https://doi.org/10.1007/s12105-021-01356-y CASE REPORTS
Clear Cell Carcinoma in the Oral Cavity with Three Novel Types of EWSR1‑ATF1 Translocation: A Case Report
Satoshi Nojima1 · Masaharu Kohara1 · Hiroshi Harada2 · Hitomi Kajikawa3 · Katsutoshi Hirose4 · Shin‑Ichi Nakatsuka2 · Yukinobu Nakagawa5 · Kaori Oya6 · Yasuo Fukuda6 · Kazuhide Matsunaga3 · Narikazu Uzawa3 · Manabu Fujimoto5 · Satoru Toyosawa4 · Eiichi Morii1
Received: 8 June 2021 / Accepted: 28 June 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Clear cell carcinoma (CCC) is a rare epithelial malignant tumor of the salivary glands. It is characterized by tumor cells with clear cytoplasm, hyalinized stroma, and most importantly the fusion genes EWSR1-ATF1, EWSR1-CREM, and EWSR1- PLAG1. Break-apart FISH has been performed for multiple CCC cases, but direct sequencing analysis has been performed in relatively few. Herein, we report an interesting case of CCC harboring three EWSR1-ATF1 translocations: EWSR1 exon 8-ATF1 exon 4, EWSR1 exon 7-ATF1 exon 4, and EWSR1 exon 7-ATF1 exon 5. This case indicates the possibility of inde- pendent EWSR1-ATF1 gene translocations, and could provide insight into CCC tumorgenesis.
Keywords Clear cell carcinoma · Hyalinizing clear cell carcinoma · Salivary duct tumor · EWSR1 · ATF1
* Eiichi Morii
firstname.lastname@example.org Satoshi Nojima
email@example.com Masaharu Kohara
firstname.lastname@example.org Hiroshi Harada
email@example.com Hitomi Kajikawa
firstname.lastname@example.org Katsutoshi Hirose
email@example.com Shin-Ichi Nakatsuka firstname.lastname@example.org Yukinobu Nakagawa
email@example.com Kaori Oya
firstname.lastname@example.org Yasuo Fukuda
email@example.com Kazuhide Matsunaga
firstname.lastname@example.org Narikazu Uzawa
email@example.com Satoru Toyosawa
1 Department of Pathology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Department of Diagnostic Pathology and Cytology, Osaka International Cancer Institute, 3-1-69 Otemae, Chuo-ku, Osaka 541-8567, Japan
3 Department of Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan
4 Department of Oral Pathology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
5 Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan
6 Clinical Laboratory, Osaka University Dental Hospital, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
Head and Neck Pathology
Clear cell carcinoma (CCC) is a low-grade malignant salivary gland tumor of epithelial origin, which is com- posed of malignant cells with clear cytoplasm and often surrounded by hyalinizing stroma . It most frequently occurs at salivary gland sites in the oral and oropharyngeal cavities.
Ewing sarcoma breakpoint region 1 (EWSR1)–acti- vating transcription factor 1 (ATF1) is a specific fusion gene in CCC . The significance of this chimeric gene in the pathogenesis of CCC has been demonstrated. Shah et al. performed EWSR1 break-apart fluorescence in situ hybridization (FISH) in a variety of salivary gland tumors and detected EWSR1 rearrangement in 87% of CCCs .
In addition, sequence analyses have been performed in five CCC cases to date; EWSR1 exon11-ATF1 exon 3 chimeric type was identified in three cases [2, 4, 5]. EWSR1 exon15- ATF1 exon 5  and EWSR1 exon8-ATF1 exon 4  were identified in one case each.
The fusion gene EWSR1–cAMP response element mod- ulator (CREM) has been identified in one case . Moreo- ver, the novel fusion gene EWSR1–pleomorphic adenoma gene 1 (PLAG1) was discovered via retrospective analyses of clear cell myoepithelial carcinoma by Skálová et al. .
EWSR1 gene rearrangement is involved in other tumors, including clear cell odontogenic carcinoma (CCOC), clear cell sarcoma (CCS), and clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLTGT). FISH for EWSR1 demonstrated that 62.5% of CCOCs show EWSR1 rear- rangement . EWSR1-ATF1 and EWSR1-CREB1 chi- meric transcripts have been identified in soft parts of CCS . Both EWSR1-ATF1 and EWSR1–cAMP responsive element binding protein 1 (CREB1) fusion genes have been reported in CCSLTGT [11, 12]. Therefore, translocations
in EWSR1 are crucial in the pathogenesis of various tumors, particularly those with a clear-cell phenotype.
We describe an interesting case of CCC with EWSR1 exon 8-ATF1 exon 4, EWSR1 exon 7-ATF1 exon 4, and EWSR1 exon 7-ATF1 exon 5 and review the relevant literature.
A 54-year-old Japanese male developed a mouth ulcer of the right oral mucosa after biting his cheek. The ulcer persisted and he became aware of swelling and contact pain in the right lower lip. Intraoral examination revealed an elastic, hard swelling with ulceration, approximately 33 × 31 mm in size. Computed tomography (CT) revealed an ill-defined heterogeneous mass with exophytic growth (Fig. 1A).
T1-weighted (T1w) magnetic resonance imaging (MRI) showed that lesion intensity was approximately equivalent to surrounding muscles, but the lesion showed heterogene- ous high intensity in T2-weighted (T2w) MRI (Fig. 1B, C).
About 9 months after first awareness of symptoms, an inci- sional biopsy was performed under a provisional diagno- sis of malignant tumor originating from the minor salivary gland or an appendage of the skin.
Histopathological examination of a biopsy specimen showed the proliferation of atypical epithelial cells present- ing a cord- or nest-like structure (Fig. 2A, B). Some of the atypical cells had small round nuclei and clear to eosino- philic cytoplasm (Fig. 2C). Fibrous stroma was often seen between nests of atypical cells. Immunohistochemistry dem- onstrated that atypical cells were positive for cytokeratin (CK) AE1 + AE3 (Fig. 2D), and focally positive for CK5/6, EMA, and p63 (Fig. 2E–G). By contrast, atypical cells were negative for smooth muscle actin (SMA) and glial fibrillary acidic protein (GFAP) (Fig. 2H, I). Therefore, the lesion
Fig. 1 A Computed tomography (CT) image in horizontal view. Arrowhead indicates tumor mass lesion. B T1-weighted (T1w) magnetic reso- nance imaging (MRI) scan of the tumor lesion. C T2-weighted (T2w) MRI scan of the tumor lesion
was diagnosed as malignant epithelial tumor and a surgical tumorectomy was performed.
The tumor lesion in the surgical specimen presented as a grayish-white solid mass occupying space between the skin and oral mucosa (Fig. 3A, B). Also in histopathological eval- uation, the tumor presented as a nodular lesion occupying nearly the whole of this space. Margin of the tumor lesion did not show continuity with epidermis, skin appendages, or oral mucosa (Fig. 3C–E). Histopathological examination of
the tumor showed proliferation of atypical cells with clear to eosinophilic cytoplasm in a nest, solid sheet, or trabecu- lar pattern (Fig. 3F, G). Hyalinized or sclerotic stroma sur- rounding tumor nests was often seen. These atypical cells were positive for CK5/6, EMA, and p63 in immunohisto- chemistry (Fig. 3H–J). In addition, a small part of the tumor lesion was composed of spindle-shaped atypical cells sug- gesting poor differentiation (Fig. 3K, L). Perineural inva- sion was also observed (Fig. 3M). Ki-67 labeling index (LI)
Fig. 2 A–C Histological findings of the biopsy specimen. D–I Immunohistological findings for cytokeratin (CK) AE1 + AE3, CK5/6, EMA, p63, smooth muscle actin (SMA), and glial fibrillary acidic protein (GFAP)
Head and Neck Pathology
was approximately 30% in the highest region whereas most tumor cells showed less than 1% of Ki-67 LI (Fig. 3N, O).
Genetic analysis using the tissue from this surgical spec- imen revealed that the tumor harbored the EWSR1-ATF1 fusion gene. Three types of fusion gene were identified by direct sequence analysis: EWSR1 exon 8-ATF1 exon 4, EWSR1 exon 7-ATF1 exon 4, and EWSR1 exon 7-ATF1 exon 5 (Fig. 4A, B). The CRTC1-MAML2 and CRTC3-MAML2 fusion genes were not identified. Therefore, we made a final diagnosis of CCC derived from the minor salivary gland. At 20 months after surgical resection, there was no evidence of recurrence.
CCC is a low-grade salivary gland carcinoma that is consist- ently associated with a EWSR1-ATF1 fusion gene. Sequence analysis of EWSR1-associated fusion genes has been per- formed in several cases [2, 4–7]. The EWSR1 exon 11-ATF1 exon 3 fusion type has been reported in three cases [2, 4, 5].
Hirose et al. described the EWSR1 exon 15-ATF1 exon 5
fusion gene . Heft Neal et al. reported a case of CCC har- boring EWSR1 exon 8-ATF1 exon 4 . In addition, various EWSR1-CREM fusion genes (EWSR1 exon 14 with CREM exon 6, EWSR1 exon 14 with CREM exon 5, or EWSR1 exon 14 with CREM exon 6, EWSR1 exon 11 with CREM exon 6) were identified by Chapman et al. via Sanger sequencing .
EWSR1 plays a crucial role in development and is involved in the pathogenesis of various soft tissue tumors.
This gene consists of 17 exons, the first 7 of which encode the N-terminal transactivation domain. The C-terminal exons of EWSR1 encode the putative RNA-binding domain and the domains encoded by these exons, particularly exons 15–17, repress its N-terminal activation domain [13, 14]. ATF1 encodes an activating transcription factor protein of the ATF subfamily and the basic-region leucine zipper (bZIP) family. ATF1 regulates cell growth, survival, and other activities . Exon 3 in ATF1 encodes an acti- vation domain and phosphorylation at Ser63 in this exon is essential for regulating cell proliferation . Although the mechanisms by which EWSR1-ATF1 gene transloca- tion contributes to oncogenesis are unclear, the EWSR1 N-terminal domain likely constitutively activates the ATF1
Fig. 3 A, B Gross appearance of the tumor lesion in the surgical specimen. C–G Histological findings of the surgical specimen. H–J Immunohistological findings for CK5/6, EMA, and p63. K, L Histo-
logical findings of poorly differentiated component. M Histological finding of perineural invasion. N, O Immunohistological findings for Ki-67 with low- or high-labeling indices
DNA-binding domain in fusion transcripts, and loss of the EWSR1 C-terminal domain could enhance this inappro- priate transactivation. Indeed, CCC with the EWSR1 exon 11-ATF1 exon 3 fusion gene typically has a good prog- nosis, whereas CCSs harboring the EWSR1 exon 8-ATF1 exon 4 fusion gene show aggressive clinical behavior .
A study using integrative sequencing demonstrated that
CCCs with the EWSR1-ATF1 fusion show a gene signature enriched with the ATF1-motif .
In the present case, the EWSR1 exon 8-ATF1 exon 4, EWSR1 exon 7-ATF1 exon 4, and EWSR1 exon 7-ATF1 exon 5 fusion genes were identified in CCC. This suggests that the tumor cells have a triplet chromosome harboring the EWSR1-ATF1 fusion. This is the first case of a single
Fig. 4 A Genetic analysis using the surgical specimen. Direct sequencing showed three types of EWSR1-ATF1 fusion gene. B Diagram of the EWSR1-ATF1 fusion gene variants in this case
Head and Neck Pathology
CCC lesion harboring three types of EWSR1-ATF1 fusion gene. This result further suggests that at least one of the translocations must have occurred after duplication of the chromosome. Previous studies with molecular cell biol- ogy demonstrate that aneuploidy promotes chromosomal aberrations and tumorigenesis . Carcinogenesis in this case might depend on such mechanisms. Among the fusion genes in this case, EWSR1 exon 8-ATF1 exon 4 are almost always found in CCS but were recently identified in a case of CCC . Both EWSR1 exon 7-ATF1 exon 4 and EWSR1 exon 7-ATF1 exon 5 are found only in cases of CCS, and this is the first report of these types of fusion genes in CCC.
Notably, such types of fusion gene were found in a malig- nant tumor of epithelial phenotype. Interestingly, Tsuka- moto et al. reported that four types of EWSR-ATF1 fusions were detected in the metastatic site of CCS, whereas only single EWSR1-ATF1 fusion was found in the primary site . Coexistence of multiple EWSR1-ATF1 fusion genes might cause increased metastatic ability of tumor cells. If this hypothesis is applicable to CCC, careful follow-up for metastasis will be needed. From the viewpoint of molecular mechanisms, because the transcripts encoded by the three types of EWSR1-ATF1 translocations contain only a part of the suppressive EWSR1 C-terminal domain, this case might have a poor prognosis. However, there was no recurrence at 20 months after resection.
In summary, we report a case of CCC with a novel pattern of EWSR1-ATF1 gene translocations. This is the first case in which a single tumor lesion harbors three different types of EWSR1-ATF1 fusion gene, all of which are reported mainly in CCSs. Further studies of the gene translocations and their inter-relationships will provide insight into the carcinogen- esis of CCC.
Authors Contribution EM designed this study. SN, HH, KH, SIN, KO, YF, ST, and EM interpreted histological and immmunohistochemical findings. SN also interpreted genetic findings and wrote the manuscript.
MK performed direct sequence analysis. HK, YN, KM, NU, and MF contributed to patient management. All authors read and approved the final manuscript.
Funding The authors have no funding, financial relationship.
Data Availability The data in this study are available from the corre- sponding author, Eiichi Morii, upon reasonable request.
Conflict of interest All authors declare that they have no conflict of interest.
Ethical Approval The study in this case report was performed in accordance with the ethics committee requirement of Osaka University and with Declaration of Helsinki.
Informed Consent Informed consent for publication of clinical details and images was obtained from the patient.
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