At the crossroad between Ewing sarcoma and neuroblastoma: a report of two cases of Ewing sarcoma with post treatment neuroblastoma-like differentiation

Abstract

Background

Ewing sarcoma (ES) is the second most frequent sarcoma of bone, often affecting young patients and pursuing an aggressive clinical course. Among therapeutic choices, radio- and chemotherapy are employed in neoadjuvant setting, and they yield variable histological changes in neoplastic tissue, which mainly include necrosis and fibrosis. Cytodifferentiation is seldom observed in pediatric tumors such as nephroblastoma and rhabdomyosarcoma following treatment. Nevertheless, it represents an extremely rare phenomenon in ES.

Case presentation

In this study we present the clinico-pathologic and molecular features of two cases of ES undergoing neuroblastoma-like differentiation after treatment. Both tumors were primarily located in bone and presented the histologic and immunohistochemical features of classic ES in needle biopsies. They were treated with standard chemotherapy protocols followed by surgical resection. The resection specimens of the primary tumor of patient 1 and pleural metastases of patient 2 presented foci of eosinophilic fibrillary stroma resembling neuropil and containing cellular elements with wide granular eosinophilic cytoplasm, eccentric nuclei containing vesicular chromatin and prominent nucleoli, reminiscent of ganglion cells. These cells were positive for chromogranin, synaptophysin and CD56, while CD99 was negative. Molecular confirmation of EWSR1 rearrangement was provided in both cases by next generation sequencing and FISH analysis.

Conclusions

Evidence of neural differentiation in ES unravels interesting hints about its controversial histogenesis. Furthermore, awareness of this event must be increased to avoid misdiagnosis with neuroblastoma, which shows significant morphological overlap.

Background

First described by James Ewing in 1921, Ewing’s Sarcoma (ES) is an aggressive round cell neoplasm of uncertain differentiation, mostly affecting children and young adults and characterized by recurrent t(11;22)(q24;q12) translocation, involving EWSR1 and a member of the ETS family of transcription factors [1, 2]. It accounts for the second most common tumor of bone in children after osteosarcoma, with a peak incidence at 15 years old [3]. Although the diaphysis of long bones is the most common site of occurrence, extraskeletal locations are possible, mainly represented by extremities and paravertebral regions [4]. Classical histopathology of ES is characterized by sheets of uniform round cells with stippled chromatin and small nucleoli [5, 6]. However, ES bears a certain heterogeneity in histopathological presentation and occasionally appears under morphological variants such as Peripheral Neuroectodermal Tumor (PNET), or more rarely large cell ES, ES/PNET with vascular-like features, synovial sarcoma-like ES, sclerosing ES and adamantinoma-like ES [6]. PNET is characterized by an overt neuroectodermal differentiation, displaying variable amounts of Homer-Wright pseudorosettes in addition to the typical features of ES [6, 7].

Among round cell tumors entering the differential diagnosis, undifferentiated neuroblastoma is worth considering due to its monotonous histological appearance strictly resembling ES [8]. Immunohistochemistry can be helpful since ES is diffusely and strongly positive for CD99, whereas neuroblastoma is negative [9–10]. The latter is strongly positive for CD56, which can be expressed by ES though, correlating with a better prognosis. Neural markers such as NSE and synaptophysin can be positive in both tumors, thus not being useful in their distinction [11].

Notwithstanding the histopathological similarities, ES and NBT represent two well distinct entities, differing by biology, clinical features, and management. Herein, we discuss two cases of ES revealing peculiar histological features after radio- and chemotherapy. Molecular investigation demonstrated the presence of EWSR1 rearrangement in both cases confirming the diagnosis of ES.

Case presentation

Case 1

A 10-year-old girl presented at another Institution with increasing pain in her right arm associated with swelling of the shoulder. MRI and PET revealed a large scapular mass infiltrating the adjacent soft tissues (Fig. 1). A biopsy was performed, and histopathological evaluation was conclusive for the diagnosis of ES. Therefore, she underwent two cycles of chemotherapy (IE and VAC). She came to our attention three months after the onset of symptoms. Radiological studies showed a partial response to treatment. The histological review of biopsy confirmed the initial diagnosis of ES. Hence, the patient continued her treatment with a cycle of neoadjuvant chemotherapy with VIDE followed by VAC according to the ISG/AIEOP EW-1 protocol. After treatment, PET, thoracic CT and MRI of the right shoulder showed a further decrease in size of the mass (Fig. 1).

Fig. 1

MRI images comparison between pre-chemotherapy (a-c) and post-chemotherapy (d-f, h) showing the reduction of the large expanding mass of the right scapular region with sign of resorption of the spina of the scapula and infiltration of the surrounding muscles. The lesion shows intermediate signal on T2w image (a, d), slightly hyperintense on T2w FatSat images (b, e, f), and high enhancement on post-contrast T1w FatSat GRE image (c). The highly cellular lesion is depicted on the ADC maps, available only in the exams performed during chemotherapy, as hypointense with an increase in ADC values from July (g) to September (h) (see ROIs measurements on the top right of g and h images)

The patient underwent total scapulectomy, and in the resection specimen the residual viable tumor accounted for approximately 60% of the whole lesion. Hence the patient was considered a poor responder and was treated with high-dose chemotherapy with VAC, CE and IE. In perspective of myeloablative Busulfan/Melphalan (BuMel) consolidation, hematopoietic stem cell apheresis was performed. Eventually, she underwent another cycle of VAC and BuMel high-doses chemotherapy with autologous HSCs rescue. The patient was disease free at two-year follow-up. The functionality of the affected arm appeared to be fully restored.

The pre-treatment biopsy showed a monotonous proliferation of round cells arranged in discohesive sheets. At higher magnification neoplastic cells displayed a high nucleus/cytoplasmic ratio, with finely dispersed chromatin and inconspicuous nucleoli. Immunohistochemistry showed intense CD99 positivity and negative stain for CD20 and CD3. Unstained sections and paraffin blocks were not provided, preventing from further immunohistochemical and molecular studies. In the resection specimen viable neoplastic cells were intermixed with extensive areas of fibrosis and regression. At low power a striking biphasic appearance was noted, with a predominant component made of uniform round cells growing in solid and pseudo-alveolar architecture, displaying the classical features of ES. Intermingled with the main population, several Homer-Wright rosettes were present imparting an overt neuroectodermal phenotype (Fig. 2). The neoplastic proliferation was partly divided by thin fibrovascular septa lined by variable layers of cells depicting a pseudopapillary pattern of growth (Fig. 2). The second component consisted of scattered foci of eosinophilic stroma showing a fibrillary quality with a close resemblance to neuropil. This matrix engulfed unexpected cellular elements with wide granular eosinophilic cytoplasm, eccentric nuclei containing vesicular chromatin and prominent nucleoli, with occasional binucleation, reminiscent of ganglion cells (Fig. 2). The latter findings were consistent with a differentiating neural component. Immunohistochemically, the round cell population stained diffusely positive for CD99 and S100, whereas neural markers such as chromogranin, synaptophysin and CD56 were negative (Fig. 3). In a specular fashion, the ganglion-like cells were positive for chromogranin, synaptophysin and CD56, while CD99 was negative and S100 showed a focal and weak positivity (Fig. 3). Notably, synaptophysin and CD56 expression were positive in the fibrillary matrix (Fig. 3). Markers of muscular differentiation such as desmin, myogenin and MyoD1 were all negative. Comparison between the histological features of the biopsy specimen and resection specimen was highly suggestive for neural cytodifferentiation induced by treatment. Next Generation Sequencing with FusionPlex Expanded Sarcoma (Archer FusionPlex Sarcoma v2 Panel) performed on the resection specimen, detected a EWSR1::FLI1 fusion (breakpoints: chr22:29683123,chr11:128675261). The presence of EWSR1 rearrangement was confirmed by FISH with break apart probe for EWSR1.

Fig. 2

Post chemotherapy Ewing sarcoma showing areas with a pseudopapillary growth pattern (A) and foci rich in eosinophilic fibrillary stroma resembling neuropil (B). These areas contained several cellular elements with wide granular eosinophilic cytoplasm, eccentric nuclei with vesicular chromatin and prominent nucleoli, reminiscent of ganglion cells (C)

Fig. 3

Representative images of the immunohistochemical stainings. A: Diffuse membrane staining of round cells for CD99. B: S100 positivity is limited to the round cell component. C: CD56 is positive only in the areas rich in ganglion cells. D: Synaptophysin positivity is detected only in areas with fibrillary stroma and ganglion cells

Case 2

A 19-year- old girl received a diagnosis of ES localized in sacrum with metastases to lungs at another institution. After resection of the sacral tumor, she underwent several cycles of radio- and chemotherapy consisting of Vincristine, Doxorubicin, Cyclophosphamide, Ifosfamide, Etoposide (VDC/IE), Vincristine, Actinomycin D and Ifosfamide (VAI), Gemcitabine, Docetaxel (GEM-DOC) and Cyclophosphamide due to multiple pulmonary recurrences. She then came to our center presenting with bilateral pleural metastatic disease at CT investigation (Fig. 4) and received six further cycles of radio- and chemotherapy, including Vincristine, Irinotecan, Temozolomide (VIT), High-Dose Ifosfamide, High-Dose Methotrexate, Cisplatin, and Doxorubicin (HDIFO), Carboplatin, Etoposide (CE), followed by surgical excision of residual pulmonary lesions. At five-months follow-up she was free of disease.

Fig. 4

CT images showing the pleural secondary localization with central necrotic area and peripheral enhancement of the lesions, progressively and gradually increasing through time and phases. A: non contrast; B: arterial phase; C: venous phase; D: equilibrium phase

Histopathologic investigation on surgical specimen of pleural metastases showed a double population, the predominant one being composed of uniform round cells associated with branching fibrovascular septa, in keeping with ES. This population was intimately associated with islands of granular and eosinophilic matrix reminiscent of neural stroma (Fig. 5). Moreover, ganglion-like cells with abundant cytoplasm and binucleation were occasionally embedded in these stromal foci, reflecting a neural phenotype (Fig. 5). Immunohistochemical stains mirrored the zonation observed by morphology, with CD99 and S100 positivity in the typical ES component and neural markers including chromogranin, synaptophysin and neurofilaments being expressed in areas with evidence of neural differentiation. Again, these features were indicative for a phenomenon of cytodifferentiation purportedly due to treatment. FISH analysis with break apart probe for EWSR1 demonstrated the presence of the rearrangement in both components.

Fig. 5

Pleural metastasis of Ewing sarcoma. Uniform round cells associated with branching fibrovascular septa, associated with islands of fibrillary eosinophilic matrix reminiscent of neural stroma (A). Large ganglion-like cells with abundant cytoplasm and binucleation were occasionally embedded in these stromal foci (B)

Discussion and conclusions

Cytodifferentiation in tumors is a well-documented phenomenon. In some pediatric tumors, especially neuroblastoma, it can occur spontaneously, which led Bolander to label this event as ‘the oncogenic grace period’ [12]. Anyway, maturation of tumor cells is far more common in post-treatment settings and has been extensively studied in some pediatric tumors such as rhabdomyosarcomas, where the extent of cytodifferentiation seems to be correlated with a favorable prognosis [13]. Likewise, embryonal tumors like nephroblastoma, medulloblastoma and neuroblastoma often undergo maturation after treatment [14], whereas this phenomenon is very uncommon for ES [15], where it usually undertakes neuroectodermal differentiation, reflecting the presumed histogenesis of ES. Albeit rare, the possibility of neural differentiation in ES must be considered to avoid misdiagnosis which may lead to suboptimal treatment and reduced survival.

Here, we report two cases of ES which acquired peculiar morphological features after chemotherapy, likely representing a phenomenon of post-treatment maturation. In case 1, the pre-operative biopsy showed a classical morphology for ES, whereas in the resection specimen unexpected features were noted, including the presence of ganglion-like cells embedded in a neuropil stroma which were highly suggestive for neural differentiation and closely resembled neuroblastoma. The latter finding was also seen in the post-treatment specimen of case 2 in a pleural metastasis. It is worth noting that evidence of neuroblastoma-like differentiation in ES has not been previously reported in the metastatic setting.

Rare cases of ES with post-treatment neuroblastoma-like maturation have been reported [16,17,18,19]. They occurred in a young population ranging from 8 to 17 years and showed a biphasic appearance with undifferentiated small cell population intermingled with varying amount of a ganglioneuroblastomatous component. Each case was eventually investigated with either FISH or RT-PCR which confirmed the presence of the EWSR1::FLI1 translocation. Among them, two cases primarily involved extraskeletal locations, namely soft tissues of forearm [16] and retroperitoneum [17], while the remainders were in bones, notably femur, radius and ilium. In all cases except one [18] molecular confirmation was carried out both before and after therapy, thus strengthening the correlation between neural cytodifferentiation and treatment-induced effect [17]. The case reported by Knezevich et al. failed to demonstrate the EWS gene rearrangement in the post-treatment specimen, possibly due to the outgrowth of a neoplastic clone devoid of the typical translocation [16]. Interestingly, the study by Salet et al. highlighted the presence of EWS rearrangement both in the typical round cell population and in the ganglioneuroblastoma-like component by FISH analysis [19].

The phenomenon of post-treatment neuroectodermal maturation can be explained by two possible mechanisms: (1) Selection and subsequent expansion of a pre-existing minor neural component after elimination of undifferentiated neoplastic cells by chemotherapy; (2) Chemotherapy directly inducing maturation of selective neoplastic clones which are prone to neural differentiation [18]. Although exceptionally, cases of ES with neural differentiation without prior treatment have also been described [20]. All of them notably involved extraskeletal locations, including intraspinal canal, retroperitoneum, cervix, lung and testis, and affected patients aged from 14 to 35 years. Besides, they were all confirmed by molecular investigation for the presence of EWSR1::FLI1 translocation. The cases reported by Weissferdt et al. were remarkable for the unusual sites of onset (cervix, lung, testis) along with the exceptional histopathological features observed in one case, which displayed an organoid and nested pattern reminiscent of an ependymomatous differentiation [20]. Considering these data, ES seems to intrinsically hold the capability for a true neural differentiation regardless of treatment, although much controversy still hangs over the histogenesis of this tumor. Another matter of debate had been long questioning the existence of PNET and its putative relationship with ES, until the finding of the same genetic abnormality in the two entities, leading to the conclusion that they represent the same disease [21–23].

Contemporary studies on ES/PNET gene expression profile suggest the neural crest stem cell genetic program to be involved in the tumorigenesis [24]. Therefore, the current opinion is that ES/PNET can derive from malignant transformation of mesenchymal stem cells originating either from mesodermal or neural crest [25]. Albeit the controversy concerning its neuroectodermal rather than mesenchymal origin is still pending, this theory provides a rationale to justify the phenotypic plasticity which may be occasionally observed in this tumour [26]. In this regard, our cases clearly demonstrated the potential for multiform differentiation beyond the classical round cell appearance, apparently unmasked by the effect of treatment. While the presence of a neuroblastoma-like component is a rare but well-documented finding in ES, the presence of fibrovascular septa configuring a pseudo-papillary and alveolar architecture displayed by case 1 is something worth commenting. In a case series reported by Hasegawa et al., they examined the histological, immunohistochemical and ultrastructural features of eleven round cell tumors and described four cases with atypical neuroectodermal differentiation featuring large polygonal cells or PNET morphology [27]. Among these atypical cases, they mentioned the presence of fibrovascular septa along with Homer Wright rosettes and ganglion-like cells, which closely resemble the features observed in our case, except for the pseudopapillary and alveolar-like pattern of growth that was lacking [27].

In conclusion, we report two cases of ES which acquired uncommon morphologic features after treatment, possibly representing a histopathologic diagnostic pitfall. The diagnosis was supported by molecular studies which confirmed the presence of EWSR1::FLI1 translocation and/or EWSR1 rearrangement in both cases. The awareness of this possible histological presentation in ES is important since it can be misleading in absence of proper clinical information and genetic studies. Finally, it could be of interest to explore any possible correlation between this phenomenon and clinical outcome in patients affected by ES.