Detection of specific gene rearrangements by fluorescence in situ hybridization in 16 cases of clear cell sarcoma of soft tissue and 6 cases of clear cell sarcoma-like gastrointestinal tumor

Clear cell sarcoma of soft tissue (CCSST) is a malignant mesenchymal tumor that mostly affects young adults and tends to affect the lower extremities, close to the tendon and aponeuroses [1]. Histologically, CCSSTs have epithelioid tumor nests accompanied by some spindling areas, and wreath-like multinucleated giant cells. CCSSTs present with a melanocytic differentiation and often express melanocytic markers including S-100 protein, melanoma antigen (Melan-A), human melanoma black 45 (HMB45), microphthalmia-associated transcription factor (MITF), and SRY-Box 10 (SOX-10) on immunohistochemistry (IHC). Ultrastructurally, CCSST has premelanosomes in the cytoplasm of tumor cells and shares some characteristic features with malignant melanomas (MMs). MMs genetically have BRAF mutations, although CCSST lacks this mutation. Clear cell sarcoma-like gastrointestinal tumor (CCSLGT) is also a malignant mesenchymal tumor that shares some pathological features with CCSST and arises from the gastrointestinal tract, such as the small and large intestine, and stomach. CCSLGT was originally reported to be an “osteoclast-rich tumor of the gastrointestinal tract with features resembling clear cell sarcoma of the soft parts” [2] and the first case of CCSLGT was reported by Alpers et al. [3] as a “malignant neuroendocrine tumor of the jejunum with osteoclast-like giant cells” in 1985. Subsequently, the term CCSLGT was first used by Kosemehmetoglu et al. [4] in their review, which included 13 CCSLGT cases. However, some authors have proposed using the term “malignant gastrointestinal neuroectodermal tumor,” because CCSLGTs lack melanocytic differentiation on IHC and ultrastructural examination and appear to have poorer prognosis [5]. Although CCSLGT has a similar histology to CCSST in some respects, such as a clear cytoplasm and epithelioid cells, there are some differing characteristics. CCSLGT has a pseudo-papillary growth pattern and many osteoclast-type giant cells, and the tumor cells tend to be positive for S-100 protein but show less expression of melanocytic markers on IHC [6]. Genetically, CCSST and CCSLGT usually have characteristic chimeric fusions of Ewing sarcoma breakpoint region 1 (EWSR1) with cAMP response element-binding protein (CREB) gene family members, EWSR1-activating transcription factor 1 (ATF1) and EWSR1-CREB1, which were derived from each translocation of t(12;22)(q13;q12) and t(2;22)(q34;q12), respectively [7‐10]. EWSR1-ATF1 fusion is much more frequent than EWSR1-CREB1 fusion, but EWSR1-CREB1 fusion of CCSLGT is comparatively often observed.

The novel finding of the study was that we discovered CREM rearrangement in a few CCSs. Kao et al. [14‐16] stated that an EWSR1-CREM fusion was previously detected by RNA sequencing in 2 melanoma cell lines (CHL-1 and COLO 699) and proposed that these cell lines may have originated from CCS because of the histological and immunohistochemical overlap between malignant melanoma and CCS. On the other hand, EWSR1-CREM fusion was found in a unique myxoid mesenchymal tumor that was recently described as a new entity [14, 17]. This myxoid tumor is thought to have an intracranial location, and 8 cases have previously been reported, of which 7 occurred in intracranial lesions like meninges, brain tumors, and ventricles, and one case arose in the pelvic/perirectal region. A genetic study revealed EWSR1 fusions with CREB family genes in all of these tumors. Among the 8 tumors, 3 had EWSR1-CREM fusion, 4 had EWSR1-CREB1 fusion, and one tumor showed EWSR1-ATF1 fusion. However, histological and immunohistochemical findings completely differed between this particular myxoid mesenchymal tumor and CCSST/CCSLGT, and interestingly, these genetic results corresponded to those of CCSST and CCSLGT.

CCSLGT was originally reported as an “osteoclast-rich tumor of the gastrointestinal tract with features resembling clear cell sarcoma of the soft parts” [2]. However, some authors prefer to refer to CCSLGT as a “malignant gastrointestinal neuroectodermal tumor” (GNET), because these tumors lack evidence of melanocytic differentiation [5]. A recent review discussed the relationship between clear cell sarcoma of the gastrointestinal tract (CCS-GIT) with GNET. There were differences in morphology and IHC findings between CCS-GIT and GNET. GNET tended to show a wider spectrum of growth patterns, including a pseudo-papillary growth pattern, and exhibited no evidence of melanocytic differentiation. Clinically, CCS-GIT affected males more often than females, and GNET occurred in younger patients although no significant differences existed in their biological behaviors [18]. It has been reported that GNET has poorer prognosis than CCS-GIT [5], but additional studies are needed to clarify the relationship between the two entities.

While a previous study revealed that EWSR1-ATF1 fusion was identified by RT-PCR in 91% of CCSST cases [1], we detected EWSR1-ATF1 fusion in 13 of 16 CCSST (81.3%) both by FISH and RT-PCR. Moreover, after excluding 1 case of EWSR1-CREM fusion, 13 of 15 CCSST cases (86.7%) had EWSR1-ATF1 fusion. The percentage of positive cases in the present study nearly reached that of the previous study. CCSLGT has been identified as having EWSR1-ATF1 or EWSR1-CREB1 fusion, with CCSLGT more frequently having EWSR1-CREB1 fusion. These fusions have also been detected in angiomatoid fibrous histiocytoma and primary pulmonary myxoid sarcoma despite the morphological and immunohistochemical differences between CCSST/CCSLGT and these tumors. To detect specific fusions, FISH is an effective and useful tool using formalin-fixed, paraffin-embedded sections in routine pathological work. In this study, we successfully detected EWSR1, ATF1, CREB1, and CREM rearrangements by FISH in the majority of cases. Some cases showed only one specific rearrangement and did not reveal any rearrangement of partner genes. In such cases, it is expected that certain unknown partner genes can form some novel chimeric genes, and that powerful analytic tools such as next-generation sequencing will be useful for detecting these novel fusions.

Rearrangements of EWSR1 and ATF1 or EWSR1-ATF1 fusion were predominantly found in CCSST, whereas those of EWSR1 and CREB1 or EWSR1-CREB1 tended to be detected in CCSLGT. We detected a novel CREM rearrangement in surgical CCSST specimens by FISH. Although this novel rearrangement occurred in a minority of CCSST cases, further studies are needed to elucidate its pathological significance.