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A novel fusion gene of collagen type I alpha 1 (exon 31) and platelet-derived growth factor B-chain (exon 2) in dermatofibrosarcoma protuberans

European Journal of Dermatology. Volume 17, Number 3, 217-9, May-June 2007, Investigative report

DOI : 10.1684/ejd.2007.0151

Summary

Author(s) : Gen Nakanishi, Song-Nan Lin, Kenji Asagoe, Norihiro Suzuki, Akiko Matsuo, Ryo Tanaka, Eiichi Makino, Wataru Fujimoto, Keiji Iwatsuki , Department of Dermatology, Okayama University Medical School, 2-5-1, Shikata-Cho, Okayama, 700-8558, Japan, Department of Dermatology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192, Japan.

Summary : Dermatofibrosarcoma protuberans (DFSP) is an uncommon, slow growing, sarcoma of dermal and subcutaneous tissue with an infiltrative growth pattern. Although DFSP has a high rate of local recurrence, it rarely metastasizes. DFSP is characterized by a chromosomal translocation involving the collagen type I a 1 (COL1A1) gene on chromosome 17 and the platelet-derived growth factor B-chain (PDGFB) gene on chromosome 22. Various exons of the COL1A1 gene have been reported to be involved in the fusion with exon 2 of the PDGFB gene. In this study, we examined the COL1A1-PDGFB fusion transcripts using frozen specimens from three patients with DFSP. The molecular biology study with reverse transcriptase polymerase chain reaction (RT- PCR) and sequencing showed that the ends of exons 25, 31, and 45 in the COL1A1 gene were fused with PDGFB. The exon 2 of the PDGFB gene fused with exon 31 of the COL1A1 gene was a novel fusion gene.

Keywords : COL1A1, dermatofibrosarcoma protuberans, fusion gene, PDGFB

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ARTICLE

Auteur(s) : Gen Nakanishi¹, Song-Nan Lin¹, Kenji Asagoe¹, Norihiro Suzuki¹, Akiko Matsuo², Ryo Tanaka², Eiichi Makino², Wataru Fujimoto², Keiji Iwatsuki¹

¹Department of Dermatology, Okayama University Medical School, 2-5-1, Shikata-Cho, Okayama, 700-8558, Japan
²Department of Dermatology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192, Japan

accepté le 3 Janvier 2007

Dermatofibrosarcoma protuberans (DFSP) is a mesenchymal neoplasm of both dermal and subcutaneous tissue commonly occurring on the trunk of young to middle aged adults. DFSP is characterized as a low grade sarcoma because it has the potential to be locally aggressive, it often recurs following excision, but it rarely metastasizes. Histopathologically, DFSP is composed of a monomorphous storiform pattern of spindle cells often with a honeycomb pattern of infiltration in the subcutaneous fat. Typically, DFSPs are positive for the immunohistochemical stain anti-CD34, however, some dermatofibroma cases may only demonstrate focal staining for CD34, and a few DFSP cases, especially fibrosarcomatous DFSPs, are negative for CD34 staining. The COL1A1-PDGFB fusion protein produced by a reciprocal translocation, t(17;22)(q22;q13), is the functional key molecule for DFSP [1]. The tumorigenic potential of the COL1A1-PDGFB fusion product has been demonstrated [2]. We therefore chose to examine the COL1A1-PDGFB gene fusion transcript by using the reverse transcriptase polymerase chain reaction (RT-PCR).

Methods

Clinical features

Case 1

A 30-year-old woman was evaluated in August 2004, at the Okayama University Medical School Department of Dermatology for a 5 cm white cicatrix on her lower back which contained two erythematous, indurated nodules each measuring 1 × 1 cm. Seven years prior to her first visit to our dermatology department a pigmented tumor was biopsied and diagnosed as a dermatofibroma by a medical practitioner. In September of 2004, we excised this area on the lower back and hematoxylin and eosin stained sections were obtained. Microscopic findings demonstrated a storiform pattern of monomorphous spindle cells in the reticular dermis extending into the subcutaneous fat with overlying mild epidermal hyperplasia (figures 1A and 1B). Immunohistochemical findings of these sections strongly showed CD34; however, the initial biopsy was negative for CD34 immunostaining (unpublished data).

Case 2

A 51-year-old man presented to the Kawasaki Medical School Department of Dermatology with a 5 × 6 cm erythematous plaque with a 1 × 1 cm nodule on the right clavicle. Fifteen years prior to this visit, the lesion was biopsied by a medical practitioner and diagnosed as hemangioma. The patient was treated by surgical removal of the tumor, and a split-thickness skin graft was used to cover the skin defect. Histological examination of the tumor showed a proliferation of spindle-shaped cells with a storiform arrangement (figures 1C and D). Immunohistochemical staining revealed that most of the tumor cells were CD34 positive.

Case 3

A 33-year-old man presented to the Kawasaki Medical School Department of Dermatology with a 6 month history of a raised, enlarging tumor on the right thigh. Cutaneous examination revealed a 5 × 7 cm firm, erythematous plaque on the right thigh. The patient was treated with a wide excision. Histopathologic examination of the tumor was notable for a dense infiltrate of spindle and oval cells within the dermis and infiltrating deeply into the subcutaneous fat (figures 1 E and F). The tumor cells were CD34 positive.

Mutation analysis

Total RNA was extracted from frozen tissues sections of all three cases using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and reverse transcription was performed using a SuperscriptIII cDNA synthesis kit (Invitrogen Corp., Carlsbad, CA, USA). To detect the expression of the COL1A1-PDGFB gene, polymerase chain reaction was carried out using sixteen COL1A1 forward primers and a specific PDGFB reverse primer according to Wang et al. [3]. The PCR product was directly sequenced using an ABI373A automated DNA sequencer. Informed consent was obtained from all participants.

Results

Figure 2A shows the results of the RT-PCR analysis of the COL1A1-PDGFB fusion transcript from Case 1. PCR products were obtained by amplification with the COL1A1 exon 23 primer and PDGFB exon 2 primer, but not with the COL1A1 exon 26 primer or PDGFB exon 2 primer. As shown in figure 2B, sequence analysis of the PCR product revealed that the end of the COL1A1 gene, exon 25, was fused with the start of exon 2 of the PDGFB gene. RT-PCR analysis showed amplified DNA from case 2 in a ladder pattern between the primer for exon 5 of the COL1A1 gene and the primer for exon 26-27 (figure 3A) and amplified DNA from case 3 between the primer for exon 10-11 of the COL1A1 gene and the primer for exon 46 (figure 4A). Sequence analysis of the PCR products revealed that the end of the COL1A1 gene, exon 31 in case 2 and exon 46 in case 3, was fused with the start of exon 2 in the PDGFB gene (figures 3B and 4B).

Discussion

Chromosomal translocation has been detected in many different types of sarcomas. As a result of such translocation, two protein-coding regions are fused in frame, producing a chimeric fusion protein. In terms of DFSP and giant cell fibroblastoma, the chromosomal translocation results in COL1A1-PDGFB fusion mRNA, in which the PDGFB exon 1 is deleted and replaced by a variable segment of the COL1A1 gene. Various exons such as 7, 8, 10, 11, 18, 19, 22, 23, 24, 25, 26, 27, 29, 32, 33/34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47, of the COL1A1 gene have been shown to be involved in the fusion with PDGFB gene [1, 4-12]. In this study we have identified a novel fusion of COL1A1 (exon 31)-PDGFB (exon 2) in DFSP.

It has been shown that fusion gene transcripts are translated into functional proteins that contribute to tumor development, but there have been no reports indicating that different kinds of fusion gene products have different oncogenic activity. In our study, COL1A1 (exon 31)-PDGFB (exon 2) was a novel fusion gene, but histopathological examination was not able to demonstrate any specific characteristics. Moreover, there was no relationship between different COL1A1-PDGFB fusion gene products and clinical features noted in our 3 patients.

It is sometimes difficult to detect fusion transcripts, especially from paraffin blocks, and it takes quite a bit of manpower and expense to perform RT-PCR analysis and sequencing in the laboratory. It has been shown that the COL1A1-PDGFB fusion gene was expressed as a protein, and thus it is theoretically possible to produce antibodies against it. However, it would be quite difficult to produce antibodies against all the different kinds of COL1A1-PDGFB fusion proteins. On the other hand, in terms of receptors, PDGF receptors are expressed in non-neoplastic and neoplastic cells, and thus the existence of PDGF receptors cannot prove the malignant tumorigenic status. Immunohistochemical detection of activated, phosphorylated PDGF receptors in DFSP by a specific antibody could provide presumptive support for diagnosis.

In conclusion, we have evaluated three patients with DFSP clinically and histopathologically in our dermatology clinics. All of these cases were further examined for the COL1A1-PDGFB gene fusion transcript by using the reverse transcriptase polymerase chain reaction (RT-PCR). Our analysis identified a novel fusion gene of collagen type I alpha 1 (exon 31) and platelet-derived growth factor B-chain (exon 2) in dermatofibrosarcoma protuberans.

Acknowledgements

Financial support: none. Conflict of interest: none.

References

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