Sarcoma With CIC-DUX4 Gene Fusion

Introduction

Nephroblastoma is the most common malignant renal tumor in childhood. Other malignant tumors have a predilection for the kidney, such as clear cell sarcoma and rhabdoid tumor.^1,2 While most nephroblastomas are easily recognized by their characteristic combination of epithelial, blastemal, and stromal elements, approximately 5% of these tumors are composed exclusively of undifferentiated blastemal or stromal elements. Wilms tumors (WTs) usually present as large abdominal masses which can extend to the renal vein and the inferior vena cava. They occur mainly between 1 and 5 years of age. ³ Rhabdoid tumors of the kidney are very aggressive tumors, usually found in younger children; they lack integrase interactor 1 (INI-1) expression. ⁴ Ewing sarcoma is a primitive, poorly differentiated, highly cellular round cell sarcoma originally defined by its primitive neural features. ⁵ CD99 expression was initially considered as a reliable marker for Ewing sarcoma; but development of molecular biology showed the possibility of absence of specific rearrangements (EWS-FLI1…) requiring research of other genetic abnormalities. Study of alternative molecular alterations in EWSR1-negative undifferentiated small round cell sarcomas is clinically relevant, since CIC-DUX4 sarcomas seem to be a very aggressive subset with poor response to the presently used therapeutic regimens. ⁶

We report a case of renal sarcoma with CIC-DUX4 gene fusion in a 12-year-old boy.

Case Report

A 12-year-old boy consulted for fever and abdominal pain. The abdominal ultrasound revealed a tumor of the lower pole of the right kidney with a thrombus in the renal vein and extending into the inferior vena cava. Computed tomography (CT) scan confirmed the tumor size (70 × 78 × 72 mm), revealed a rupture of the renal capsule (Figure 1), and an infiltration of the peri-renal fat and psoas. Chest CT scan showed multiple micro and macro pulmonary nodules bilaterally with a tree in bud pattern. The fine needle biopsy, indicated in this case because of an unusual renal tumor (age, size, and the aspect of metastasis), was postponed because of the hemorrhagic risk and extensive vena cava thrombosis. The patient received an emergency first line of chemotherapy, according to the SIOP 2001 protocol for stage 4 nephroblastoma with a 3-drug combination (Vincristine, Actinomycin D, and Doxorubicin). The biopsy performed 2 weeks later showed small undifferentiated cells; a WT could not be excluded. Ultrasound and CT scan evaluations during preoperative chemotherapy showed a partial regression of the tumor and the thrombus, allowing complete nephrectomy and thrombectomy 6 weeks after diagnosis. In the nephrectomy analyses, the presence of CD99 positivity led to a diagnosis of Ewing sarcoma. In light of this diagnosis of Ewing sarcoma and of the lack of regression of the lung metastases, treatment was upgraded to a 5-drug combination (Vincristine, Doxorubicin, Cyclosphosfamide, Etoposide, and Ifosfamide). After 2 courses, the chest CT scan showed a discordant response with regression of some lesions and progression of others. Excision of lung metastases was decided to confirm the diagnosis. Pulmonary tumoral growth was noted after 2 courses of chemotherapy consisting of Temozolomide and Irinotecan. Subsequently, a combination of Gemcitabine and Docetaxel was administrated. Initially, an objective tumor response was observed, but the CT scan performed after 8 courses showed the growth of previously noted metastases and revealed a new pulmonary nodule. Because of this progression, the patient had a complete resection of the right lower lobe, a partial resection of the right middle lobe, and metastasectomy of the left lung. Then, a new chemotherapy regimen using Cyclophosphamide, Navelbine, and Celebrex was introduced. A week later, a brain CT scan was performed because of frequent vomiting, and showed multiple brain metastases with brain herniation. Unfortunately, he died a few days later. Informed consent for case reports was obtained from the patient’s parents.

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Figure 1.

Initial CT scan showing a tumor of the right kidney, with a rupture of the renal capsule and an infiltration of the peri-renal fat and the psoas (Arrow).

All specimens were fixed in 10% formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin, eosin, and saffron.

The first biopsy revealed nests of small undifferentiated cells associated with large areas of necrosis which could correspond to a blastematous form of WT.

The radical nephrectomy demonstrated a unifocal 6 cm tumor that had not invaded the perirenal fat nor the hilum but extending into the renal vein as a tumoral thrombus. The cut surface had a lobulated periphery and was heterogenous with approximately 50% necrosis. Histologically, a spindle cell component composed of cells with coarse nuclear chromatin was noted (Fig. 2a). The predominant round cells were focally set in a myxoid stroma (Figure 2(B)). Finally, several nests of cells presented a rhabdoid morphology with vesicular nuclei and prominent nucleoli (Figure 2(C)). The mitotic activity was high (45 per 10 high-power fields). Renal hilar lymph nodes were negative for metastases. The lung metastases presented the same histological aspect as the primary tumor with only focal necrosis.

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Figure 2.

A, The hematoxylin and eosin section shows a spindle cell component; original magnification ×200. B, round cell component with focal myxoid stroma is noted; original magnification ×400. C, Cells with prominent nucleoli and more abundant cytoplasm, pushing the nuclei at the periphery giving a rhabdoid appearance, original magnification ×400. D, Immunohistochemical stains demonstrate diffuse membranous expression of CD99, original magnification ×400; and E, nuclear expression N-terminal of WT1, original magnification ×400; F, FISH analysis using a CIC-DUX4 fusion probe on an formalin-fixed paraffin embedded section. And 18.8% of the nuclei analyzed showed a fusion signal (overlapping or closely juxtaposed red and green signals, arrow), original magnification ×1000.

Immunohistochemistry was performed on paraffin-embedded sections using Ultraview Universal DAB detection kit (Ventana, Ventana medical system, Tuscon, AR) and antibodies against CD99, desmin, myogenin, synaptophysin, chromogranin, S-100 protein, WT1, and INI-1. Immunohistochemical stains demonstrated membranous CD99 positivity in approximately 90% of neoplastic cells (Figure 2(D)). Desmin, myogenin, synaptophysin, chromogranin, and S-100 were negative. Subsequently, diffuse nuclear positive Immunohistochemical staining with WT1 (N terminal) (Figure 2(E)) and INI were demonstrated.

On fresh-frozen tissue, molecular analysis of CIC-DUX4 was performed by the polymerase chain reaction (PCR) direct sequencing method. The primer sets used for PCR amplification and direct sequencing were obtained from the DNA sequencing core facility at the Institut Curie as previously described. ⁷ On the lung metastases, the reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated a CIC-DUX4 translocation.

Fluorescence in situ hybridization (FISH) analyses of interphase nuclei with regard to rearrangement of the EWSR1 and CIC-DUX4 was carried out on unstained 3–4 µm formalin-fixed paraffin-embedded tumor tissue sections using a paraffin pretreatment reagent kit (Vysis, Abbott Molecular, Rungis, France) and with EWSR1 commercial break-apart probes (Vysis, Downers Grove, IL, USA) and a CIC-DUX4 fusion probe (Empire Genomics, Interchim, Montluçon, France). Studies were performed and interpreted according to the manufacturer’s guidelines. At least 100 nuclei were analyzed. A result was considered positive when a fusion signal (overlapping or closely juxtaposed red and green signals) was detected in >10% tumor cells.^8,9

FISH studies showed an absence of EWSR1-translocation. But the CIC-DUX4 fusion probe demonstrated that 18.8% of the nuclei analyzed showed a fusion signal (overlapping or closely juxtaposed red and green signals) (Figure 2(F)).

Discussion

Ewing-like sarcomas are a heterogenous group of tumors that frequently affect pediatric and young adult patients. ¹⁰ A subset of Ewing-like sarcomas harboring the CIC-DUX4 or BCOR-CCNB3 fusions has been described. Both sarcoma types showed not only a small round cell component but also heterogeneous components, including spindle cells with low mitotic activity. ¹¹ Sarcomas with CIC-DUX4 gene fusion are preferentially located in soft tissues. ¹² Only 2 other cases have been described in kidney—only 1 was in an adult ¹³ and the other had a novel variant of CIC-DUX4 gene fusion. ¹⁴ These tumors preferentially affect young adults with a median age at diagnosis of 27–30 years. The most common site of involvement is the trunk but some cases have been described in the head and neck and extremities^12,15; Tumors with CIC-DUX4 are highly aggressive neoplasms with a rapid growth.^12,15,16 In the patients who died of the disease, the majority presents pulmonary distant metastases and they develop rarely brain metastases.^8,17 Histologically, these tumors are characterized by a proliferation of undifferentiated small round cells interrupted by extensive areas of geographic necrosis. The tumor cells contain nuclei with prominent nucleoli and abundant eosinophilic cytoplasm with a rhabdoid appearance, in contrast to Ewing sarcoma. The majority of the tumors lack spindle cell differentiation. The mitotic rate is high. Immunohistochemical studies show membranous mostly focal CD99 staining unlike Ewing sarcoma that shows diffuse membranous CD99 expression.^6,8,15–18 WT1 staining is positive whereas it is negative in Ewing sarcoma.^15,19 These characteristics can be helpful in the diagnosis of round cell sarcomas negative for EWSR1 rearrangement. Moreover, nuclear INI-1 staining is retained in these tumors whereas neural and neuroendocrine markers including S-100, GFAP, synaptophysin, and chromogranin are negative.^6,8,15–18 The lack of any lineage-specific markers can make diagnosis and classification difficult. Molecular analysis by either FISH or RT-PCR represents the gold standard to establish a definitive diagnosis of CIC-DUX4 round cell sarcomas. By FISH, these tumors are negative for the EWSR1 gene rearrangement. CIC-DUX4 gene fusion involves the fusion of CIC with DUX4. ¹⁵ Kawamura-Saito et al. ²⁰ showed that CIC–DUX4 functions as a dominant oncogene, and the chimeric protein may act as an oncogenic transcription factor. The addition of the DUX4 C-terminus to CIC, modifies CIC’s transcriptional activity. CIC-DUX4 might interfere with the regulation of CIC downstream targets. CIC-DUX4 leads to an up-regulation of the expression of many genes, in particular, members of the polyoma enhancer activator 3, a subclass of the ETS transcription factor gene family which might play an important role in tumorigenesis. Moreover, up-regulation of polyoma enhancer activator 3 family genes by CIC-DUX4 fusion may represent a molecular change equivalent to EWSR1-ETS fusion, suggesting a possible pathogenetic link between CIC-DUX4 sarcoma and Ewing sarcoma. ¹⁵ For these rare tumors, no standard therapy exists. In general, surgery, radiation therapy, and chemotherapy are used. ¹²

Here, we describe a case of renal Ewing-like sarcoma characterized by a specific CIC-DUX4 translocation as detected by molecular analysis and confirmed by FISH. Correct recognition of these entities is facilitated by cytogenetic or molecular genetic studies. Fundamental knowledge of this entity may drive to the development of new targeted therapies.