Background

CIC-rearranged sarcoma (CRS) is an undifferentiated round cell sarcoma that was that was first reported by Kawamura-Saito et al. in 2006 [1]. Because the cellular morphology and immunophenotype associated with this disease overlap with those of Ewing sarcoma, CRS is also called Ewing-like sarcoma. CRS is the most common type of Ewing-like sarcoma, and it exhibits a more aggressive clinical behavior than Ewing sarcomas [2].

The CIC gene is located on chromosome 19p13 [3] and encodes capicua transcriptional repressor, which is a high-mobility group (HMG) box transcription factor. Established fusion partners for the CIC gene mainly include DUX4 [4] and FOXO4 [5]. However, a recent transcriptomic study of tumors of the central nervous system has identified NUTM1 as a novel fusion partner of CIC. Importantly, NUTM1 rearrangements are known to underlie NUT carcinoma (NC), a highly aggressive type of undifferentiated carcinoma that most commonly occurs in the thorax, head, and neck region along the midline. Almost 75% of cases of NC harbor a BRD4::NUTM1 fusion originating from t(15; 19)(q14; p13.1) [6].

Gene profiles of tumors harboring CIC::NUTM1 fusions cluster tightly with those of CIC::DUX4-related sarcomas, regardless of their anatomic location in the central nervous system, kidney, bone, or soft tissues [7, 8]. Compared to CIC::DUX4 sarcomas, those with the CIC::NUTM1 fusion tend to lead to a more aggressive clinical course, suggesting that this fusion is likely to be associated with worse prognoses, but additional clinical studies are needed to further investigate this hypothesis.

The morphologies of these various tumor types tend to be similar, thus potentially leading to misdiagnoses. For example, cells from sarcomas containing the CIC::NUTM1 fusion exhibit similar morphologies as those from CIC::DUX4 sarcomas, and the cellular morphology of CIC::NUTM1 sarcoma overlaps with that of NC. In both cases, tumors have been found to contain sheets of discohesive cells, which range from round-epithelioid to spindle shaped, and the tumors tend to have collagenous or abundant myxoid stroma. As NC may also occur in soft tissues and visceral organs [9] and to overexpress the NUT protein, CRS has the possibility of being misdiagnosed as NC; therefore, further confirmation by molecular pathologic techniques such as fluorescence in situ hybridization (FISH) and/or RNA sequencing is required to confirm the diagnosis. We report herein the first case of a CIC::NUTM1 sarcoma occurring in the soft tissues of the limbs. This case study identifies a new molecular subtype and raley location of the CRS.

Case presentation

An 18-year-old male patient was diagnosed with CIC::NUTM1-containing sarcoma in the soft tissues of the limbs by the Department of Pathology, Renmin Hospital of Wuhan University. The diagnosis was confirmed independently by two senior professional pathologists.

The patient experienced pain in the arms for more than 6 months prior to hospital readmission due to persistent pain with progressive enlargement of a mass. Computerized tomography imaging (CT) showed a soft tissue mass at the posterior part of the right distal humerus with smooth boundaries and no obvious destruction of adjacent bones (Fig. 1A). Magnetic resonance imaging (MR) showed soft tissue shadow signals behind the distal section of the right humerus and high intensity but uneven signals inT1-weighted and T2-weighted short tau inversion recovery imaging (Fig. 1B).

Fig. 1
figure 1

CIC::NUTM1 sarcomas. (A), CT exam showed a soft tissue mass at the posterior part of the right distal humerus. (B), MR imaging showed soft tissue signal shadows behind the distal section of the right humerus. The boundary was clear from the surrounding fat space, and no bone erosion was observed. (C), The tumor consisted of solid sheets with a vague lobular form separated by fibrosis septa. (D), Myxoid stromal changes areas were focally present, classified as a “myoepithelial-like” growth pattern, with variable round and epithelioid appearance. (E), Under high power, the tumor cells exhibited scant cytoplasm, and the nuclei contained vesicular chromatin with prominent single nucleoli, and mitotic activity was brisk. (F), Large patchy necrosis was observed in the center of the nest. (G), Tumor cells displayed spindles and were arranged in a storiform pattern. H to J, Immunohistochemistry showed diffuse nuclear expression of NUT (H) and nuclear and cytoplasmic expression of ETV4 (I), while staining was weakly positive for nuclear WT1cter (J). K and L, FISH using a CIC break-apart probe (K) and a NUTM1 break-apart probe (L)

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The tumor size was approximately 6.0 cm × 1.9 cm × 4.8 cm, and it contained irregular nodules. The boundary was clear from the surrounding fat space, and no bone erosion was noted, leading to it classification as a neoplastic lesion. Thoracic CT demonstrated multiple masses in both lungs, which were suspected of being metastatic lesions.

Morphology and immunohistochemical characteristics

Formalin-fixed paraffin-embedded tissue blocks were cut into 4-µm-thick sections and stained with hematoxylin and eosin using a standard protocol.

Upon gross examination, resection specimens presented as a solid mass that was grayish-white with hemorrhagic foci. The tumor margins were clear with focal fibrous pseudocapsules, and a local push-like infiltration into the surrounding skeletal muscle tissue was observed.

Histologically, the tumor presented an obvious nodular appearance, with nodules separated by thick fibrotic septa (Fig. 1C). The tumor consisted of uniformed small to medium sized round-to-ovoid cells with nuclei that were typically monotonous. The cells were arranged in a variety of patterns, mainly solid sheets or nests, with microcystic foci or loose reticular patterns in the chondroid or myxoid stromal background; these areas were similar in morphology to myoepithelial carcinoma in terms of the presence of loose or discohesive tumor cells (Fig. 1D).

The tumor cells exhibited scant cytoplasm that was transparent or mildly eosinophilic. The nuclei harbored hyperchromatic chromatin and vesicular chromatin with distinct single nucleoli. Cells exhibiting mitotic activity and apoptosis were relatively common, mitosis with approximately 19 observed per 10 HPFs (Fig. 1E). Large patchy necrosis was common (Fig. 1F).

The local area was composed of spindle cells arranged in herring bone or storiform patterns with rough nuclear chromatin was rough (Fig. 1G). The tumor cells expressed NUT (Fig. 1H) and ETV4 (Fig. 1I) proteins in a diffuse but strong and homogeneous pattern, and they were stained positive for WT-1 with weak to moderate intensity (Fig. 1J). The cell membranes exhibited multifocal strongly positive staining for CD99. Staining for P40, AE1/AE3, EMA, NKX2.2, myogenin, and MyoD1 were all negative. The Ki67 proliferation index was approximately 40%.

FISH

Upon FISH examination of the tumors, CIC (Fig. 1K) and NUTM1 (Fig. 1L) gene break-apart signals.

Discussion

CRS is the most common subgroup of the Ewing-like sarcoma family. This subtype presents as an undifferentiated round cell neoplasm, characterized by the occurrence CIC gene rearrangements [4]. This gene encodes an HMG box transcription factor. DUX4, the most common CIC fusion counterpart, is a double homeodomain gene located on chromosome 4q35 or 10q 26.3 [2, 10]. The function of DUX4 in normal cells remains unknown, but its expression is restrained in normal differentiated cells. The other, more rare, fusion gene partners ( including FOXO4, LEUTX, NUTM1, and NUTM2A) appear in approximately 5% of cases [5, 11, 12].

CRS mainly occurs in the soft tissues of young adults, especially in the soft tissues of the limbs and trunk. It also occurs rarely in older patients, where it is associated with a more aggressive clinical course than Ewing sarcomas [2]. CRS also shows a poor response to chemotherapy options that are used for Ewing sarcoma, and the 5-year overall survival rate is only 43%, which is significantly lower than that of Ewing sarcoma (77%) [2]. This biological evidence demonstrates that CRS represents a unique variant distinctive from Ewing sarcoma and it was acknowledged as an independent entity in the 2020 World Health Organization Classification of Bone and Soft Tissue Tumors (5th edition) [13].

The morphologic features of CRS include a lobular architecture divided by core fibrous septa, focal spindle cells, variable myxoid stroma, and extensive patchy necrotic areas. Compared with Ewing sarcoma, CRS is less monotonous and more pleomorphic. In particular, areas with vesicular nuclei and distinctive nucleoli are present in CRS. In rare cases, the neoplastic cells assume an epithelioid morphology, occasionally with rhabdoid cells or obvious cytoplasmic changes. The mitotic count is generally high. Most examples (85%) of CRS are found to express CD99 in various intensities [2], but in only 23% of cases is diffuse expression of CD99 observed. ETV4 [14] and WT1 (both WT1nter and WT1cter) are consistently positive [15].

NUT midline carcinoma is a clinically aggressive solid malignancy that mainly affects the axial structures of younger patients, and it typically results in death within a few months after diagnosis. The mediastinum and the upper aerodigestive tract are the most commonly affected anatomic locations [16]. With a propensity for early metastases to lymph nodes and pulmonary, the average overall survival of NC is only 6.7 months, and the prognosis is extremely poor [17]. NUTM1 gene rearrangement is the typical pathogenic cause of NC, with the most common fusion partner of NUTM1 being BRD4 [16]. As a result, overexpression of the NUT protein is seen in the vast majority of NC cases, and it has been determined to be highly specific for NC [18]. Despite it is named “carcinoma”, the tumor may occur at any anatomic location, including soft tissue [19].

NC has recently been found to exhibit a wider morphologic spectrum than that previously associated with the disease. NC cells include small cells, epithelioid cells, and round cells of the myxoid and loose reticular type [9]. Nuclei are monotonous, showing a round-ovoid form. Mitotic activity is brisk, and necrosis is frequently present. Dickson et al. [9]. reported six cases of primary NUTM1-rearranged undifferentiated tumors that occurred in the soft tissue of the extremities, kidney, stomach, and brain, most of which contained sheets of discohesive cells ranging from round-rhabdoid-epithelioid to spindle shaped. The stroma in NC is collagenous or abundant myxoid, and these features have more morphologic overlaps with undifferentiated round sarcomas, including CRS and malignant myoepithelial tumors. No potent squamous keratinization has been found in any of these cases. Of the six cases reported by Dickson et al., four cases expressed cytokeratin, but only two cases were significantly positive, which differed from classical NUT middle carcinoma. The relationship between NUT-associated tumors of the soft tissue or viscera and NUT midline carcinoma remains unclear.

CIC::NUTM1 sarcoma was initially identified in the central nervous system, but it may also occur in viscera, such as the kidney [20]. The sarcoma in the present case was located in the upper limbs, which was a rarely reported location. Nevertheless, bone involvement, especially spinal cord involvement [21], seems to be more frequent in CIC::NUTM1 sarcoma when compared to CIC::DUX4 sarcoma. Furthermore, patients with CIC::NUTM1 sarcoma tend to be younger, mainly in the pediatric population, with a median age of 6 years [12] for NUTM1 as compared to an average age of 21.6 years for patients with CIC::DUX4 (Table 1).

Tumors derived from this NUT fusion variant resemble NUTM1-rearranged undifferentiated tumors in soft tissue, and they tend to be associated with an aggressive clinical course. Antonescu et al. reported six cases of CIC::NUTM1 sarcoma, one of which was in a locally advanced stage, with an average survival of 18.6 months, which was significantly shorter than the mean survival associated with CIC::DUX4 sarcomas 139 months [2].

Our case progressed rapidly, with multiple bilateral lung metastases only 6 months after the first consultation. However, our case was found to be responsive to chemotherapy, as the primary tumor size shrank, and the metastatic pulmonary lesions were undetectable 6 months after the onset of chemotherapy. The primary tumor in the humerus also disappeared after 14 months of therapy, according to MR imaging. Therefore, while the CIC::NUTM1 fusion type may be associated with worse prognoses, our experience with enhanced sensitivity to chemotherapy may indicate that future cases will have better prognoses than have previously reported cases. However, it is worth noting that the present study was limited to a single case, and the follow-up period was also limited. Accordingly, we believe that it will be important to accumulate additional cases and to extend the duration of follow-up in order to obtain a more comprehensive understanding of long-term outcomes and to draw more robust conclusions.

Table 1 Summary of clinicopathologic and molecular features of CIC::NUTM1 sarcomas cases
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CIC::NUTM1 sarcoma exhibits a similar morphology to CIC::DUX4 sarcoma, including uniform small round cells arranged in a lobular pattern, with focal spindle cells and mucoid changes in the stroma. These characteristics are reminiscent of the myoepithelial-like features of CIC::DUX4 sarcoma, and both exhibit distinct nucleoli [22]. Some cases of CIC::NUTM1 are likely distinct from NC and are better classified as sarcomas, including those of the central nervous system or soft tissues [8]. Moreover, gene expression profiling performed in a large series of round cell sarcomas demonstrated that tumors with the CIC::NUTM1 fusion clustered tightly with CIC::DUX4 and CIC::FOXO4-positive sarcomas and separately from NCs [7], indicating a likely biologic relationship between these CIC-rearranged neoplasms; accordingly, the morphologies of these tumors more closely resemble those of classical CIC-fusion sarcomas. Our case showed classical morphologic features resembling those of CIC::DUX4 sarcoma.

The immunohistochemical staining of NUTM1 normally exhibits diffuse and uniform nuclear staining, but it is negative in round cell sarcomas except for those involving CIC::NUTM1, and thus acts as a specific marker for this form of sarcoma. CIC::DUX4 tumors consistently express ETV4 and WT1cter [15], because the CIC::DUX4 fusion transcript induces upregulation of polyoma enhancer activator 3 (PEA3/ETV4/E1AF) at both the RNA and protein levels. Le Guellec [14] thus found that ETV4 could be used as a marker to identify CRS from other undifferentiated round cell sarcomas. Likewise, in a series of cases reported by Le Loarer, ETV4 was positive in all but one case involving CIC::DUX4, while it was negative in NC. Distinct from CIC::DUX4 sarcomas, WT1cter is scattered positive in CIC::NUTM1 sarcomas. Our case was consistent with CIC::NUTM1 sarcomas: although the morphologic features presented similar to those of CIC::DUX4 sarcomas, the immunophenotype showed weak intensity positive for WT1cter and diffuse nuclear staining of NUTM1 (Table 2).

NC may display a myoepithelioid appearance or the presence of chondroid differentiation in soft tissues, overlapping with the morphological features of CIC-NUTM1 sarcomas; moreover, the immunophenotypes of both are NUTM1-positive, making distinguishing these tumors challenging.

Table 2 Differential diagnosis of CIC::NUTM1 sarcomas
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Positive staining for keratins and P40 does not necessarily permit a diagnosis of carcinoma over sarcoma. Moreover, cytokeratin is expressed consistently in several sarcomas, including epithelioid sarcoma and synovial sarcoma. Meanwhile, cytokeratin or P40 may be occasionally absent in NC [23]. Previous molecular studies have demonstrated that the CIC::NUTM1 fusion (n = 3) clustered tightly with CIC::DUX4 and CIC-FOXO4-fusion sarcomas and was split from NCs harboring the BRD3/4::NUTM1 fusion, suggesting that all CIC-rearranged tumors belong to a unique family of tumors [7, 8]. Although NUTM1 is positive in both CIC::NUTM1 and NC, the staining pattern is different. The diffuse homogeneous nuclear staining pattern of NUTM1 seems to be distinct from the dotted nuclear staining pattern in most NC (Fig. 2), although this distinction needs to be further examined in larger series. Therefore, the expression of ETV4 and homogeneous staining pattern of NUT leads to a preferential diagnosis of CIC::NUTM1 sarcoma rather than a NUT carcinoma (Table 2).

Fig. 2
figure 2

Differential diagnosis of NUT carcinoma versus CIC::NUTM1 sarcoma. (A), NUT carcinoma in the mediastinum of a 23 year old female patient, with tumor cells arranged in sheets of epithelioid monomorphic cells with myxoid stroma. (B), NUT carcinoma typically exhibits a punctate-positive nuclear pattern. (C), CIC::NUTM1 sarcoma with uniform small to medium cells of scant or clear cytoplasm. (D), NUT exhibits diffuse expression with a homogenous pattern in CIC::NUTM1, which is completely different from its expression pattern in NUT carcinoma

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Conclusion

We report here an extremely rare case of a CIC::NUTM1 sarcoma. This is the first case reported to occur in the soft tissues of the extremities, with genetic and clinicopathologic features distinct from those of CIC-rearranged sarcoma and NCs. Though the morphologic features of the tumors strongly resembled those of CIC-rearranged sarcomas, this entity showed a unique preference for axial bone and was associated with a more aggressive course, leading to a worse prognosis compared with classic CIC-rearranged sarcomas; therefore, we conclude that CIC::NUTM1 sarcoma is a unique entity from classical CRS, and it should be considered to be different from other CRS. When a tumor shows the typical morphology of CRS but stains positive for ETV4 and shows a uniform nuclear staining pattern for NUTM1, clinicians should consider a diagnosis of CIC::NUTM1 sarcoma. However, further confirmation by molecular pathologic techniques such as FISH or RNA sequencing should be employed to confirm the diagnosis.