／【Ｋ：】Ｓｅｒｖｅｒ／１８０１０６５５ 新潟厚生連農業協同組合／本文／３０ｅ

厚生連医誌

第２７巻

五十嵐０４６‐０４８

2018.02.22 20.29

１号 ４６〜４８ ２０１８Case report

A case of low-grade fibromyxoid sarcoma diagnosed
by FUS-CRREB３L２fusion transcript using reverse
transcription-polymerase chain reaction in formalin-fixedparaffin-embedded tissue specimen
Nagaoka Central General Hospital, Department of Pathology,
Department of Genetic diagnosis（Chu-etsu genetic diagnosis study group）; Pathologist

Toshihiko Ikarashi
Background : Genetic analysis is useful for the differentiation of soft part tumors. We could diagnose acase of low-grade fibromyxoid sarcoma（LGFMS）
by detecting FUS-CREB３L２fusion transcript（FUS-CREB３L２）and reported in this paper.
Case report : The ７
６ years old female case had noticed
an egg-shaped tumor in left groin subcutis forthree years. At enucleation of tumor, the tumor
was５．
５cm in size and accompanied a central mucinous lake. Microscopically the tumor consistedof fibrous tumor cells of weak atypism and intercellular myxoid stroma. The mitotic count was lessthan １％/２ mm２ and there were no characteristic
findings immunohistochemically. The FP３-RP３
type of FUS-CREB３L２ was detected by reverse
transcription-polymerase chain reaction（RT-PCR）
using formalin-fixed paraffin-embedded specimens（FFPE）and LGFMS was able to be diagnosed.
Conclusion : In the case that the pathological differentiation among fibrous soft part tumors was difficult,the genetic screening using FFPE was very effective, especially of LGFMS.
Key words : low-grade fibromyxoid sarcoma（LGFMS）,
FUS gene on chromosome １６p１
１．
１（FUS）, forward primer FP２-fc２ on in exon ６ of FUS（FP２）,forward primer FP３-fc２ in exon ６ of FUS（FP３）,
CREB３L２ gene on chromosome ７q３４（CREB３L２）, reverse primer RP１-fc２ in exon ５ of CREB３L２（RP１）, reverse primer RP２-fc２ between exon
５ and exon ６ of CREB３L２（RP２）, reverse primer
RP３-fc２ in exon ６ of CREB３L２（RP３）, FUSCREB３L２ fusion transcript（FUS-CREB３L２）, reverse transcription-polymerase chain reaction（RTPCR）, formalin-fixed paraffin-embedded tissue
specimen（FFPE）, deoxyribonucleic acid（DNA）,ribonucleic acid（RNA）

Background
Genetic analysis is useful for the differentiation ofsoft part tumors because each histological type shows thespecific genetic fusion. LGFMS reveals the FUS-CREB３
L２ fusion transcript.（１）A formalin fixation is an essential process in an examination of histopathology, and thegenetic resolution with the formalin disturbs the subsequent genetic examination using FFPE specimens. We examined the possibility of the clinical genetic screening in
LGFMS using the FFPE specimen and reported in this paper.

Case report
The patient first noticed tumor three years ago, diagnosed as fibroma, and tumor was extirpated in her ７
６years old at ３ years later after noticing her tumor mass,
The left inguinal subcutaneous lambda-shaped oval tumor
was expansive growth and ５．
５x４．
３x３cm in size. Microscopically tumor consisted of compact fibrous cells of
weak atypism, low mitotic index of １％，and prominentintercellural myxoid change with mucinous retention in
the center of tumor mass. There was no immunohistochemical characteristics, no imaging evidence of infiltration and metastasis, and no involvement of femoral nerve.
Her histopathological findings suggested the mixed tumorof fibrous and myxoid elements without any aggressivemalignancy. The genetic examination of the fusion pattern
was accomplished as a definitive diagnosis as following ３
steps.（１，２）（i）Total ribonucleic acid（RNA）was extracted from１
０％ neutral formalin-FFPE with NucleoSpin!total RNA FFPE（Takara, Tokyo）, （ii）RT-PCR wasdone with PrimeScript TM One Step RT-PCR Kit Ver.２（Takara, Tokyo）. The reaction mixture was produced
based on Takara s commercial manual, The amplification
profile of PCR consisted of ３
５ cycls of denaturation at９４℃ for ３０ , annealing and elongation at ６０℃ for ３０and ７２℃ for ６０ rspectively, and followed by a final ex-― 46 ―

／【Ｋ：】Ｓｅｒｖｅｒ／１８０１０６５５ 新潟厚生連農業協同組合／本文／３０ｅ

五十嵐０４６‐０４８

2018.02.22 20.29

A case of low-grade fibromyxoid sarcoma diagnosed by FUS-CRREB３L２fusion transcript using reverse
transcription-polymerase chain reaction in formalin-fixed paraffin-embedded tissue specimen

tension at ７２℃ for １０ . The forward and reverse primers
were same as those in the previous report and our selection of primers limited to the most frequent five ones inLGFMS（１）; namely, primers consisted of forward primer
FP２-fc２on in exon ６ of FUS（FP２）, forward primer FP３-fc２ in exon ６ of FUS（FP３）, reverse primer RP１-fc２ inexon ５ of CREB３L２（RP１）, reverse primer RP２-fc２ between exon５and exon６of CREB３L２（RP２）, and reverseprimer RP３-fc２in exon６of CREB３L２（RP３）. （iii）FUS-CREB３L２fusion products by PCR,７２-２０３bp, were identified by polyacrylamide gel electrophoresis. This caseshowed FUS-CREB３L２ fusion transcript as FP３-RP３ pattern between forward primer FP３-fc２ in exon ６ of FUS（FP３）and reverse primer RP３-fc２ in exon ６ of CREB３L２（RP３）and could finally be diagnosed as LGFMS. Poly
-acrylamide gel electrophoresis（PAGE）was done under
２００V, ３０ with Mini-gel Slab-type electrophoresis of １０６x１００x１mm in plate size（Nihn-eido, Tokyo）.

lishment of the most suitable condition in reverse transcription- polymerase chain reaction（RT-PCR）of SYT-SSX gene -. Niigata-ken Koseiren Med J ２０００；１１：
３０−４．

和 文 抄録

症例報告
ホルマリン固定・パラフィン包埋病理標本（FFPE）を使った逆転写ポリメラーゼ連鎖反応（RT-PCR）による FUS-CRREB３L２融合転写物（FUS-CREB３L２）を検出することによって診断された低悪性線維粘液肉腫（LGFMS）の１症例
長岡中央綜合病院、病理科、遺伝子診断室；病理医
いか らしとしひこ

五十嵐俊彦
Discussion

背景：線維性軟部腫瘍の診断鑑別には遺伝子診断が有用である。今回、FUS-CREB３L２を検出することにより LGFMS と診断された症例を経験したので報告した。
症例内容：症例は７６才女性で、３年来、左の鼠径部皮下に卵型腫瘍を自覚した。腫瘍摘出時、腫瘍は５．
５x４．
３x３cm で中心性粘液性停留を示し、顕微鏡所見上粘液背景の強い線維性腫瘍細胞の細胞異型は弱く、１％の低い分裂指数で、免疫組織化学的特徴でな か っ た。FFPE か ら 抽 出 し た
RNA を使った RT-PCR により FUS-CREB３L２のFP３-RP３型が検出され、LGFMS と診断できた。
結論：線維性軟部腫瘍の病理学的鑑別が困難な症例において、FFPE を使った遺伝子検査は有効な手段であった。

An examination for RT-PCR using FFPE materials
included two serious problems derived from a gene destruction by formalin fixation ; i.e., the inability of gene
amplification and the nonspecific amplification bands. In
this case the amplification of objective gene was possibleby short fixation period with neutral formalin in spite offewer reliable primers, but we were troubled with the nonspecific amplification bands. Eliminating these nonspecificextra-band, we tried to reduce the number of cycles of thePCR and increase the annealing temperature. We ultimately coped with this problem by the alteration of PAGE
condition from１５０V to２００V.
Although it was very difficult to differentiate
LGFMS from other fibromatous tumors, the genetic
analysis of FUS-CREB３L２ fusion was the most reliable
technique to diagnose LGFMS.

キーワード：低悪性線維粘液肉腫（LGFMS）、染色体１６p１１．
１の FUS 遺 伝 子（FUS）、FUS 遺 伝 子 エクソン６の順行プライマー FP２-fc２（FP２）、FUS
遺 伝 子 エ ク ソ ン６の 順 行 プ ラ イ マ ー FP３-fc２（FP３）、染色体７q３４の CREB３L２遺伝子（CREB３L２）、CREB３L２遺伝子エクソン５の逆行プラ
イマー RP１-fc２（RP１）、CREB３L２遺伝子エクソン５・６間の逆行プライマー RP２-fc２（RP２）、
CREB３L２遺伝子エクソン６の逆行プライマー
RP３-fc２（RP３）、FUS-CREB３L２融合転写（FUSCREB３L２）、逆転写ポリメラーゼ連鎖反応（RTPCR）、ホルマリン固定・パラフィン包埋病理標本（FFPE）、デオキシリボ核酸（DNA）、リボ核酸（RNA）

References
１．Matsuyama A et al. Molecular detection of FUSCREB３L２ fusion transcripts in low-grade fibromyxoid
sarcoma using formalin-fixed, paraffin-embedded tissue
specimens. Am J Surg Patho２００６；３０：１０７７−８４．
２．Ikarashi T et al. Molecular detection of SYT-SSX fusion gene transcription in monophasic type synovial
sarcoma with the use of formalin-fixed paraffin-embedded specimens − Case report guaranteed by the chromosomal analysis of incubated cells and and an estab-― 47 ―／【Ｋ：】Ｓｅｒｖｅｒ／１８０１０６５５ 新潟厚生連農業協同組合／本文／３０ｅ

厚生連医誌

第２７巻

五十嵐

０４６‐０４８2018.02.22 20.29

１号 ４６〜４８ ２０１８

List of abbreviation

Fig１. Electrophoresis of FUS-CREB３L２fusion transcript（FUS-CREB３L２）.
FP３-RP３fusion pattern was found in the６th lane.
０th lane : １００bp markers, １st lane : FP２-RP１ fusion, ２nd lane : FR２-RP２ fusion, ３rd lane : FR２RP３fusion,４th lane : FR３-RP１fusion,５th lane : FR３-RP２fusion,６th lane : FR３-RP３fusion.
（２０１
８／０２／１
２受付）― 48 ―