoptosis and G1 cell cycle arrest through antagonizing the PI 3′-kinase/Akt-mediated cell growth pathway.17 An aggressive phenotype in some types of tumors is reported to be associated with alterations of this gene.14 These findings raise the possibility that the loss of PTEN/MMAC1 function is responsible for the tumorigenesis of some STS because the loss of chromosome 10q was also frequently reported in STS.1, 2, 3, 4 However, neither PTEN/MMAC1 gene mutation nor the PI 3′-kinase/PTEN/Akt kinase pathway in the field of STS has been elucidated. Although no specific recurrent chromosomal aberrations have been identified in STS, with several exceptions of synovial sarcoma and similar abnormalities, the losses and gains of some chromosome segments have been detected. We here focused on the loss of chromosome 10q and PTEN/MMAC1 gene mutation in STS.
The purpose of our study was to investigate the frequency of PTEN/MMAC1 gene mutation and to evaluate the role of the PTEN/MMAC1 gene in the tumorigenesis of STS without specific balanced translocations. We report here that LMS derived from the intra-abdominal cavity might have different tumorigenesis from those of an extremity or the trunk from the viewpoint of PTEN/MMAC1 inactivation, although PTEN/MMAC1 gene mutations are rare events in STS.
MATERIAL AND METHODS
Materials and DNA preparation
Snap-frozen tumor samples from 51 cases of STS were obtained from the collection of soft-tissue tumors registered in the Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences Kyushu University, Japan. Fresh samples were carefully dissected from the tumors in order not to include the surrounding normal tissue, and the samples were frozen in liquid nitrogen immediately and stored at −80°C. Diagnosis in each of the 51 cases was based on light microscopic examinations with hematoxylin-eosin staining of paraffin blocks. Immunohistochemical analysis, using conventional markers of differentiation routinely used in the diagnosis of soft-tissue neoplasms, was performed in almost all of the cases. Histologic diagnosis of 51 cases of STS in our study revealed 22 cases of MFH (16 cases of pleomorphic type and 6 cases of myxoid type), 13 cases of LMS, 13 cases of MPNST, 2 cases of solitary fibrous tumor (SFT), 1 of which was malignant, and 1 case of low-grade fibromyxoid sarcoma. Tumors originating from visceral organs (e.g., the gastrointestinal tract and uterus) or bone were excluded from our study. The following tumors were included as being of soft tissue origin: 6 of the tumors from the retroperitoneum, and single tumors from the inferior vena cava, posterior mediastinum and spinal cord. The anatomical distribution of these tumors is summarized in Table I. Genomic DNA was purified by using standard proteinase K digestion and phenol/chloroform extraction after homogenization, as previously described.18 In addition, genomic DNA from corresponding non-tumoral tissue of paraffin-embedded material was also extracted in each tumor with PTEN/MMAC1 mutation in the tumor-derived DNA, in order to rule out the possibility of single nucleotide polymorphisms (SNP), and then analyzed subsequently.
|Malignant fibrous histiocytoma||22|
|Inferior vena cava||1|
|Malignant peripheral nerve sheath tumor||13|
|Solitary fibrous tumor||2|
|Low-grade fibromyxoid sarcoma||1|
Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and mutational analysis of the PTEN/MMAC1 gene
PCR-SSCP was carried out for the PTEN/MMAC1 gene from exon 5 to exon 9 using previously described pairs of primers.12, 19 Although 9 exons are known to code for the PTEN/MMAC1 gene, the PTEN/MMAC1 mutations are reported to be relatively rare from exon 1 to exon 4;11 therefore, we evaluated the PTEN/MMAC1 gene mutations from exon 5 to exon 9. PCR was carried out in a Gene Amp PCR System 9600 (Perkin Elmer, Foster City, CA) for 40 cycles after initial denaturing at 96°C for 5 min in a total volume of 20 μl of reaction mixture containing 50 ng of genomic DNA of each sample. Each cycle consisted of denaturation at 96°C for 1 min, at 58°C for 1 min and at 72°C for 1 min. After the final cycle of amplification, the extension was continued for an additional 7 min at 72°C. Annealing temperatures were the same for each pair of primers. Human genomic DNA (Clontech), which was confirmed to have the same base sequences as those of Genebank (Accession Number AF000730-734), was used as a positive control for each PCR and the subsequent reactions. SSCP was performed using a gel containing 12.5% acrylamide (GenePhor™, Amersham Pharmacia Biotech, Uppsala, Sweden) and a DNA fragment analyzer (GenePhor™, Amersham Pharmacia Biotech) at 600 V, 25 mA, 15 W and 5°C for 120 min, and then the DNA bands were visualized by a DNA Silver Staining Kit (GenePhor™, Amersham Pharmacia Biotech). The direct sequences were performed for samples with aberrantly migrating bands by SSCP. The sequence data were collected by ABI Prism 310 Collection Software and were analyzed by Sequencing Analysis and Sequence Navigator Software (Perkin Elmer).
PTEN/MMAC1 gene mutation
The results are summarized in Table II and Figure 1. We screened PTEN/MMAC1 gene mutations from exon 5 to exon 9 in 51 cases of STS and found 2 mutations in 2 (3.9%) of these specimens. In case 30 (43 years old/female), we observed a missense mutation at codon 401 (ACA to ATA), which resulted in a substitution of Ile for Thr (Fig. 1a). In case 31 (55 years old/male), a T to C change between the intron 8 and exon 9 boundary was detected, which can cause a splicing variant (Fig. 1b). No mutations were detected in DNA derived from normal tissue in these 2 cases, indicating that these mutations were somatic. Both tumors with PTEN/MMAC1 gene mutation were shown histologically to be LMS. As a result, LMS harbored PTEN/MMAC1 gene mutations with a frequency of 15.4% (2/13). Furthermore, these 2 tumors were derived from intra-abdominal cavity: they were single tumors from the inferior vena cava and retroperitoneum, respectively. Karyotypic information was available for 1 of these 2 cases (case 30), and the karyotype was normal.
|Case number||Location||Position||Nucleotide change||Mutation||Karyotype|
|30||Inferior vena cava||Exon 9 (codon 401)||ACA to ATA||Missense (Thr to Ile)||Normal|
|31||Retroperitoneum||Intron 8 to exon 9||tctttctctag/GTGAAG to tcttcctctag/GTGAAG||Splicing variant?||N.A.1|
The region on chromosome 10q that contains the PTEN/MMAC1 gene and other tumor suppressor genes, such as the MXI1 gene,20 is known to be hemizygously deleted in many human cancers, with a frequency reaching 60–80% in prostate cancers, endometrial cancers and malignant glioblastomas.11 In tumors with hemizygous deletions at chromosome 10q23, the PTEN/MMAC1 gene mutations have been reported to occur at various levels of frequency.11, 13, 14, 15 LOH of chromosome 10q has also been reported in bone and soft tissue sarcomas.1, 2, 3, 4, 21 Sixty-seven percent of tumors in chondrosarcoma show LOH of chromosome 10q.21 In LMS and MFH, losses in DNA copy numbers at 10q have also been detected in 23—69%1, 2, 3, 4 and 26%2 of tumors, respectively. More recently, PTEN/MMAC1 mutation has been reported to be a rare event, being present in only 1 of 40 samples of chondrosarcoma tumors and cell lines.22 However, PTEN/MMAC1 gene mutations have not yet been reported in the field of soft tissue sarcomas. We here for the first time have reported that although it is at a lower frequency of 3.9%, the PTEN/MMAC1 gene mutation also occurs in soft tissue sarcoma, especially in intra-abdominal LMS. Furthermore, to be sure that SSCP analysis is not missing some mutations, we have sequenced 20 cases of STS that were negative for SSCP analysis from exon 5 to 9 of the PTEN/MMAC1 gene. However, as a result, we could not find PTEN/MMAC1 mutations in any of these specimens.
According to Knudson's 2-hit hypothesis,23 tumor suppressor gene function is lost by independent inactivation events of both parental alleles. Although loss of chromosome 10q has been frequently detected in LMS, we could not obtain evidence of LOH of chromosome 10q in tumors with PTEN/MMAC1 mutation. Furthermore, SSCP analysis in our study revealed aberrantly migrating bands, together with dense normal bands, as shown in Figure 1a,b. The presence of normal bands in mutation-positive tumors may indicate that the remaining allele is still intact. It is not clear whether the PTEN/MMAC1 gene is potentially inactivated by the “2-hit hypothesis” in LMS tumors with the PTEN/MMAC1 gene mutation, although we did prove that detected mutations were somatic ones.
Loss of the 10q22 locus, on those neighboring site the PTEN/MMAC1 gene is located, is less common in soft tissue LMS, when compared to gastrointestinal and uterine LMS, although loss of this locus is generally frequent in LMS.3 In our study, the PTEN/MMAC1 gene mutation was observed in 2 of 3 cases of intra-abdominal LMS, whereas the mutation did not occur in 10 cases of the remaining extra-abdominal LMS. These findings may reflect that the degree of genetic susceptibility in that locus differs somewhat between these 2 types of LMS. Furthermore, it has also been demonstrated that the PTEN/MMAC1 gene mutations occur in uterine LMS with a low frequency, leading to the estimation that the PTEN/MMAC1 mutations rarely contribute to the pathobiology of uterine LMS.24, 25 These findings may also suggest that intra-abdominal LMS and uterine LMS have different pathogenesis.
It has also been demonstrated that MFH and LMS share quite similar recurrent chromosomal imbalances, such as losses of chromosome 13q and 10q 4, 5. Furthermore, both tumors occur in patients with hereditary bilateral retinoblastoma at a high frequency.26 These findings suggest that MFH do not represent a specific sarcomatous entity but may instead represent a common oncogenic pathway in the progression of LMS.4 However, because we were focused on the PTEN/MMAC1 gene located on chromosome 10q23.3, our observations differed from those of others. We screened for PTEN/MMAC1 gene mutation in a large series of soft tissue sarcomas including MFH and LMS in our study; however, only 2 cases of intra-abdominal LMS harbored mutations. Taken together, these results suggest that, at the least, intra-abdominal LMS have a different tumorigenic pathway from those of MFH or of LMS with extra-abdominal origin.
On the other hand, the PTEN/MMAC1 gene mutation could not be detected in MPNST, SFT or low-grade fibromyxoid sarcoma. That result is in concordance with the previous cytogenetic studies that losses of chromosome 10q are quite rare in these tumors.6, 27 Therefore, the PTEN/MMAC1 gene could not be considered as the primary target for inactivation in the tumorigenesis of these tumors.
STS are also known to have p53 gene mutations with some frequency; however, the prognostic impact of the p53 gene mutations in STS is controversial. Recently, it has been demonstrated that the PTEN promoter contains a functional p53 binding site and that PTEN mRNA and protein levels increase in response to stimuli that result in p53 induction.28 It has also been shown that PTEN is required for p53-mediated apoptosis in immortalized mouse embryonic fibroblasts.28 Moreover, it has been reported that U87 glioblastoma cell line, which lacks functional PTEN, is resistant to γ irradiation-induced cell killing despite strong induction of wild-type p53 and undergo cell cycle arrest following this treatment.29 These findings might suggest that the PTEN inactivation is partially responsible for the uncontrolled growth in STS, especially in LMS of intra-abdominal origin. PTEN also should be considered as one of the molecular targets in the design of future gene therapies of STS.
In conclusion, a large series of STS without specific balanced translocations were screened for the presence of PTEN/MMAC1 gene mutation, though mutations were detected in only 2 cases of LMS, and mutation was not observed in any of the other tumor types examined in our study. Two cases of LMS with PTEN/MMAC1 gene alteration were derived from the retroperitoneum and inferior vena cava, respectively. These results may provide evidence that inactivation of the PTEN/MMAC1 gene has an important role to play in the tumorigenesis of intra-abdominal LMS and that LMS derived from these regions have different tumorigenesis from those of an extremity or the trunk, although the PTEN/MMAC1 gene is not the primary target for inactivation in STS without specific balanced translocations.
We thank KN International for revising the English used in this article.