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Introduction

Ewing sarcoma is a round cell sarcoma commonly found in younger patients (children and young adults), and standard therapy includes vincristine, doxorubicin, and cyclophosphamide alternating with ifosfamide and etoposide (VDC/IE) chemotherapy in combination with surgical resection and/or radiation therapy (1). Meanwhile, round cell sarcomas which resemble Ewing sarcoma are referred to as ‘Ewing-like sarcomas’. According to the latest edition of the World Health Organization classification, in undifferentiated round cell sarcomas of the bones and soft tissues, Ewing-like sarcoma has been classified into three categories: round cell sarcoma with EWSR1::non-ETS fusions, capicua transcriptional repressor (CIC)-rearranged sarcoma, and sarcoma with B cell lymphoma 6 corepressor (BCOR) genetic alterations (2). CIC-rearranged sarcomas and sarcomas with BCOR genetic alterations are well-established clinicopathologically, whereas round cell sarcomas with EWSR1::non-ETS fusions are a heterogeneous and premature group.

Since the discovery of EWSR1::NFATC2 sarcoma as an EWSR1::non-ETS sarcoma by Szuhai et al (3), new clinical and pathological information on this sarcoma has accumulated. Yoshida et al (4) reported that, in addition to the co-expression of CD99, NKX2-2, and PAX7, NKX3-1 is a useful immunohistochemical marker of EWSR1::NFATC2 sarcomas, similar to Ewing sarcoma (5–7). In addition, Perret et al (8) demonstrated the utility of aggrecan immunohistochemistry for the identification of NFATC2-rearranged sarcomas, including EWSR1::NFATC2 and FUS::NFATC2 fusions. Furthermore, unlike Ewing sarcoma, these sarcomas often form focal nests, cords, or trabeculae within a fibrotic, hyalinized, or myxoid stromal background, mimicking myoepithelial tumors (4,9).

EWSR1::NFATC2 sarcoma is very rare but is considered to be the most common subtype of ESWR1::non-ETS sarcoma. Regarding pathological diagnosis, the histological features of EWSR1::NFATC2 sarcoma are similar to those of other sarcomas such as Ewing-like adamantinoma, which was first reported by Lipper et al (10) as a variant of adamantinoma of the long bones. Notably, Makise et al (11) reported the case of a patient previously diagnosed with Ewing-like adamantinoma who was finally diagnosed with EWSR1::NFATC2 sarcoma using fluorescence in situ hybridization (FISH)-amplified fusion signals of EWSR1 and NFATC2.

EWSR1::NFATC2 sarcoma is thought to have a more indolent clinical course than Ewing sarcoma, despite poor responses to Ewing sarcoma chemotherapy regimens (7); therefore, metastatic EWSR1::NFATC2 sarcoma is very rare. Thus, there is insufficient information regarding chemotherapeutic treatments for metastatic EWSR1::NFATC2 sarcoma. Herein, we report a case of a man with EWSR1::NFATC2 sarcoma, initially diagnosed as Ewing-like adamantinoma, who received a series of chemotherapy treatments of for Ewing sarcoma, including eribulin, trabectedin, and pazopanib.

Case report

A 55-year-old male patient with Ewing-like adamantinoma, who had a durable response with trabectedin monotherapy as third-line therapy, was recently definitively diagnosed as having EWSR1::NFATC2 sarcoma using direct sequencing. The patient was admitted to Department of Medical Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research (Tokyo, Japan) in July 2022. He initially experienced pain in his left lower leg and consulted a local clinic at 38 years of age. The patient also exhibited disc space narrowing and osteolytic changes in the proximal tibia. The patient had no significant medical history. One month later, he underwent biopsy of the tumor in the proximal part of the left tibia at our hospital. The pathological diagnosis was ‘epithelioid malignant tumor of the left tibia suggesting Ewing-like adamantinoma’. A month thereafter, he underwent wide excision of the tumor in his left tibia with left total knee arthroplasty and a medial gastrocnemius muscle flap. The patient survived with no evidence of recurrent or metastatic disease. However, five years after surgery, the patient underwent right thyroid lobectomy and lymph node dissection for papillary thyroid cancer. In addition, 14 years after surgery, multiple new lesions were detected radiographically in the left lung, left pleural dissemination, and mediastinal, left hilar, and juxtaesophageal lymph nodes. The lesion, which was biopsied by transbronchial lung biopsy (TBLB), was diagnosed as ‘Ewing-like adamantinoma’. At 52 years of age, he started systemic chemotherapy with five cycles of vincristine, doxorubicin, and cyclophosphamide (VDC) combination chemotherapy, with the maximum doxorubicin dosage, resulting in stable disease (SD). Next, he received eight cycles of vincristine, actinomycin D, and cyclophosphamide (VAC) combination chemotherapy. In addition, he received radiation therapy (60 Gy/30 fractions) for the tumor in the left lower lobe. However, he developed radiation pneumonitis and steroid therapy was initiated at a dose of 40 mg prednisolone per day. The prednisolone dosage was gradually reduced, and once a dosage of 5 mg/day was reached, the patient started receiving eribulin monotherapy as second-line therapy. Prednisolone was administered until the completion of 16 cycles of eribulin. Microsatellite instability (MSI) tests using the TBLB specimens yielded negative results. In addition, a BCL2L1 amplification, previously reported in EWSR1::NFATC2 sarcoma, was identified using FoundationOne® CDx (12). Although EWSR1 rearrangement was not detected using FoundationOne® CDx, FISH analysis using an EWSR1 break apart probe revealed amplification of the 5′-end of EWSR1, indicating the rearrangement of the EWSR1 gene, compatible with EWSR1::NFATC2 sarcoma. Reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers (forward: 5′-GAGAGAACCGGAGCATGAGTG-3′ and reverse: 5′-CTTGGGCTGCACCTCGATCCGC-3′) followed by direct sequencing revealed an in-frame EWSR1::NFATC2 fusion where exon 8 of EWSR1 (ENST00000397938.7) was fused to exon 3 of NFATC2 (ENST00000371564.8), as previously shown by Sadri et al (9). We also used a pair of primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse: 5′-GAAGATGGTGATGGGATTTC-3′) as internal control for RT-PCR. In addition, the tumor mutational burden (TMB) was found to be 1 Mut/Mb. Three months later, he had progressive disease (PD): left pleural dissemination progressed, left pleural effusion increased, and peritoneal dissemination in the left paracolic gutter was suspected. The next treatment was trabectedin monotherapy. Although he also had a bone infection in the postoperative region of the left tibia, he continued the trabectedin monotherapy with concurrent oral antibiotics. The patient remained stable for 16 months, however, after 18 cycles of trabectedin monotherapy, the patient developed PD. He was subsequently administered pazopanib (800 mg/day). After 2 months, he once again had PD due to the progression of the pleural effusion.

Discussion

In recent years, EWSR1::NFATC2 sarcoma has become recognizable using various pathological and genetic tests. Our patient was diagnosed by additional immunohistochemical staining for NKX3-1, FISH, and RT-PCR followed by direct sequencing. EWSR1::NFATC2 sarcoma is classified as ESWR1::non-ETS sarcoma, a wastebasket diagnosis; however, it comprises the majority of this class of Ewing-like sarcomas, and the number of case reports and studies have been accumulating.

EWSR1::NFATC2 sarcoma often occurs in the diaphyseal medulla of long bones, similar to Ewing sarcoma. In the present case, the tumor was located at the proximal site of the metaphysis of the left tibia (Fig. 1A and B). Notably, no tibial tumors, including those in our case, occurred on the anterior side of the cortex, which is the most common site of classic adamantinoma of the long bones, as shown in Table SI (4,8,13–17). EWSR1::NFATC2 sarcomas tend to grow slowly and are relatively indolent in nature. The metastatic tumors in our case were also characteristic because of their slow growth.

Figure 1. Sagittal magnetic resonance imaging of the primary tibial tumor. The tumor was present in proximal site of the metaphysis of the left tibia. The periosteal or intracortical envelopes from the tibial metaphysis to the epiphysis show (A) a lytic lesion of vague low intensity on T1-SE and (B) mild hyperintensity on T2-FSE (arrow heads pointing to the right), involving soft tissue mass protruding to the dorsal side of the lytic lesion (arrow heads pointing to the left).

Ewing-like adamantinoma is composed of relatively uniform, small round or epithelioid cells arranged in nests, cords, or trabeculae with fibrous or myxoid stroma. Our patient had an epithelioid malignant tumor of the left tibia, suggesting a Ewing-like adamantinoma 19 years previously. This case was similar to the EWSR1::NFATC2 sarcoma case reported by Makise et al (11).

When we reanalyzed the tissue samples, hematoxylin and eosin staining revealed relatively uniform round cells in a trabecular arrangement within a fibrous background (Fig. 2A) and epithelioid tumor cells with abundant clear cytoplasm, forming nests or trabeculae (Fig. 2B). Immunohistochemical staining revealed that the tumor cells were weakly and focally positive for AE1/AE3 (Fig. 2C), weakly positive for CD99 (Fig. 2D), and diffusely positive for NKX3.1 (Fig. 2E).

Figure 2. (A and B) Histopathological examination and (C-E) immunohistochemical examination of the tissue samples. Hematoxylin and eosin staining shows (A) relatively uniform round cells in a trabecular arrangement within a fibrous background and (B) epithelioid tumor cells with abundant clear cytoplasm also forming nests or trabeculae (scale bars, 100 µm). Immunohistochemical staining shows the tumor cells are (C) weakly and focally positive for AE1/AE3, (D) weakly positive for CD99 and (E) diffusely positive for NKX3.1 (scale bars, 100 µm).

The main radiological characteristics of EWSR1::NFATC2 sarcoma are as follows: tendency to arise at the diaphysis of long bones, cortical expansion with buttressing-type thickening, and frequent bone surface involvement with saucer-like erosion without cortical destruction. Adamantinoma is a primary low-grade malignant bone tumor of epithelial origin (18). Although predominantly localized in the mid-tibial diaphysis, cases of synchronous or isolated lesions in the fibula have been reported. It is a rare neoplasm, comprising only 0.1–0.5% of all primary bone tumors (19). Our tibial EWSR1::NFATC2 sarcoma case is very rare and appears to be different from classic adamantinomas in terms of localization.

EWSR1::NFATC2 sarcoma carries a t(20;22)(q13;q12) chromosomal translocation (3). This type of sarcoma is translocation sarcoma (TRS). In 2015, trabectedin was approved in Japan for the treatment of patients with soft tissue sarcoma (STS) after a clinical trial targeting TRS (20). We previously demonstrated that the median progression-free survival was been 7.3 months in our single-institution cohort (21). Kobayashi et al (22) reported an overall median progression-free survival (PFS) of 3.7 months in a cohort of 140 patients who underwent trabectedin treatment at 29 Japanese Musculoskeletal Oncology Group institutions. With respect to the histological type in their study, the median PFS was 17.4 months for myxoid liposarcoma, 4.9 months for leiomyosarcoma, 5.6 months for synovial sarcoma, and 3.7 months for dedifferentiated liposarcoma, respectively. As the PFS was 16.4 months in our patient during trabectedin administration, he showed a noteworthy response to trabectedin without any severe adverse events. However, he developed PD after 18 cycles of trabectedin as the left pleural lesion grew bigger and the pleural effusion increased (Fig. 3A and B), and received best supportive care. Notably, the patient also had stable disease for more than a year with eribulin treatment. A future trial with eribulin in these patients may therefore be worthwhile.

Figure 3. Computed tomography imaging of the left metastatic lung and pleural lesions. The left metastatic pleural lesions have shrunk slightly and newly developed (yellow arrowhead) between the period (A) before start of trabectedin monotherapy and (B) after 18 cycles of trabectedin monotherapy. An increase in pleural effusion was observed.

With regard to drug therapy, several reports have suggested pazopanib as an effective treatment (18). A summary of metastatic cases treated with chemotherapy is presented in Table I. Seligson et al (12) demonstrated the mammalian target of rapamycin (mTOR) pathway as a potential therapeutic target in EWSR1::NFATC2 sarcomas using multiscale-omic assessment. They also presented the case of a 58-year-old male patient with metastatic EWSR1::NFATC2 sarcomas who achieved 47 months of disease stabilization when treated with a combination of the mTOR inhibitor, everolimus, and the vascular endothelial growth factor receptor-tyrosine kinase inhibitor, pazopanib. EWSR1::NFATC2 sarcomas are molecularly distinct entities with an overactive mTOR signaling pathway that may be therapeutically targetable. Gouda et al (18) reported a case of a patient with EWSR1::NFATC2 sarcoma in which exceptional tumor control was achieved using pazopanib and surgery, for an overall duration exceeding 5 years. In addition, Machado et al (23) suggested the possibility of exploring an immunotherapy approach because the transcriptomes of EWSR1::NFATC2 sarcomas should be enriched in genes associated with inflammatory and immune responses (13).

Table I. Clinicopathological features of reported cases of metastatic EWSR1::NFATC2 sarcoma.

Table I.

Clinicopathological features of reported cases of metastatic EWSR1::NFATC2 sarcoma.

First author, year	Age, years/Sex	Primary site	Surgery	Metastatic site	Gene alteration	Chemotherapy	Outcome/duration or follow-up	(Refs.)
Seligson ND, 2021	58/M	Intraperitoneal	Surgical excision	Retroperitoneum,	SPV in FANCE	ACT (VDC followed by IE)	Little benefit	(12)
		mass	of the mass	lung		Pazo alone	Little CB
						Pazo + CPT-11	Little CB
						Pazo + Eve	SD/26 mo
						Pazo + Pembro	SD/10 mo
						Nivo + Ipi	SD/4 mo
						Pazo + Eve (re-challenge)	SD/21 mo
Gouda MA, 2023	30sa/M	Left leg	Wedge resection;	Lung, heart	mTOR E1799K	PCT (VDC/IE)	NA	(18)
			surgical resection		mut, TOP1 amp	ACT (high-dose IFM alternating	NED/24 mo
			of the cardiac			with ADR and CDDP)
			metastasis			TMZ + CPT-11	MR
Present study	38a/M	Metaphyseal	Wide resection	Lung, LNs, pleura	BCL2L1 amp	VDC/VAC	SD/9 mo	-
		cortex of tibia		(RT 60 Gy/30 fr		Eribulin	SD/15 mo
		(left)		after VDC/VAC)		Trabectedin	SD/16 mo
						Pazopanib	PD/2 mo

a Age at initial diagnosis. ACT, adjuvant chemotherapy; ADR, adriamycin; amp, amplification; CB, clinical benefit; CDDP, cisplatin; CPT-11, irinotecan; IFM, ifosfamide; fr, fractions; LN, lymph node; M, male; mo, months; mut, mutation; MR, minimal response; NA, not applicable; NED, no evidence of disease; PCT, preoperative chemotherapy; RT, radiation therapy; SD, stable disease; SPV, single pathogenic variant; VAC, vincristine, actinomycin D, and cyclophosphamide; VDC, vincristine, doxorubicin, and cyclophosphamide.

In conclusion, we described a case of metastatic EWSR1::NFATC2 sarcoma that was initially diagnosed as a Ewing-like adamantinoma arising from the left tibia. Reconsideration during the durable response to trabectedin revealed a definitive diagnosis of a translocation-related sarcoma, EWSR1::NFATC2 sarcoma. Accordingly, physicians should consider EWSR1::NFATC2 sarcoma in patients who have been previously diagnosed with Ewing-like adamantinoma with a slow clinical course. It is important to diagnose it via FISH analysis using an EWSR1 break-apart probe. Amplification of the 5′-end of EWSR1 indicates the rearrangement of the EWSR1 gene, which is consistent with EWSR1::NFATC2 sarcoma. In addition, this sarcoma may have a prolonged response to eribulin and trabectedin in preventing or delaying the progression of additional metastasis after adriamycin-based chemotherapy. Further research is required to identify additional active agents or sequences for chemotherapy for this type of sarcoma.

Supplementary Material

Supporting Data

Acknowledgements

The authors would like to thank Ms. Satoko Baba (Division of Pathology, Cancer Institute of Japanese Foundation for Cancer Research) for their technical assistance with fluorescence in situ hybridization.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

TU drafted the manuscript. TU, MO, TT and AO contributed to the management of the clinical case. TU, MO, KY, TT, KT, SM and KA contributed to conception and design, and analysis and interpretation of clinical data. KY performed histological assessment and immunohistochemistry. KY and YT performed RT-PCR and direct sequencing. HS, RO, XW, NF, YS, KN and JT contributed to the patient's care and acquisition of data. TN, MS and KH contributed to the acquisition of data. MO, KY, TT and ST contributed to interpretation of clinical data and reviewed the manuscript. TU and MO confirm the authenticity of all the raw data. KT, SM, KA and ST supervised this study. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.

Competing interests

The authors declare that they have no competing interests.

Glossary

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References