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Ossifying fibromyxoid tumor presenting EP400-PHF1 fusion gene
Human Pathology (2013) 44, 2603–2608
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Case study
Ossifying fibromyxoid tumor presenting EP400-PHF1 fusion gene☆,☆☆,★ Makoto Endo MD, PhD a,1 , Kenichi Kohashi MD, PhD a,1 , Hidetaka Yamamoto MD, PhD a , Takeaki Ishii MD a , Tatsuya Yoshida MD b , Tomoya Matsunobu MD, PhD b , Yukihide Iwamoto MD, PhD b , Yoshinao Oda MD, PhD a,⁎ a
Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
b
Received 3 January 2013; revised 27 February 2013; accepted 1 April 2013
Keywords: Ossifying fibromyxoid tumor; EP400; PHF1; Fusion gene; RT-PCR
Summary Ossifying fibromyxoid tumor is a rare soft tissue tumor of borderline malignancy and uncertain differentiation. Recently, a novel fusion gene, EP400-PHF1, was discovered in ossifying fibromyxoid tumor; however, its relation to this type of tumor has been uncertain because the EP400PHF1 fusion gene has been successfully detected in only 1 case. We present an ossifying fibromyxoid tumor case with the EP400-PHF1 fusion gene detected by reverse transcriptase polymerase chain reaction, along with compatible cytogenetic data showing a t(6;12)(p21;q24.3) translocation. Our results suggest that the EP400-PHF1 fusion gene is a reproducible finding in ossifying fibromyxoid tumor. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Ossifying fibromyxoid tumor (OFMT) is an uncommon soft tissue tumor classified as a tumor of uncertain differentiation with intermediate malignancy (rarely metastasizing) [1]. Histologically, OFMT is composed of uniform round-to-ovoid cells, which has led to speculation that OFMT ☆ Y.O. is supported by a Grant-in-Aid for Scientific Research (B) (No. 21390107) from the Japan Society for the Promotion of Science. M.E. is a Japan Society for the Promotion of Science research fellow. ☆☆ This case was presented in part at the 8th Japanese-Korean Joint Slide Conference of Bone and Soft Tissue Pathology in Nagoya, Japan; November 2009. ★ Conflicts of interest: None. ⁎ Corresponding author. Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: [email protected] (Y. Oda). 1 M.E. and K.K. contributed equally.
0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.04.003
is a translocation-associated sarcoma [2]. Recently, GebreMedhin et al [3] reported a novel fusion gene, EP400-PHF1, and recurrent rearrangement of the PHF1 gene in OFMT. This would be quite a significant discovery for the diagnosis of OFMT; however, the EP400-PHF1 fusion gene was found in only 1 case in their study [3]. Here, we present the second case of OFMT with the EP400-PHF1 fusion gene definitely confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and direct sequencing, along with the compatible cytogenetic data of translocation, t(6;12)(p21;q24.3).
2. Case report A 71-year-old woman was referred to our hospital, presenting with a mass on the little-finger side of the right palm, which had grown very slowly over the previous 20 years. Her health was good, except for diabetes mellitus, for which she took medication. Physical examination revealed a
2604
M. Endo et al. was detected with the Dako EnVision Detection System (Dako). The sections were then reacted with a 3,3′diaminobenzidine peroxytrichloride substrate solution and counterstained with hematoxylin.
3.2. Cytogenetic analysis Viable tumor sample was obtained immediately after operation, disaggregated, cultured, harvested, and karyotyped with Giemsa banding, as described previously [5].
Fig. 1 Conventional radiograph of the right hand (A) and its magnification (B) reveal an oval lobulated mass with focal thin and curved mineralization (arrows) at the base of the little finger.
3-cm elastic hard mass that was negative for both tenderness and Tinel sign. Conventional radiographs revealed an oval lobulated mass (Fig. 1), measuring about 3 × 2.5 cm, with focal thin and curved mineralization (Fig. 1, arrows) in the subcutaneous area of the palm. The mass was removed by surgery. As of 3 years after surgery, the patient has remained free of any evidence of recurrence or metastasis. The patient was informed that data from the case would be submitted for publication, and she agreed.
3.3. RT-PCR and direct sequencing for EP400-PHF1 fusion gene Total RNA was extracted from the frozen sample using a TRIzol reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) to prepare the first-strand complementary DNA. Primers used for RT-PCR are listed in Table 1. PCR protocols are available on request. Each PCR product was loaded onto 2% agarose gel with ethidium bromide and visualized under UV illumination. The PCR products were also evaluated by direct sequencing using the Big-Dye terminator method (version 1.1; Applied Biosystems, Foster City, CA) to confirm the breakpoints of fusion transcripts.
4. Results 3. Materials and methods 4.1. Pathologic and immunohistochemical findings 3.1. Immunohistochemistry Immunohistochemistry for vimentin (1:25; Dako, Glostrup, Denmark), AE1/AE3 (1:1000; Dako), CAM5.2 (1:20; Becton Dickinson, Franklin Lakes, NJ), EMA (1:400; Dako), α-SMA (1:5000; Sigma-Aldrich, St Louis, MO), desmin (1:100; Dako), muscle-specific actin (HHF35) (1:50; Enzo Life Sciences, Farmingdale, NY), CD10 (1:100; Leica Biosystems, Nussloch, Germany), CD34 (1:50; Leica Biosystems), CD99 (1:100; Dako), S100 protein (1:400; Dako), NSE (HISTOFINE SAB-PO(R) kit; Nichirei Bioscience, Tokyo, Japan), NCAM (1:50; Leica Biosystems), GFAP (1:400; Dako), Leu7 (1:200; Becton Dickinson), SMARCB1 (also known as INI1; 1:250; BD Transduction Laboratories, San Diego, CA), and Ki-67 (1:100; Dako) was performed as described previously [4]. The immune complex Table 1
Primers used for RT-PCR
Primer set 1 2
Grossly, the cut surface of the tumor was whitish in color and well demarcated in the subcutaneous tissue. Histologically, round-to-ovoid tumor cells proliferated in myxoid or fibrous stroma, presenting a multinodular growth pattern (Fig. 2A-C). Neither a hypercellular area nor mitotic figures were observed. A small, shell-like bone formation was found at the periphery of the tumor (Fig. 2D). Immunohistochemically, the tumor cells were positive for vimentin, S100 protein (Fig. 2E), CD10, and NSE, but negative for cytokeratins (AE1/AE3, CAM5.2), EMA, α-SMA, desmin, musclespecific actin (HHF35), CD34, Leu7, NCAM, GFAP, and CD99. SMARCB1 (INI1) expression was reduced in a mosaic pattern (Fig. 2F). Based on these histologic and immunohistochemical analyses, the tumor was diagnosed as typical (nonatypical, nonmalignant) OFMT.
EP400ex37-F PHF1ex2-R EP400ex38-F PHF1ex2-R
Sequence
Product size in our case (bp)
5′-CAGGACGACAGCGACATCTA-3′ 5′-CAAAGTGAGGAGGCACCAGA-3′ 5′-CCAACTTTTGCCAAACCCAC-3′ 5′-CAAAGTGAGGAGGCACCAGA-3′
431 140
OFMT presenting EP400-PHF1 fusion gene
2605
Fig. 2 A, Histologically, the tumor shows a multinodular growth pattern. Round-to-ovoid tumor cells proliferate in myxoid (B) or fibrous (C) stroma. D, A small shell-like bone formation is found at the periphery of the tumor. E, Immunohistochemically, the tumor cells show positivity for S100 protein. F, SMARCB1 (INI1) expression is reduced in a subpopulation of tumor cells, showing a mosaic pattern.
4.2. Cytogenetic analysis
4.3. RT-PCR and direct sequencing
Cytogenetic analysis revealed a karyotype with a 46,XX, t(6;12)(p21;q24.3)[6],46,sl,add(1)(q42)[14] (Fig. 3).
A 431-base pair (bp) product was found by RT-PCR with primer set 1, which was designed for detecting EP400 exon
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Fig. 3 Cytogenetic analysis reveals 46,XX,t(6;12)(p21;q24.3) (arrows) and add(1)(q42) (arrowhead).
37 and the PHF exon 2 fusion gene reported in the previous article (Fig. 4A) [3]. Using primer set 2, a product of 140 bp was detected (Fig. 4B). Direct sequencing of the fragment revealed an EP400-PHF1 chimeric transcript, in which exon 38 of EP400 and exon 2 of PHF1 were fused with a 10-bp insertion between them (Fig. 5).
M. Endo et al. p400, which interacts with a histone acetyl transferase and helicases, regulating chromatin structure and gene transcription [8]. Meanwhile, the PHF1 gene located at 6p21.3 encodes the PHD finger protein 1 (PHF1), which is involved in chromatin structure regulation in a different mechanism from p400 [9]. In detail, PHF1 forms Polycomb repressive complex 2 (PRC2) with EZH1, EZH2, SUZ12, and others, and regulates histone H3 at lysine 27 (H3K27) methylation, resulting in a variety of biological processes including differentiation and cell proliferation [9]. PHF1 is also known as a counterpart of JAZF1-PHF1 and EPC1PHF1 chimeric genes in endometrial stromal sarcomas [10]. It is reasonable to assume that EP400 and PHF1 play an important role in the pathogenesis of OFMT; however, further biological investigation is necessary to draw a firmer conclusion. There are some previous articles presenting karyotypes of OFMT as listed in Table 2 [2,3,11-13]. Chromosome 6p21 encoding the PHF1 gene is a frequently altered point shown in cases 4, 5LR, 5M, 6, and 7 and our case. The EP400PHF1 fusion gene was successfully detected only in case 6 and our case, which both presented karyotypes of t(6;12)(p21;q24.3 or q24). From these cytogenetic data, case 1 presenting add(12)(q24.3) and case 4 presenting der(6)t(6;12)(p21;?) are also potential candidates for having the EP400-PHF1 fusion gene. Meanwhile, a counterpart of 6p21 in cases 5LR, 5M, and 7 is Xp11, implying the
5. Discussion Since OFMT was first described by Enzinger et al [6] in 1989, subsequent studies have clarified its clinicopathologic and immunohistochemical characteristics [7]. OMFT is likely to occur in adults, predominantly in men, and mostly in the subcutis of the extremities [7]. Most cases have metaplastic bone, and the detection of calcification by imaging studies aids in diagnosis. Cytologically, the tumor cells are relatively uniform in shape and size, indicating the possible involvement of translocation in its histogenesis [2]. Recently, Gebre-Medhin et al [3] reported a discovery of recurrent rearrangement of the PHF1 gene in OFMT. They showed the rearrangement of the PHF1 locus in all 4 typical, 2 of 3 atypical, and 1 of 6 malignant OFMTs [3]. In addition, the EP400-PHF1 fusion gene was successfully detected in 1 of the 3 OFMTs examined for it. At first, we tried to detect the reported variant of the fusion gene, EP400 exon 37 and PHF1 exon 2, with primer set 1, and found a larger product than expected. Using the redesigned forward primer (primer set 2), a product of 144 bp was successfully detected, and the subsequent direct sequencing revealed a new variant of the EP400-PHF1 chimeric transcript, which has not been reported before. The EP400 gene locating in 12q24.33 encodes the E1A-binding protein
Fig. 4 A, RT-PCR using EP400ex37 and PHF1ex2 primers reveals a 435-bp product. B, With EP400ex38 and PHF1ex2 primers, a 144-bp product is detected.
OFMT presenting EP400-PHF1 fusion gene
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Fig. 5 Direct sequencing reveals an EP400-PHF1 chimeric transcript, in which exon 38 of EP400 and exon 2 of PHF1 are fused with a 10bp insertion between them.
possibility of a fusion type other than EP400-PHF1 in OFMT. Interestingly, Xp11 is also known as a counterpart of translocation in renal cell carcinoma [14], alveolar soft part sarcoma, and synovial sarcoma [15]. SMARCB1 (also known as INI1) is a core subunit of the SWI/SNF ATP-dependent chromatin remodeling complex, encoded at 22q11.2 [16]. Loss of SMARCB1 expression is observed in a variety of malignancies including malignant rhabdoid tumor, epithelioid sarcoma, and OFMT [17]. Interestingly, loss of SMARCB1 expression in OFMT results in a “mosaic pattern,” which means that SMARCB1-positive and SMARCB1-negative tumor cells are mingled randomly. Such a mosaic pattern of SMARCB1 expression has also been reported in gastrointestinal stromal tumor [18] and schwannoma associated with familial schwannomatosis [19]. There is little understanding about molecular processes leading to the mosaic-pattern loss of SMARCB1 expression and its involvement in pathogenesis. Especially in OFMT, the biological relationship between the
Table 2
EP400-PHF1 fusion gene and loss of SMARCB1 expression is unclear; however, it is interesting that all EP400, PHF1, and SMARCB1 gene products altered in OFMT are related to chromatin structure and its dynamics. Further molecular biological research is expected for OFMT. Histologically, OFMT should be differentiated from its mimics including mixed tumor/myoepithelioma, malignant peripheral nerve sheath tumor, superficial acral fibromyxoma, sclerosing epithelioid fibrosarcoma, and extraskeletal osteosarcoma. Overlapping histologic and immunohistochemical features of the above entities sometimes make a correct diagnosis difficult. Detection of the EP400-PHF1 fusion gene would be a helpful diagnostic tool in such difficult cases. In summary, we describe a case of OFMT with the EP400-PHF1 fusion gene, along with the compatible translocation, t(6;12)(p21;q24.3), in the karyotyping. Our results support that the EP400-PHF1 fusion gene is a reproducible finding in OMFT.
Cytogenetic findings of OFMT
Case no.
Karyotype
Reference
1 2ML
45,XY,der(14)t(6;14)(p10;q10),add(12)(q24.3)[10]/46,XY[1] 72~74,XXY,−5[7],+6[5],+del(8)(p21),del(9)(p22),+10,der(11)t(3;11)(p21;p15),del(12)(q13),der(13)t(5;13) (q13;q34),+18[6],+19,+20,−22[5][cp10] 46,XY, add(3)(p11),+der(3)t(3;?;11) (3qter3→p11::?::11q13→11qter),−5, del(8)(p21),add(9)(q22),del(9) (p22),der(11)t(3;11)(p21;p15), del(12)(q13),+der(13)t(5;13)(q13;q34),−22 A complex karyotype including t(11;19)(q11;q13) and abnormalities of chromosomes 1 and 3 42~46,XY,−Y[7],add(1)(q42)[5],add(6)(p21)[7],t(10;18)(q26;q11)[5],der(11)t(11;15)(q23;q15)[7], add(12)(q13)[7],ins(14;?)(q13;?)[6],−15[7],+mar[7][cp16] 46,Y,ins(X;6)(p11;p21p25),t(2;12)(q31;q22) 47,Y,ins(X;6)(p11;p21p25),t(2;12)(q31;q22),+12 44,XY,6,der(7)inv(7)(p?11q22)ins(7;6)(q?22;p21p2?5)t(6;12)(p21;q24),der(12)ins(12;6)(q24;p21p1?1),−13 45,Y,add(X)(p11),del(1)(p35),der(3)t(3;16)(p12;p11),der(6)t(6;7)(p21;p15),der(7)t(X;7)(p2?;p15),t(X;6) (p11;p21),del(13)(q?21),der(17)t(?13;17)(q2?2;q2?5),20 46,XX,t(6;12)(p21;q24.3)[6],46,XX,t(6;12)(p21;q24.3),add(1)(q42)[14]
[11] [12]
2MK 3 4a 5LR 5M 6 7b Our case
Abbreviations: ML, metastasis to lung; MK, metastasis to kidney; LR, local recurrence; M, metastasis. a SKY analyses indicated that add(6) was equivalent to der(6)t(6;12)(p21;?). b In case 7, the same karyotype was found in the primary lesion and in 3 relapses, spanning 4.5 years.
[12] [2] [13] [3] [3] [3] [3]
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Acknowledgment We appreciate Dr Jun Nishio, Department of Orthopaedic Surgery, Fukuoka University, for the fruitful discussion. We also thank Naomi Tateishi for her excellent technical assistance and KN International for revising the manuscript.
References [1] Rubin BP, Stenma G. Ossifying fibromyxoid tumour. In: Fletcher CDM, Unni KK, Mertens F, editors. World Health Organization classification of tumours: pathology and genetics of tumours of soft tissue and bone. Lyon: IARC Press; 2002. [2] Folpe AL, Weiss SW. Ossifying fibromyxoid tumor of soft parts: a clinicopathologic study of 70 cases with emphasis on atypical and malignant variants. Am J Surg Pathol 2003;27:421-31. [3] Gebre-Medhin S, Nord KH, Moller E, et al. Recurrent rearrangement of the PHF1 gene in ossifying fibromyxoid tumors. Am J Pathol 2012;181:1069-77. [4] Setsu N, Kohashi K, Endo M, et al. Inhibin-alpha and synaptophysin immunoreactivity in synovial sarcoma with granular cell features. HUM PATHOL 2012;43:850-7. [5] Kobayashi C, Oda Y, Takahira T, et al. Chromosomal aberrations and microsatellite instability of malignant peripheral nerve sheath tumors: a study of 10 tumors from nine patients. Cancer Genet Cytogenet 2006;165:98-105. [6] Enzinger FM, Weiss SW, Liang CY. Ossifying fibromyxoid tumor of soft parts: a clinicopathological analysis of 59 cases. Am J Surg Pathol 1989;13:817-27. [7] Miettinen M, Finnell V, Fetsch JF. Ossifying fibromyxoid tumor of soft parts—a clinicopathologic and immunohistochemical study of 104 cases with long-term follow-up and a critical review of the literature. Am J Surg Pathol 2008;32:996-1005.
M. Endo et al. [8] Mattera L, Courilleau C, Legube G, et al. The E1A-associated p400 protein modulates cell fate decisions by the regulation of ROS homeostasis. PLoS Genet 2010;6:e1000983. [9] Margueron R, Reinberg D. The Polycomb complex PRC2 and its mark in life. Nature 2011;469:343-9. [10] Micci F, Panagopoulos I, Bjerkehagen B, Heim S. Consistent rearrangement of chromosomal band 6p21 with generation of fusion genes JAZF1/PHF1 and EPC1/PHF1 in endometrial stromal sarcoma. Cancer Res 2006;66:107-12. [11] Sovani V, Velagaleti GV, Filipowicz E, Gatalica Z, Knisely AS. Ossifying fibromyxoid tumor of soft parts: report of a case with novel cytogenetic findings. Cancer Genet Cytogenet 2001;127:1-6. [12] Nishio J, Iwasaki H, Ohjimi Y, et al. Ossifying fibromyxoid tumor of soft parts. Cytogenetic findings. Cancer Genet Cytogenet 2002;133: 124-8. [13] Kawashima H, Ogose A, Umezu H, et al. Ossifying fibromyxoid tumor of soft parts with clonal chromosomal aberrations. Cancer Genet Cytogenet 2007;176:156-60. [14] Komai Y, Fujiwara M, Fujii Y, et al. Adult Xp11 translocation renal cell carcinoma diagnosed by cytogenetics and immunohistochemistry. Clin Cancer Res 2009;15:1170-6. [15] Oda Y, Tsuneyoshi M. Recent advances in the molecular pathology of soft tissue sarcoma: implications for diagnosis, patient prognosis, and molecular target therapy in the future. Cancer Sci 2009;100: 200-8. [16] Hollmann TJ, Hornick JL. INI1-deficient tumors: diagnostic features and molecular genetics. Am J Surg Pathol 2011;35:e47-63. [17] Graham RP, Dry S, Li X, et al. Ossifying fibromyxoid tumor of soft parts: a clinicopathologic, proteomic, and genomic study. Am J Surg Pathol 2011;35:1615-25. [18] Yamamoto H, Kohashi K, Tsuneyoshi M, Oda Y. Heterozygosity loss at 22q and lack of INI1 gene mutation in gastrointestinal stromal tumor. Pathobiology 2011;78:132-9. [19] Patil S, Perry A, Maccollin M, et al. Immunohistochemical analysis supports a role for INI1/SMARCB1 in hereditary forms of schwannomas, but not in solitary, sporadic schwannomas. Brain Pathol 2008;18:517-9.
www.elsevier.com/locate/humpath
Case study
Ossifying fibromyxoid tumor presenting EP400-PHF1 fusion gene☆,☆☆,★ Makoto Endo MD, PhD a,1 , Kenichi Kohashi MD, PhD a,1 , Hidetaka Yamamoto MD, PhD a , Takeaki Ishii MD a , Tatsuya Yoshida MD b , Tomoya Matsunobu MD, PhD b , Yukihide Iwamoto MD, PhD b , Yoshinao Oda MD, PhD a,⁎ a
Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
b
Received 3 January 2013; revised 27 February 2013; accepted 1 April 2013
Keywords: Ossifying fibromyxoid tumor; EP400; PHF1; Fusion gene; RT-PCR
Summary Ossifying fibromyxoid tumor is a rare soft tissue tumor of borderline malignancy and uncertain differentiation. Recently, a novel fusion gene, EP400-PHF1, was discovered in ossifying fibromyxoid tumor; however, its relation to this type of tumor has been uncertain because the EP400PHF1 fusion gene has been successfully detected in only 1 case. We present an ossifying fibromyxoid tumor case with the EP400-PHF1 fusion gene detected by reverse transcriptase polymerase chain reaction, along with compatible cytogenetic data showing a t(6;12)(p21;q24.3) translocation. Our results suggest that the EP400-PHF1 fusion gene is a reproducible finding in ossifying fibromyxoid tumor. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Ossifying fibromyxoid tumor (OFMT) is an uncommon soft tissue tumor classified as a tumor of uncertain differentiation with intermediate malignancy (rarely metastasizing) [1]. Histologically, OFMT is composed of uniform round-to-ovoid cells, which has led to speculation that OFMT ☆ Y.O. is supported by a Grant-in-Aid for Scientific Research (B) (No. 21390107) from the Japan Society for the Promotion of Science. M.E. is a Japan Society for the Promotion of Science research fellow. ☆☆ This case was presented in part at the 8th Japanese-Korean Joint Slide Conference of Bone and Soft Tissue Pathology in Nagoya, Japan; November 2009. ★ Conflicts of interest: None. ⁎ Corresponding author. Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: [email protected] (Y. Oda). 1 M.E. and K.K. contributed equally.
0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.04.003
is a translocation-associated sarcoma [2]. Recently, GebreMedhin et al [3] reported a novel fusion gene, EP400-PHF1, and recurrent rearrangement of the PHF1 gene in OFMT. This would be quite a significant discovery for the diagnosis of OFMT; however, the EP400-PHF1 fusion gene was found in only 1 case in their study [3]. Here, we present the second case of OFMT with the EP400-PHF1 fusion gene definitely confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and direct sequencing, along with the compatible cytogenetic data of translocation, t(6;12)(p21;q24.3).
2. Case report A 71-year-old woman was referred to our hospital, presenting with a mass on the little-finger side of the right palm, which had grown very slowly over the previous 20 years. Her health was good, except for diabetes mellitus, for which she took medication. Physical examination revealed a
2604
M. Endo et al. was detected with the Dako EnVision Detection System (Dako). The sections were then reacted with a 3,3′diaminobenzidine peroxytrichloride substrate solution and counterstained with hematoxylin.
3.2. Cytogenetic analysis Viable tumor sample was obtained immediately after operation, disaggregated, cultured, harvested, and karyotyped with Giemsa banding, as described previously [5].
Fig. 1 Conventional radiograph of the right hand (A) and its magnification (B) reveal an oval lobulated mass with focal thin and curved mineralization (arrows) at the base of the little finger.
3-cm elastic hard mass that was negative for both tenderness and Tinel sign. Conventional radiographs revealed an oval lobulated mass (Fig. 1), measuring about 3 × 2.5 cm, with focal thin and curved mineralization (Fig. 1, arrows) in the subcutaneous area of the palm. The mass was removed by surgery. As of 3 years after surgery, the patient has remained free of any evidence of recurrence or metastasis. The patient was informed that data from the case would be submitted for publication, and she agreed.
3.3. RT-PCR and direct sequencing for EP400-PHF1 fusion gene Total RNA was extracted from the frozen sample using a TRIzol reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) to prepare the first-strand complementary DNA. Primers used for RT-PCR are listed in Table 1. PCR protocols are available on request. Each PCR product was loaded onto 2% agarose gel with ethidium bromide and visualized under UV illumination. The PCR products were also evaluated by direct sequencing using the Big-Dye terminator method (version 1.1; Applied Biosystems, Foster City, CA) to confirm the breakpoints of fusion transcripts.
4. Results 3. Materials and methods 4.1. Pathologic and immunohistochemical findings 3.1. Immunohistochemistry Immunohistochemistry for vimentin (1:25; Dako, Glostrup, Denmark), AE1/AE3 (1:1000; Dako), CAM5.2 (1:20; Becton Dickinson, Franklin Lakes, NJ), EMA (1:400; Dako), α-SMA (1:5000; Sigma-Aldrich, St Louis, MO), desmin (1:100; Dako), muscle-specific actin (HHF35) (1:50; Enzo Life Sciences, Farmingdale, NY), CD10 (1:100; Leica Biosystems, Nussloch, Germany), CD34 (1:50; Leica Biosystems), CD99 (1:100; Dako), S100 protein (1:400; Dako), NSE (HISTOFINE SAB-PO(R) kit; Nichirei Bioscience, Tokyo, Japan), NCAM (1:50; Leica Biosystems), GFAP (1:400; Dako), Leu7 (1:200; Becton Dickinson), SMARCB1 (also known as INI1; 1:250; BD Transduction Laboratories, San Diego, CA), and Ki-67 (1:100; Dako) was performed as described previously [4]. The immune complex Table 1
Primers used for RT-PCR
Primer set 1 2
Grossly, the cut surface of the tumor was whitish in color and well demarcated in the subcutaneous tissue. Histologically, round-to-ovoid tumor cells proliferated in myxoid or fibrous stroma, presenting a multinodular growth pattern (Fig. 2A-C). Neither a hypercellular area nor mitotic figures were observed. A small, shell-like bone formation was found at the periphery of the tumor (Fig. 2D). Immunohistochemically, the tumor cells were positive for vimentin, S100 protein (Fig. 2E), CD10, and NSE, but negative for cytokeratins (AE1/AE3, CAM5.2), EMA, α-SMA, desmin, musclespecific actin (HHF35), CD34, Leu7, NCAM, GFAP, and CD99. SMARCB1 (INI1) expression was reduced in a mosaic pattern (Fig. 2F). Based on these histologic and immunohistochemical analyses, the tumor was diagnosed as typical (nonatypical, nonmalignant) OFMT.
EP400ex37-F PHF1ex2-R EP400ex38-F PHF1ex2-R
Sequence
Product size in our case (bp)
5′-CAGGACGACAGCGACATCTA-3′ 5′-CAAAGTGAGGAGGCACCAGA-3′ 5′-CCAACTTTTGCCAAACCCAC-3′ 5′-CAAAGTGAGGAGGCACCAGA-3′
431 140
OFMT presenting EP400-PHF1 fusion gene
2605
Fig. 2 A, Histologically, the tumor shows a multinodular growth pattern. Round-to-ovoid tumor cells proliferate in myxoid (B) or fibrous (C) stroma. D, A small shell-like bone formation is found at the periphery of the tumor. E, Immunohistochemically, the tumor cells show positivity for S100 protein. F, SMARCB1 (INI1) expression is reduced in a subpopulation of tumor cells, showing a mosaic pattern.
4.2. Cytogenetic analysis
4.3. RT-PCR and direct sequencing
Cytogenetic analysis revealed a karyotype with a 46,XX, t(6;12)(p21;q24.3)[6],46,sl,add(1)(q42)[14] (Fig. 3).
A 431-base pair (bp) product was found by RT-PCR with primer set 1, which was designed for detecting EP400 exon
2606
Fig. 3 Cytogenetic analysis reveals 46,XX,t(6;12)(p21;q24.3) (arrows) and add(1)(q42) (arrowhead).
37 and the PHF exon 2 fusion gene reported in the previous article (Fig. 4A) [3]. Using primer set 2, a product of 140 bp was detected (Fig. 4B). Direct sequencing of the fragment revealed an EP400-PHF1 chimeric transcript, in which exon 38 of EP400 and exon 2 of PHF1 were fused with a 10-bp insertion between them (Fig. 5).
M. Endo et al. p400, which interacts with a histone acetyl transferase and helicases, regulating chromatin structure and gene transcription [8]. Meanwhile, the PHF1 gene located at 6p21.3 encodes the PHD finger protein 1 (PHF1), which is involved in chromatin structure regulation in a different mechanism from p400 [9]. In detail, PHF1 forms Polycomb repressive complex 2 (PRC2) with EZH1, EZH2, SUZ12, and others, and regulates histone H3 at lysine 27 (H3K27) methylation, resulting in a variety of biological processes including differentiation and cell proliferation [9]. PHF1 is also known as a counterpart of JAZF1-PHF1 and EPC1PHF1 chimeric genes in endometrial stromal sarcomas [10]. It is reasonable to assume that EP400 and PHF1 play an important role in the pathogenesis of OFMT; however, further biological investigation is necessary to draw a firmer conclusion. There are some previous articles presenting karyotypes of OFMT as listed in Table 2 [2,3,11-13]. Chromosome 6p21 encoding the PHF1 gene is a frequently altered point shown in cases 4, 5LR, 5M, 6, and 7 and our case. The EP400PHF1 fusion gene was successfully detected only in case 6 and our case, which both presented karyotypes of t(6;12)(p21;q24.3 or q24). From these cytogenetic data, case 1 presenting add(12)(q24.3) and case 4 presenting der(6)t(6;12)(p21;?) are also potential candidates for having the EP400-PHF1 fusion gene. Meanwhile, a counterpart of 6p21 in cases 5LR, 5M, and 7 is Xp11, implying the
5. Discussion Since OFMT was first described by Enzinger et al [6] in 1989, subsequent studies have clarified its clinicopathologic and immunohistochemical characteristics [7]. OMFT is likely to occur in adults, predominantly in men, and mostly in the subcutis of the extremities [7]. Most cases have metaplastic bone, and the detection of calcification by imaging studies aids in diagnosis. Cytologically, the tumor cells are relatively uniform in shape and size, indicating the possible involvement of translocation in its histogenesis [2]. Recently, Gebre-Medhin et al [3] reported a discovery of recurrent rearrangement of the PHF1 gene in OFMT. They showed the rearrangement of the PHF1 locus in all 4 typical, 2 of 3 atypical, and 1 of 6 malignant OFMTs [3]. In addition, the EP400-PHF1 fusion gene was successfully detected in 1 of the 3 OFMTs examined for it. At first, we tried to detect the reported variant of the fusion gene, EP400 exon 37 and PHF1 exon 2, with primer set 1, and found a larger product than expected. Using the redesigned forward primer (primer set 2), a product of 144 bp was successfully detected, and the subsequent direct sequencing revealed a new variant of the EP400-PHF1 chimeric transcript, which has not been reported before. The EP400 gene locating in 12q24.33 encodes the E1A-binding protein
Fig. 4 A, RT-PCR using EP400ex37 and PHF1ex2 primers reveals a 435-bp product. B, With EP400ex38 and PHF1ex2 primers, a 144-bp product is detected.
OFMT presenting EP400-PHF1 fusion gene
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Fig. 5 Direct sequencing reveals an EP400-PHF1 chimeric transcript, in which exon 38 of EP400 and exon 2 of PHF1 are fused with a 10bp insertion between them.
possibility of a fusion type other than EP400-PHF1 in OFMT. Interestingly, Xp11 is also known as a counterpart of translocation in renal cell carcinoma [14], alveolar soft part sarcoma, and synovial sarcoma [15]. SMARCB1 (also known as INI1) is a core subunit of the SWI/SNF ATP-dependent chromatin remodeling complex, encoded at 22q11.2 [16]. Loss of SMARCB1 expression is observed in a variety of malignancies including malignant rhabdoid tumor, epithelioid sarcoma, and OFMT [17]. Interestingly, loss of SMARCB1 expression in OFMT results in a “mosaic pattern,” which means that SMARCB1-positive and SMARCB1-negative tumor cells are mingled randomly. Such a mosaic pattern of SMARCB1 expression has also been reported in gastrointestinal stromal tumor [18] and schwannoma associated with familial schwannomatosis [19]. There is little understanding about molecular processes leading to the mosaic-pattern loss of SMARCB1 expression and its involvement in pathogenesis. Especially in OFMT, the biological relationship between the
Table 2
EP400-PHF1 fusion gene and loss of SMARCB1 expression is unclear; however, it is interesting that all EP400, PHF1, and SMARCB1 gene products altered in OFMT are related to chromatin structure and its dynamics. Further molecular biological research is expected for OFMT. Histologically, OFMT should be differentiated from its mimics including mixed tumor/myoepithelioma, malignant peripheral nerve sheath tumor, superficial acral fibromyxoma, sclerosing epithelioid fibrosarcoma, and extraskeletal osteosarcoma. Overlapping histologic and immunohistochemical features of the above entities sometimes make a correct diagnosis difficult. Detection of the EP400-PHF1 fusion gene would be a helpful diagnostic tool in such difficult cases. In summary, we describe a case of OFMT with the EP400-PHF1 fusion gene, along with the compatible translocation, t(6;12)(p21;q24.3), in the karyotyping. Our results support that the EP400-PHF1 fusion gene is a reproducible finding in OMFT.
Cytogenetic findings of OFMT
Case no.
Karyotype
Reference
1 2ML
45,XY,der(14)t(6;14)(p10;q10),add(12)(q24.3)[10]/46,XY[1] 72~74,XXY,−5[7],+6[5],+del(8)(p21),del(9)(p22),+10,der(11)t(3;11)(p21;p15),del(12)(q13),der(13)t(5;13) (q13;q34),+18[6],+19,+20,−22[5][cp10] 46,XY, add(3)(p11),+der(3)t(3;?;11) (3qter3→p11::?::11q13→11qter),−5, del(8)(p21),add(9)(q22),del(9) (p22),der(11)t(3;11)(p21;p15), del(12)(q13),+der(13)t(5;13)(q13;q34),−22 A complex karyotype including t(11;19)(q11;q13) and abnormalities of chromosomes 1 and 3 42~46,XY,−Y[7],add(1)(q42)[5],add(6)(p21)[7],t(10;18)(q26;q11)[5],der(11)t(11;15)(q23;q15)[7], add(12)(q13)[7],ins(14;?)(q13;?)[6],−15[7],+mar[7][cp16] 46,Y,ins(X;6)(p11;p21p25),t(2;12)(q31;q22) 47,Y,ins(X;6)(p11;p21p25),t(2;12)(q31;q22),+12 44,XY,6,der(7)inv(7)(p?11q22)ins(7;6)(q?22;p21p2?5)t(6;12)(p21;q24),der(12)ins(12;6)(q24;p21p1?1),−13 45,Y,add(X)(p11),del(1)(p35),der(3)t(3;16)(p12;p11),der(6)t(6;7)(p21;p15),der(7)t(X;7)(p2?;p15),t(X;6) (p11;p21),del(13)(q?21),der(17)t(?13;17)(q2?2;q2?5),20 46,XX,t(6;12)(p21;q24.3)[6],46,XX,t(6;12)(p21;q24.3),add(1)(q42)[14]
[11] [12]
2MK 3 4a 5LR 5M 6 7b Our case
Abbreviations: ML, metastasis to lung; MK, metastasis to kidney; LR, local recurrence; M, metastasis. a SKY analyses indicated that add(6) was equivalent to der(6)t(6;12)(p21;?). b In case 7, the same karyotype was found in the primary lesion and in 3 relapses, spanning 4.5 years.
[12] [2] [13] [3] [3] [3] [3]
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Acknowledgment We appreciate Dr Jun Nishio, Department of Orthopaedic Surgery, Fukuoka University, for the fruitful discussion. We also thank Naomi Tateishi for her excellent technical assistance and KN International for revising the manuscript.
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