BRAF V600E Mutation Is a Potential Therapeutic Target for a Small Subset of Synovial Sarcoma

Modern Pathology (2020) 33:1660–1668 https://doi.org/10.1038/s41379-020-0530-3

ARTICLE

BRAF V600E mutation is a potential therapeutic target for a small subset of synovial sarcoma

1,11 2 3,4 4,5 2 6 Sho Watanabe ● Akihiko Shimomura ● Takashi Kubo ● Masaya Sekimizu ● Takuji Seo ● Shun-Ichi Watanabe ● 7,8 9 2 3,10 3,4 Akira Kawai ● Noboru Yamamoto ● Kenji Tamura ● Takashi Kohno ● Hitoshi Ichikawa ● Akihiko Yoshida 1,8

Received: 28 October 2019 / Revised: 12 March 2020 / Accepted: 12 March 2020 / Published online: 1 April 2020 © The Author(s), under exclusive licence to United States & Canadian Academy of Pathology 2020

Abstract Synovial sarcoma (SS) is an aggressive tumor that most often affects the deep soft tissues in young adults. Intrathoracic SS is rare and is associated with poor outcome, highlighting the urgent need for a novel therapeutic strategy. In the process of clinical sequencing, we identiﬁed two patients with intrathoracic SS harboring the BRAF V600E mutation. The patients were women aged 32 and 23 years, and both presented with SS18–SSX2-positive monophasic SS in the thoracic cavity. BRAF V600E mutations were detected by next generation sequencing, and validated immunohistochemically by diffuse intense ﬁ

1234567890();,: 1234567890();,: positivity to BRAF V600E mutation-speci c antibodies. The phosphorylated ERK (pERK) immunohistochemistry result was also positive. One patient received a combination therapy of dabrafenib and trametinib, which led to tumor shrinkage. However, the tumor growth progressed 7.5 months later with an additional NRAS Q61K mutation. Immunohistochemical screening of 67 archival SS tumor samples failed to identify additional samples with BRAF V600E mutation. However, 32% of BRAF V600E-negative cases was positive for pERK, and one of the six tumors showing the highest pERK expression harbored an FGFR2-activating mutation. This is the ﬁrst report of targetable BRAF mutation in a small subset of SS. Our study suggests involvement of the mitogen-activated protein kinase pathway and the potential clinical implication of BRAF mutation screening in SS.

Introduction

These authors contributed equally: Sho Watanabe, Akihiko Synovial sarcoma (SS) is an aggressive tumor that most Shimomura often affects young adults, and accounts for 5–10% of soft- tissue sarcomas. The majority of tumors arise in the deep Supplementary information The online version of this article (https:// doi.org/10.1038/s41379-020-0530-3) contains supplementary soft tissues of extremities, with the remaining occurring in material, which is available to authorized users. the head, neck, trunk wall, and internal trunk, including the

* Akihiko Yoshida 6 Department of Thoracic Surgery, National Cancer Center Hospital, [email protected] Tokyo, Japan 7 Department of Musculoskeletal Oncology, National Cancer Center 1 Department of Diagnostic Pathology, National Cancer Center Hospital, Tokyo, Japan Hospital, Tokyo, Japan 8 Rare Cancer Center, National Cancer Center Hospital, 2 Department of Breast and Medical Oncology, National Cancer Tokyo, Japan Center Hospital, Tokyo, Japan 9 Department of Experimental Therapeutics, National Cancer Center 3 Division of Translational Genomics, Exploratory Oncology Hospital, Tokyo, Japan Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan 10 Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan 4 Department of Clinical Genomics, National Cancer Center Research Institute, Tokyo, Japan 11 Present address: Division of Cancer Immunology, Research Institute/Exploratory Oncology Research & Clinical Trial Center, 5 Department of Orthopaedic Surgery, Showa University School of National Cancer Center East, Chiba, Japan Medicine, Tokyo, Japan BRAF V600E mutation is a potential therapeutic target for a small subset of synovial sarcoma 1661 thoracic cavity [1]. SS can be categorized as a more com- and the reported translocated introns of ~100 genes. The mon monophasic spindle cell type and biphasic type with genes targeted by the assay are listed in Supplementary epithelial nests/glands. A poorly differentiated pattern may Table 1. This assay is currently used in clinical practice be present in a subset. It is genetically defined by SS18 gene and a detailed protocol has been published previously fusions including SS18–SSX1, SS18–SSX2, and SS18–SSX4 [14, 15]. [1]. The SS18–SSX protein exerts oncogenic activity through various mechanisms that disrupt epigenetic control. Immunohistochemistry For example, the fusion protein binds to the SWI/SNF chromatin remodeling complex, resulting in the displace- A paraffin section of 4-μm thickness was cut from the ment of native SS18 and eviction of BAF47 (SMARCB1). representative block of each tumor. Heat-induced epitope It also interacts with KDM2B to bring together the SWI/ retrieval was performed using Targeted Retrieval Solu- SNF complex and PRC1.1 on the unmethylated CpG tion pH9 (Dako, Glostrup, Denmark). The endogenous islands to aberrantly reactivate the expression of devel- peroxidase activity was blocked using 3% hydrogen opmentally regulated genes that are otherwise repressed by peroxide. The primary antibodies used were BRAF PRC2 [2, 3]. The SMARCB1 eviction from the SWI/SNF V600E mutation-specific antibody (clone VE1, dilution complex can be visualized as an immunohistochemical 1:200; Spring Bioscience, Pleasanton, CA, USA) and reduction of SMARCB1 staining, which is a diagnostically phosphorylated ERK (pERK, #4370, dilution 1:400; Cell helpful finding because it is specifically seen in 90% of SS Signaling Technology, Danvers, MA, USA). The slides samples [4, 5]. SS is otherwise genomically silent and were incubated for 1 h at room temperature with the additional genetic alterations are rare in the primary tumors, primary antibodies and subsequently labeled using the with uncommon secondary mutations including TP53, EnVision system (Dako). Mouse LINKER (Dako) was PTEN, CTNNB1, APC, SETD2, and FBXW7 [6]. used for BRAF V600E staining. BRAF V600E staining Primary intrathoracic SS is uncommon, but SS might was considered positive when diffuse cytoplasmic represent the most common sarcoma type in the thoracic staining was observed. pERK staining was considered cavity [7, 8]. The management of intrathoracic SS has positive when nuclear staining was observed in ≥10% numerous challenges, including late detection, large tumor of cells. size, high histological grade, high patient age, and difficulty to obtain adequate surgical margin [7, 9]. In addition, cor- rect diagnosis can be delayed by a small sample size and a Results variety of histological mimics. Consequently, intrathoracic SS is associated with poorer outcome than its soft-tissue Case summary of BRAF-mutant SS counterparts, with frequent recurrence and metastasis [7– 11]. The prognosis of recurrent/metastatic SS remains poor Case 1: A 32-year-old Japanese woman presented with [12, 13], highlighting the need for a novel therapeutic chest pain resulting from a 12-cm mass in the right strategy. thoracic cavity (Fig. 1a). A thoracoscopic biopsy Herein, we describe two patients with SS that harbored revealed a monophasic SS showing a classic spindle cell BRAF V600E mutation, for the first time to our knowledge. morphology (Fig. 1b). The immunohistochemical analy- The tumors in both patients primarily involved the thoracic sis showed focal-positive staining for epithelial mem- cavity and one of them responded well to the combined brane antigen and cytokeratin, whereas SMARCB1 BRAF/MEK inhibition, until it ultimately recurred with an expression was reduced. RNA sequencing revealed an additional NRAS mutation. SS18–SSX2 fusion transcript, which was validated by fluorescence in-situ hybridization using the SS18 break- apart probe (Fig. 1c). The patient received chemotherapy Materials and methods with doxorubicin and ifosfamide, which resulted in some tumor shrinkage. She underwent tumor resection and The study was approved by the Institutional Review Board adjuvant chemotherapy with doxorubicin and ifosfamide, of the National Cancer Center, Tokyo, Japan (2012–374, leading to a complete remission. After 18 months, a 2014–089). pulmonary metastasis appeared and the resected specimen showed a similar histology. The patient was enrolled Next generation sequencing in a phase I trial of trabectedin, which led to transient disease control. Following tumor progression, she was We performed targeted next generation sequencing treated with ifosfamide monotherapy and pazopanib. (NGS) using the NCC Oncopanel to capture coding exons Clinical NGS using NCC Oncopanel v2 on the resected 1662 S. Watanabe et al.

Fig. 1 Case 1. Computed tomography image showing a large mass in rearrangement). Clinical next generation sequencing revealed a BRAF the right thoracic cavity (a, arrow). The tumor showed a classic his- V600E mutation (d), which was supported by diffuse intense immu- tology of monophasic synovial sarcoma consisting of a fascicular nohistochemical reactivity using BRAF V600E mutation-specific growth of uniform spindle cells (b). RNA sequencing revealed an antibody (e). Phosphorylated ERK immunohistochemistry was posi- SS18–SSX2 fusion, which was confirmed by positive evidence of SS18 tive (f). rearrangement using FISH (c, isolated red signals indicate SS18 pulmonary metastatic tumor revealed a BRAF V600E estimated tumor cell ratio of 75%. The tumor showed mutation (Fig. 1d), with a variant allele frequency of diffuse intense immunohistochemical reactivity using 24.8% (280/1129 reads) in case of a histologically BRAF V600E mutation-specific antibodies decorating BRAF V600E mutation is a potential therapeutic target for a small subset of synovial sarcoma 1663

Fig. 2 Case 2. Computed tomography image showing a mass in the Clinical next generation sequencing detected a BRAF V600E mutation superior thoracic cavity (a, arrow). The tumor showed classic histol- (d), which was supported by diffuse intense immunohistochemical ogy of monophasic synovial sarcoma (b). RNA sequencing revealed reactivity using BRAF V600E mutation-speciﬁc antibody (e). Phos- an SS18–SSX2 fusion, which was validated by Sanger sequencing (c). phorylated ERK immunohistochemistry was positive (f). virtually all tumor cells, both in the primary and meta- received supportive care and died of the disease static sites (Fig. 1e). Immunohistochemistry for pERK 43 months after the initial presentation. was also positive in both primary and metastatic tumors Case 2: A 23-year-old Japanese woman presented with (Fig. 1f). The patient was unable to receive BRAF inhi- Horner’s syndrome due to a 4.3-cm tumor in the superior bitors because such trials were not open at that time. She mediastinum (Fig. 2a). The tumor was complicated by a 1664 S. Watanabe et al.

Fig. 3 Efﬁcacy of BRAF/MEK inhibition against synovial sarcoma with BRAF V600E mutation (Case 2). Computed tomography image prior to the combination therapy showing a 2.1-cm recurrence in the right superior thoracic cavity (a, arrow). Following the administration of dabrafenib and trametinib, the mass shrank to 1.4 cm (−33% reduction, partial response) at 41 days. At 90 days, the mass was unmeasurable (b, arrow). The tumor recurred after 7.5 months (c, arrow). Biopsy of the recurrent tumor showed monophasic synovial sarcoma histology with BRAF V600E and a newly acquired NRAS Q61K as a mechanism of resistance.

hemothorax, which required immediate tumor resection. BRAF-mutant SS responded to BRAF/MEK inhibition The tumor displayed classic monophasic SS histology (Fig. 2b), which was supported by RNA sequencing that Patient 2 received a combination therapy of dabrafenib revealed an SS18–SSX2 fusion transcript. This was further (BRAF inhibitor, 150 mg BID) and trametinib (MEK inhi- validated by reverse transcriptase polymerase chain bitor, trametinib 2 mg QD), which led to a partial response reaction and Sanger sequencing (Fig. 2c). Five months according to RECIST version 1.1 and the tumor was not later, she presented with pain in the right arm and detectable in 3 months (Fig. 3a, b). The tumor showed shoulder due to local recurrence. She received a range of continuous remission until 7.5 months after BRAF/MEK therapies including doxorubicin and ifosfamide, local inhibition, when it locally recurred (Fig. 3c). The biopsy of irradiation, pazopanib, and ifosfamide monotherapy, all of the recurrent tumor showed similar monophasic SS histol- which induced only transient tumor response with the ogy and was immunoreactive to BRAF V600E and pERK. subsequent regrowth. Clinical NGS using NCC Oncopa- NGS of this recurrent specimen detected the BRAF V600E nel v4 performed on the resected tumor revealed a BRAF mutation; in addition, it revealed an NRAS Q61K mutation, V600E mutation (Fig. 2d), with a variant allele frequency which was undetectable in the primary specimen. of 53.8% (276/513 reads) in case of a histologically estimated tumor cell ratio of 100%. Immunohistochemi- BRAF V600E immunohistochemical screening of cally, the tumor showed diffuse intense positivity using archival SS tissues BRAF V600E mutation-specific antibodies (Fig. 2e) decorating all tumor cells, and pERK staining was also The recurrent identification of BRAF mutation in the two positive (Fig. 2f). patients with SS prompted us to undertake a retrospective BRAF V600E mutation is a potential therapeutic target for a small subset of synovial sarcoma 1665 survey of archival SS tissues. We performed BRAF V600E solid tumors in which small subsets of cases harbor this immunohistochemical analysis of 67 SS tumor tissues ori- alteration, according to historical and recent observations ginating from 67 patients. The diagnosis of all tumors was [24–26]. From a diagnostic standpoint, the BRAF V600E reviewed by a soft-tissue pathologist (AY) and confirmed mutation in spindle cell “sarcoma” is often suspected as a by histological analyses in addition to the positive evidence clue for misdiagnosed sarcomatoid (“dedifferentiated”) of SS18 rearrangement by FISH and/or reduced immu- malignant melanoma [27, 28]. Although this suspicion is noexpression of SMARCB1. The tested cases were enriched reasonable for many cases, our study shows that the for thoracic primary tumors (N = 23) arising from the lung, mutation can also rarely occur in bona fide sarcomas. Of pleura, mediastinum, or chest wall. No additional tumors note, SW982 “synovial sarcoma” cell line with BRAF positive for BRAF V600E immunoreactivity were found, V600E mutation [29] likely did not originate from SS and the estimated prevalence of BRAF V600E mutation in because it lacks SS18–SSX fusion [30]. SS was up to 2.9% (2/69) in all anatomical sites and up to Rare (<5%) but recurrent presence of BRAF V600E 8% (2/25) in the primary thoracic tumor. mutations in SS might open a potential avenue to targeted therapy. Diffuse intense immunoreactivity using BRAF pERK immunohistochemistry in the cohort lacking V600E-specific antibodies, observed in all tested samples BRAF V600E (i.e., primary, recurrence, and/or metastasis) of both patients, suggests an early driver role of BRAF mutation, With a hypothesis that some SSs lacking BRAF V600E rather than being acquired later in a small subclone, mutation might harbor other activating mechanisms of the although the mutation is likely a secondary event to initi- mitogen-activated protein kinase (MAPK) pathway, we ating oncogenic SS18–SSX fusion. Combined therapy using examined pERK immunohistochemistry in 53 available SS dabrafenib and trametinib has been proven effective for tumor tissues that lacked BRAF V600E immunoreactivity. advanced malignant melanoma, anaplastic thyroid carci- We found that 17 cases showed positive staining with a noma, and non-small cell lung carcinoma carrying BRAF range of 10–80%, whereas the remaining 68% showed mutations [31–34], and is expected to be promising in other negative (0% or <10%) staining. Of note, pERK-positive tumor types [35]. Patient 2 indeed showed response to and -negative cohorts were not significantly different with combined BRAF/MEK inhibition; however, the tumor regard to patient age, sex, tumor site (thoracic vs. non- ultimately recurred 7.5 months later, with an additional thoracic), histological type (monophasic vs. biphasic), or activating NRAS mutation. This pattern of initial response overall survival. Among the 17 pERK-positive tumors, the and subsequent progression to targeted therapy is similar to amount and quality of six specimens were adequate for the observations in other tumor types. NRAS mutation is a NCC Oncopanel v4 assay. One of the six tumors showed an known cause of resistance to BRAF/MEK inhibition in activating mutation of FGFR2 (c.C758G, p.P253R) with an melanoma and non-small cell lung carcinoma with BRAF allele frequency of 20.5% in case of a histologically esti- mutation [36, 37], and this particular NRAS Q61K has been mated tumor cell rate of 60%, whereas the remaining five reported in 2–8% of BRAF-mutant melanoma cases resistant tumors harbored no mutations in the genes that were cov- to single-agent BRAF inhibition [38–41] or combined ered by the panel. The FGFR2-mutant tumor was a biphasic BRAF/MEK inhibition [37]. Although it is presently SS in the forearm of a 29-year-old woman. unclear how NRAS mutation confers resistance to BRAF/ MEK inhibition [36], the proposed hypotheses based on preclinical NRAS-mutant melanoma models include com- Discussion pensatory signaling via other RAF family members [42] and/or activation of the PI3K pathway [43, 44]. To the best of our knowledge, this is the first report to In the present report, both BRAF-mutant SSs displayed a describe a BRAF V600E mutation in SS. BRAF mutation considerable degree of clinicopathological similarity, has not been detected in >180 SS tissues sequenced pre- including monophasic subtype, SS18–SSX2 fusion, primary viously [2, 16–23]. BRAF is a member of the RAF family intrathoracic location, and patient age and sex with both of of serine/threonine kinases that plays key roles in the them being young adult women. Whether these shared canonical MAPK cascade, which conveys signals from the features indicate any significance remains to be determined, surface receptor tyrosine kinase through RAS toward the but focusing on these particular clinicopathological aspects downstream MEK and ERK. The oncogenic BRAF muta- might be worthwhile to identify additional cases belonging tion is reported in a wide variety of human neoplasms, of to this molecular class. which BRAF V600E is the most common type accounting Both BRAF-mutant SSs displayed high pERK immu- for >90% of all reported mutations. The estimated fre- noexpression. Because ERK phosphorylation is widely quency of BRAF V600E mutation in SS is similar to other considered as a marker of MAPK pathway activation, this 1666 S. Watanabe et al.

finding is consistent with the aberrantly enhanced signal Funding This work was supported in part by National Cancer Center transduction via this pathway induced by BRAF mutation. Research and Development Fund (29-A-2, 30-A-6), Japan Agency for The finding is similar to some previous studies, in which Medical Research and Development (JP18kk0205004, JP19ck0106257, JP19lk1403003), and JSPS Grant-in-Aid for Young Scientists pERK immunohistochemistry was uniformly positive in (18K15108). tumors with known genetic abnormalities that activate the MAPK pathway, such as those involving BRAF and MEK1 Compliance with ethical standards [45, 46]. We thus hypothesized that SSs lacking BRAF V600E mutation yet overexpressing pERK might harbor Conflict of interest TKo is a recipient of a collaborative research grant mutations in genes that encode other members of the from the Sysmex Corporation. NY is a recipient of research grants fi MAPK pathway. We first showed by immunohistochem- from the Astellas, Chugai, Eisai, Taiho, BMS, P zer, Novartis, Eli Lilly, AbbVie, Daiichi-Sankyo, Bayer, Boehringer Ingelheim, Kyowa- istry that pERK is variably expressed in a third of clinical Hakko Kirin, Takeda, ONO, Janssen Pharma, MSD, and MERCK. NY tumor samples of SS, and, in one of the six such cases also serves as an advisor for Eisai, Takeda, Otsuka, Boehringer tested, we identified an activating FGFR2 mutation as a Ingelheim, Cimic, and Chugai, and as a speaker for BMS, Pfizer, potential cause of ERK activation. Although FGFR2 AstraZeneca, Eli Lilly, ONO, Chugai, and Sysmex. All remaining authors have declared no conflicts of interest. mutation has not been reported in SS [2, 16, 17, 21, 22], FGFR2 P253R is a known oncogenic mutation that has Publisher’s note Springer Nature remains neutral with regard to been recurrently detected in human tumors including jurisdictional claims in published maps and institutional affiliations. endometrial and lung carcinomas, and it is likely a gain-of- function mutation that alters extracellular domain and References enhances the affinity to ligands [47, 48]. Our study is limited by a small number of patients with 1. Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F. WHO fi BRAF-mutant SS. Although one patient responded to tar- Classi cation of tumours of Soft Tissue and Bone. Lyon: IARC; fi 2013. geted therapy, whether the response is speci c for this small 2. McBride MJ, Pulice JL, Beird HC, Ingram DR, D’Avino AR, molecular subset remains unknown. In addition, subsequent Shern JF, et al. The SS18-SSX fusion oncoprotein hijacks BAF BRAF screening was performed primarily by immunohis- complex targeting and function to drive synovial sarcoma. Cancer – tochemistry owing to a limited number of materials avail- Cell. 2018;33:1128 41. e1127. BRAF 3. Banito A, Li X, Laporte AN, Roe JS, Sanchez-Vega F, Huang able. Hence, cases with alternative mutations might CH, et al. The SS18-SSX oncoprotein hijacks KDM2B-PRC1.1 to have been missed, although the lack thereof was confirmed drive synovial sarcoma. Cancer Cell. 2018;33:527–41. e528. by NGS in six cases with the highest pERK expression. 4. Ito J, Asano N, Kawai A, Yoshida A. The diagnostic utility of Finally, it is unclear whether screening by pERK staining is reduced immunohistochemical expression of SMARCB1 in synovial sarcomas: a validation study. Hum Pathol. 2016;47:32–37. effective to identify cases with actionable gene abnormal- 5. Arnold MA, Arnold CA, Li G, Chae U, El-Etriby R, Lee CC, et al. ities within the MAPK pathway, because the correlation A unique pattern of INI1 immunohistochemistry distinguishes between gene mutation and pERK immunoexpression has synovial sarcoma from its histologic mimics. Hum Pathol. – not been consistent in the literature, and there are reports of 2013;44:881 7. BRAF 6. Nielsen TO, Poulin NM, Ladanyi M. Synovial sarcoma: recent -mutant colorectal carcinomas or melanomas that discoveries as a roadmap to new avenues for therapy. Cancer were negative for pERK immunohistochemistry [49, 50]. In Discov. 2015;5:124–34. addition, we noted that pERK staining tended to be more 7. Begueret H, Galateau-Salle F, Guillou L, Chetalille B, Brambilla enhanced at the periphery than the center of the tissue E, Vignaoud JM, et al. Primary intrathoracic synovial sarcoma: a clinicopathologic study of 40 t(X;18)-positive cases from the section, as seen in a previous report [45], suggesting a French Sarcoma Group and the Mesopath Group. Am J Surg contribution of pre-analytic parameters such as fixation time Pathol. 2005;29:339–46. and time to fixation. Further studies are required to estimate 8. Lan T, Chen H, Xiong B, Zhou T, Peng R, Chen M, et al. Primary the actual prevalence of BRAF-mutant SS and to accurately pleuropulmonary and mediastinal synovial sarcoma: a clinicopathologic and molecular study of 26 genetically confirmed measure the therapeutic effect of targeted therapy. cases in the largest institution of southwest China. Diagn Pathol. In conclusion, we discovered that BRAF V600E muta- 2016;11:62. tion is present in a small subset of intrathoracic SS, and 9. Essary LR, Vargas SO, Fletcher CD. Primary pleuropulmonary reported that one of such tumors showed transient response synovial sarcoma: reappraisal of a recently described anatomic subset. Cancer. 2002;94:459–69. to BRAF/MEK inhibition. Our study suggests a role of 10. Hartel PH, Fanburg-Smith JC, Frazier AA, Galvin JR, Lichy JH, MAPK pathway and potential clinical implication of BRAF Shilo K, et al. Primary pulmonary and mediastinal synovial sar- mutation screening in SS. coma: a clinicopathologic study of 60 cases and comparison with five prior series. Mod Pathol. 2007;20:760–9. 11. Kim GH, Kim MY, Koo HJ, Song JS, Choi C. Primary pulmonary Acknowledgements The authors thank Mses. Sachiko Miura, Toshiko synovial sarcoma in a tertiary referral center: clinical character- Sakaguchi, Chizu Kina, Sachiyo Mitani, and Erika Arakawa for superb istics, CT, and 18F-FDG PET findings, with pathologic correla- technical assistance. tions. Medicine. 2015;94:e1392. BRAF V600E mutation is a potential therapeutic target for a small subset of synovial sarcoma 1667

12. Palmerini E, Staals EL, Alberghini M, Zanella L, Ferrari C, 29. Becerikli M, Jacobsen F, Rittig A, Kohne W, Nambiar S, Mirmo- Benassi MS, et al. Synovial sarcoma: retrospective analysis of 250 hammadsadegh A, et al. Growth rate of late passage sarcoma cells is patients treated at a single institution. Cancer. 2009;115:2988–98. independent of epigenetic events but dependent on the amount of 13. Takenaka S, Ueda T, Naka N, Araki N, Hashimoto N, Myoui A, chromosomal aberrations. Exp Cell Res. 2013;319:1724–31. et al. Prognostic implication of SYT-SSX fusion type in synovial 30. Teicher BA, Polley E, Kunkel M, Evans D, Silvers T, Delosh R, sarcoma: a multi-institutional retrospective analysis in Japan. et al. Sarcoma cell line screen of oncology drugs and investiga- Oncol Rep. 2008;19:467–76. tional agents identifies patterns associated with gene and micro- 14. Sunami K, Ichikawa H, Kubo T, Kato M, Fujiwara Y, Shimomura RNA expression. Mol Cancer Ther. 2015;14:2452–62. A, et al. Feasibility and utility of a panel testing for 114 cancer- 31. Planchard D, Besse B, Groen HJM, Souquet PJ, Quiox E, Baik associated genes in a clinical setting: a hospital-based study. CS, et al. Dabrafenib plus trametinib in patients with previously Cancer Sci. 2019;110:1480–90. treated BRAF(V600E)-mutant metastatic non-small cell lung 15. Tanabe Y, Ichikawa H, Kohno T, Yoshida H, Kubo T, Kato M, cancer: an open-label, multicentre phase 2 trial. Lancet Oncol. et al. Comprehensive screening of target molecules by next- 2016;17:984–93. generation sequencing in patients with malignant solid tumors: 32. Planchard D, Smit EF, Groen HJM, Mazieres J, Besse B, Helland A, guiding entry into phase I clinical trials. Mol Cancer. 2016;15:73. et al. Dabrafenib plus trametinib in patients with previously untreated 16. Barretina J, Taylor BS, Banerji S, Ramos AH, Lagos-Quintana M, BRAF(V600E)-mutant metastatic non-small-cell lung cancer: an DeCarolis P, et al. Subtype-specific genomic alterations define open-label, phase 2 trial. Lancet Oncol. 2017;18:1307–16. new targets for soft-tissue sarcoma therapy. Nat Genet. 2010;42: 33. Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackewicz 715–21. A, Stroiakovski D, et al. Improved overall survival in melanoma 17. Joseph CG, Hwang H, Jiao Y, Wood LD, Kinde I, Wu J, et al. with combined dabrafenib and trametinib. N. Engl J Med. 2015; Exomic analysis of myxoid liposarcomas, synovial sarcomas, and 372:30–9. osteosarcomas. Genes Chromosomes Cancer. 2014;53:15–24. 34. Subbiah V, Kreitman RJ, Wainberg ZA, Cho JY, Schellens JHM, 18. Teng HW, Wang HW, Chen WM, Chao TC, Hsieh YY, Hsih CH, Soria JC, et al. Dabrafenib and trametinib treatment in patients et al. Prevalence and prognostic influence of genomic changes of with locally advanced or metastatic BRAF V600-mutant ana- EGFR pathway markers in synovial sarcoma. J Surg Oncol. plastic thyroid cancer. J Clin Oncol. 2018;36:7–13. 2011;103:773–81. 35. Salama AKS, Li S, Macrae ER, Park J, Mitchell EP, Zwiebel JA, 19. Je EM, An CH, Yoo NJ, Lee SH. Mutational analysis of PIK3CA, et al. Dabrafenib and trametinib in patients with tumors with JAK2, BRAF, FOXL2, IDH1, AKT1 and EZH2 oncogenes in BRAF V600E/K mutations: results from the molecular analysis sarcomas. APMIS. 2012;120:635–9. for therapy choice (MATCH) Arm H. J Clin Oncol. 2019; 20. Qi Y, Wang N, Pang LJ, Zou H, Hu JM, Zhao J, et al. Identifi- 37:3002. cation of potential mutations and genomic alterations in the epi- 36. Abravanel DL, Nishino M, Sholl LM, Ambrogio C, Awad MM. thelial and spindle cell components of biphasic synovial sarcomas An acquired NRAS Q61K mutation in BRAF V600E-mutant lung using a human exome SNP chip. BMC Med Genomics. 2015; adenocarcinoma resistant to dabrafenib plus trametinib. J Thorac 8:69. Oncol. 2018;13:e131–3. 21. Vlenterie M, Hillebrandt-Roeffen MH, Flucke UE, Groenen PJ, 37. Long GV, Fung C, Menzies AM, Pupo GM, Carlino MS, Hyman Tops BB, Kamping EJ, et al. Next generation sequencing in J, et al. Increased MAPK reactivation in early resistance to dab- synovial sarcoma reveals novel gene mutations. Oncotarget. rafenib/trametinib combination therapy of BRAF-mutant meta- 2015;6:34680–90. static melanoma. Nat Commun. 2014;5:5694. 22. The Cancer Genome Atlas Research Network. Electronic address 38. Johnson DB, Menzies AM, Zimmer L, Eroglu Z, F YE, Zhao S, edsc, cancer genome atlas research N. Comprehensive and inte- et al. Acquired BRAF inhibitor resistance: A multicenter meta- grated genomic characterization of adult soft tissue sarcomas. analysis of the spectrum and frequencies, clinical behaviour, and Cell. 2017;171:950–65. e928. phenotypic associations of resistance mechanisms. Eur J Cancer. 23. Xing Z, Wei L, Jiang X, Conroy J, Glenn S, Bshara W, et al. 2015;51:2792–9. Analysis of mutations in primary and metastatic synovial sarcoma. 39. Nazarian R, Shi H, Wang Q, Xiangju K, Koya RC, Lee H, et al. Oncotarget. 2018;9:36878–88. Melanomas acquire resistance to B-RAF(V600E) inhibition by 24. Allen A, Qin ACR, Raj N, Wang J, Uddin S, Yao Z, et al. Rare RTK or N-RAS upregulation. Nature. 2010;468:973–7. BRAF mutations in pancreatic neuroendocrine tumors may predict 40. Rizos H, Menzies AM, Pupo GM, Carlino MS, Fung C, Hyman J, response to RAF and MEK inhibition. PLoS ONE. 2019;14: et al. BRAF inhibitor resistance mechanisms in metastatic mela- e0217399. noma: spectrum and clinical impact. Clin Cancer Res. 2014;20: 25. Behling F, Barrantes-Freer A, Skardelly M, Nieser M, Christians 1965–77. A, Stockammer F, et al. Frequency of BRAF V600E mutations in 41. Van Allen EM, Wagle N, Sucker A, Treacy DJ, Johannesssen 969 central nervous system neoplasms. Diagn Pathol. 2016;11:55. CM, Goetz EM, et al. The genetic landscape of clinical resistance 26. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, to RAF inhibition in metastatic melanoma. Cancer Disco. 2014; et al. Mutations of the BRAF gene in human cancer. Nature. 4:94–109. 2002;417:949–54. 42. Dorard C, Estrada C, Barbotin C, Larcher M, Garancher A, 27. Agaimy A, Specht K, Stoehr R, Lorey T, Makl B, Niedobitek G, Leloup J, et al. RAF proteins exert both specific and compensatory et al. Metastatic malignant melanoma with complete loss of dif- functions during tumour progression of NRAS-driven melanoma. ferentiation markers (undifferentiated/dedifferentiated melanoma): Nat Commun. 2017;8:15262. analysis of 14 patients emphasizing phenotypic plasticity and the 43. Atefi M, von Euw E, Attar N, Ng C, Chu C, Guo D, et al. value of molecular testing as surrogate diagnostic marker. Am J Reversing melanoma cross-resistance to BRAF and MEK inhi- Surg Pathol. 2016;40:181–91. bitors by co-targeting the AKT/mTOR pathway. PLoS ONE. 28. Cipriani NA, Letovanec I, Hornicek FJ, Mullen JT, Duan Z, 2011;6:e28973. Borger DR, et al. BRAF mutation in ‘sarcomas’: a possible 44. Petit V, Raymond J, Alberti C, Pouteaux M, Gallagher SJ, method to detect de-differentiated melanomas. Histopathology. Nguyen MQ, et al. C57BL/6 congenic mouse NRAS(Q61K) 2014;64:639–46. melanoma cell lines are highly sensitive to the combination of 1668 S. Watanabe et al.

Mek and Akt inhibitors in vitro and in vivo. Pigment Cell Mel- 48. Dutt A, Salvesen HB, Chen TH, Ramos AH, Onofrio RC, anoma Res. 2019;32:829–41. Hatton C, et al. Drug-sensitive FGFR2 mutations in 45. Kao YC, Ranucci V, Zhang L, Sung Y, Athanasian EA, Swanson endometrial carcinoma. Proc Natl Acad Sci USA. 2008;105: D, et al. Recurrent BRAF gene rearrangements in myxoin- 8713–7. flammatory fibroblastic sarcomas, but not hemosiderotic fibroli- 49. Houben R, Vetter-Kauczok CS, Ortmann S, Rapp UR, Broecker pomatous tumors. Am J Surg Pathol. 2017;41:1456–65. EB, Becker JC. Phospho-ERK staining is a poor indicator of the 46. Shanmugam V, Craig JW, Hornick JL, Morgan EA, Pinkus GS, mutational status of BRAF and NRAS in human melanoma. J Pozdnyakova O. Cyclin D1 is expressed in neoplastic cells of Investig Dermatol. 2008;128:2003–12. langerhans cell histiocytosis but not reactive Langerhans cell 50. Holck S, Bonde J, Pedersen H, Petersen AA, Chaube A, Nielsen proliferations. Am J Surg Pathol. 2017;41:1390–6. HJ, et al. Localization of active, dually phosphorylated extra- 47. Helsten T, Elkin S, Arthur E, Tomson BN, Carter J, Kurzrock R. cellular signal-regulated kinase 1 and 2 in colorectal cancer with The FGFR landscape in cancer: analysis of 4,853 tumors by next- or without activating BRAF and KRAS mutations. Hum Pathol. generation sequencing. Clin Cancer Res. 2016;22:259–67. 2016;54:37–46.