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FLI1 translocation without prior chemotherapy
Human Pathology (2012) 43, 1772–1776
www.elsevier.com/locate/humpath
Case study
Extraosseous Ewing sarcoma with foci of neuroblastoma-like differentiation associated with EWSR1(Ewing sarcoma breakpoint region 1)/FLI1 translocation without prior chemotherapy K. Vali MD a , V. Kokta MD b , M. Beaunoyer MD a , R. Fetni PhD b , P. Teira MD c , Hervé Sartelet MD, PhD b,⁎ a
Department of Surgery, Hôpital Sainte-Justine, Montreal, Quebec, Canada Department of Pathology and Cytogenetic, Hôpital Sainte-Justine, Montreal, Quebec, Canada c Department of Pediatric Oncology, Hôpital Sainte-Justine, Montreal, Quebec, Canada b
Received 11 November 2011; revised 28 February 2012; accepted 2 March 2012
Keywords: Ewing sarcoma; Neuroblastoma
Summary Peripheral primitive neuroectodermal tumor/Ewing sarcoma and neuroblastoma are distinct malignant tumors belonging to the group of undifferentiated solid pediatric tumors. We report a case of a 14year-old adolescent girl who presented with a right lower quadrant mass. At surgery, a mobile retroperitoneal mass was entirely removed. Histologic evaluation revealed 2 distinct components; the first, consisting of sheets of undifferentiated cells, was CD99+ and CD56−, whereas the second, consisting of multiple foci of neuropil and maturing neuroblasts, was CD99− and CD56+. Fluorescence in situ hybridization analysis revealed the presence of EWSR1/FLI1 translocation in both histologic distinct components. MYCN (myelocytomatosis viral related oncogene, neuroblastoma derived) was not amplified. The tests for t(11;22) and t(21;22) performed by reverse transcription–polymerase chain reaction were negative. The final diagnosis corresponds to an extraosseous Ewing sarcoma with foci of neuroblastoma-like differentiation. This is the first case, documented by molecular studies, in which neuroblastoma-like differentiation has been noted in primitive neuroectodermal tumor/Ewing sarcoma without prior chemotherapy. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Peripheral primitive neuroectodermal tumor/Ewing sarcoma (PNET/ES) and neuroblastomas (NBs) belong to the group of pediatric small round cell tumors and are 2 clearly distinct malignant tumors [1]. However, the presence of morphological ⁎ Corresponding author. Department of Pathology, CHU Sainte-Justine, 3175, Côte Sainte-Catherine Montréal (QC), Canada H3T 1C5. E-mail address: [email protected] (H. Sartelet). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2012.03.006
and immunophenotypic neural differentiation suggests a link between the two. Clinically, ES is a frequent highly malignant tumor that usually affects children and young adults. In the same age group, extraskeletal PNET/ES is most frequently developed in extremities, paravertebral regions, and the retroperitoneum. Conversely, NB occurs in early childhood and is distributed along sympathetic ganglia in addition to the adrenal medulla. Approximately 80% to 90% of patients with NB have elevated levels of catecholamine metabolites (vanillylmandelic acid and homovanillic acid) in their urine [1]. PNET/ES are nonsecreting
Ewing sarcoma and neuroblastoma tumors. Pathologically, the gross appearance of both tumors varies, but in general, they are multilobulated, soft, fleshy, gray, and partially hemorrhagic tumors. Microcalcifications are rare in PNET/ES but are frequent in NB. Microscopically, NB tumors have a broad spectrum of differentiation varying between undifferentiated tumors composed only of small rounded blue cells and ganglioneuroma composed uniquely of ganglia and Schwann stromal cells [1]. NB with varying degrees of differentiation containing Schwann and ganglion cells and/or neuropil is easily distinguished from PNET/ES. In contrast, undifferentiated NB tumors and ES have overlapping histopathologic morphology. Moreover, Homer-Wright rosettes, containing a central solid core of neurofibrillary material surrounded by neuroblasts, can be found in both NB and in PNET. Immunohistochemically, the MIC2 (CD99) antigen demonstrates a membranous expression in a large majority of PNET/ES [2]. NB cells are characterized by an intense expression of the 140-kd neural cell adhesion molecule, neural cell adhesion molecule (N-CAM) (CD56) [3]. Both tumors stain
1773 positive for neural markers, including neuron-specific enolase, Leu-7, and synaptophysin. Cytogenetic and molecular analyses have revealed that 90% to 95% of PNET/ES are characterized by rearrangement of the EWSR1 (Ewing sarcoma breakpoint region 1) gene on 22q12 [4]. The most frequent translocation is t(11;22)(q24; q12), resulting in fusion of the FLI gene on 11q24. The fusion gene encodes an oncoprotein (EWS-FLI fusion protein) domain of FLI1 that generates aberrantly active transcription factors capable of DNA binding and malignant transformation [4]. Many cytogenetic markers have been described in NB: MYCN amplification is the most important prognostic factor in this tumor, whereas 11q23 deletion is associated with aggressive tumors and a poor prognosis [5]. The treatment varies greatly between the localized stages of these 2 distinct tumors. The treatment of low-risk nonmetastatic NB is uniquely surgical removal of the primary tumor. In contrast, patients with localized PNET/ES systematically receive chemotherapy before or after surgery. The chemotherapy
Fig. 1 A, Most of the tumor consisted of sheets of malignant small, uniform, round blue cells without stroma (hematoxylin-eosin-saffron, original magnification ×200). B, The second pattern consisted of poorly differentiated NB with a rich neuropil stroma, and some tumor cells had cytomorphologic features of differentiation toward ganglion cells (hematoxylin-eosin-saffron, original magnification ×400). C, In the small, round, blue cell areas, there was membranous positivity with CD99 (CD99, original magnification ×400). D, In the neuropil-rich areas, malignant cells had a strong membranous expression of CD56 and an absent CD99 expression (CD56, original magnification ×400).
1774 consists of a combination of several drugs including anthracyclines and, frequently, doxorubicin. We present the first case documented with molecular studies of an extraosseous ES with foci of NB-like differentiation without prior chemotherapy, discovered in a 14-year-old adolescent girl who presented with a retroperitoneal mass.
2. Case report 2.1. Clinical course A 14-year-old adolescent girl presented with a painless, mobile, right lower quadrant abdominal mass. No significant medical or family history was noted. The patient was well in appearance and afebrile. Physical examination was otherwise unremarkable. Hemoglobin level, white blood cells, and platelets were normal. Tumoral markers such as alpha-1fetoprotein (AFP) and human chorionic gonadotropin (βHCG) were negative. Ca 125 was mildly elevated at 67.9 U/mL (upper limit of normal, 35 U/mL). Ultrasound studies revealed a noncalcified right lower quadrant mass, in continuity with right ovary, measuring 9.6 × 5 × 6.9 cm. Centrally, it was heterogeneous with hemorrhagic elements and more solid appearing on the outer rim. No enlarged lymph nodes could be observed, and no metastatic disease was seen on the bone scan, computed tomographic scan of the chest, or bilateral bone marrow aspirate/biopsy. Based on these findings, the patient was scheduled for surgery within a week after presentation. After entering the abdominal cavity, both ovaries were seen, and both were found to be normal. Upon exploration of the abdomen, a mobile retroperitoneal mass with a short mesentery, originating at the iliac bifurcation, was identified. This entire mass was removed without any complications or spillage. The abdominal cavity was inspected, and no tumor implants or other retroperitoneal masses were found. Because of this retroperitoneal mass, she underwent whole-body positron emission tomography (PET)/computed tomographic scan, which was negative. At tumor board, the decision was made to treat the patient according to the protocol AEWS 0031 of the Children Oncology Group with 14 cycles of chemotherapy done, alternating every 2 weeks VDC cycle (vincristine/doxorubicine/cyclophosphamide) and IE cycle (ifosfamide/etoposide). She is currently 6 months posttreatment, and she does not have any sign of relapse.
K. Vali et al. of malignant small, uniform, round blue cells without any intervening stroma (Fig. 1A). Nuclei contained fine powdery chromatin and 1 or 2 small nucleoli. The cytoplasm was scanty and pale staining. Mitotic figure were rare. The second pattern, representing approximately 10% of the tumor, consisted of poorly differentiated NB with a rich neuropil (Fig. 1A and B). Less than 5% of the tumor cells in these foci had cytomorphologic features of differentiation toward ganglion cells (Fig. 1B). Indeed, some cells presented an enlarged, eccentric nucleus associated with a single prominent nucleolus. The cytoplasm was more abundant and eosinophilic in the latter. The 2 histologic patterns were always intermingled. Immunohistochemistry was performed on both of these areas. In the small, round, blue cell areas, there was membranous positivity with CD99 (dilution, 1:50, Mouse Monoclonal; Dako, Carpinteria, CA) (Fig. 1C). Synaptophysin was negative, and Ki-67 was evaluated at 50%. In the neuropil-rich areas, there was no CD99 staining
2.2. Pathologic and immunohistochemical findings The surgical resection measured 9 × 6.5 × 5 cm and was mostly solid with a few hemorrhagic cysts. Representative tissue specimen was selected for cytogenetics and molecular biology analysis, frozen samples and imprints were made, and the rest was processed for routine histologic examination. Histologic evaluation revealed 2 different morphological patterns. Firstly, most of the tumor bulk consisted of sheets
Fig. 2 Interphase FISH analysis was realized in fresh imprints on small undifferentiated cells (A, FISH, counterstain 4′,6′-diamidino2-phénylindole [DAPI], original magnification ×1000) and on paraffin section in neuroblastoma-like foci (B, FISH, original magnification ×100 and, inset, DAPI, original magnification ×200) using FLI1/EWSR1 probes. We found a FLI1/EWSR1 t(11:22) translocation in both areas.
Ewing sarcoma and neuroblastoma (Fig. 1C), but cells demonstrated a membranous expression of CD56 (dilution, 1:20, mouse Monoclonal; Vector Laboratories, Burlingame, CA) (Fig. 1D). Weak cytoplasmic staining was seen with chromogranin, and the neuropil background was highlighted with synaptophysin and neuron-specific enolase (NSE). Ki-67 index was low (b10%). Immunohistochemical studies of Friend leikemia virus integration 1 (FLI-1) demonstrated positive nuclear staining in both malignant components.
2.3. Molecular cytogenetic analysis A fresh tumor fragment and imprints were sent for cytogenetic investigation. Interphase fluorescence in situ hybridization (FISH) analysis using the 2 probes near EWSR1 and FLI1 genes (FLI1/EWSR1 TRANSLOCATION LPS007, Aquarius solid tumor probes; Cytocell, Cambridge, United Kingdom) revealed an abnormal signal pattern in 92% of the undifferentiated malignant cells that consisted of a FLI1/EWSR1 t(11:22) translocation (Fig. 2A). A second interphase FISH analysis was performed on paraffin section in the neuroblastoma-like foci and also revealed the presence of FLI1/EWSR t(11:22) (Fig. 2B). FISH analysis did not demonstrate specific cytogenetic abnormalities of NB (absence of MYCN amplification and loss of 11q23 and of 1p36). These finding were consistent with a diagnosis of PNET/ES. The tests for t(11;22)( EWS/FLI1) and t(21;22)(EWS/ERG) performed by reverse transcription– polymerase chain reaction (RT-PCR) were negative. The clinicopathologic results were, therefore, consistent with a primary ES with foci of NB-like differentiation.
3. Discussion This fascinating tumor in an adolescent girl demonstrates the association in a same tumor of an ES associated with poorly differentiated NB. This case also demonstrates that the differences between the 2 tumor types are sometimes overlapping. NB are derived from primordial neural crest cells that migrate from the mantle layer of the developing spinal cord and populate the primordia of the sympathetic ganglia and adrenal medulla [1]. The histogenesis of PNET/ ES is still controversial. For a long time, a neural crest origin was also evoked for PNET/ES based on in vitro, immunohistochemical, and morphological studies [6]. A more recent hypothesis is that it might arise from mesenchymal stem cells [7]. These cells have the ability to differentiate into multiple distinct lineages including bone, cartilage, and neural cells. Based on the 2 latter hypotheses, either neural crest cell or mesenchymal stem cell origins, PNET/ES has a very close origin similar to that of NB, which may explain a mixed tumor like those we describe in our case. Homer-Wright rosettes can be seen in both NB differentiated and PNET/ES. Moreover, several publications described
1775 PNET/ES associated with the presence of ganglion cells [8,9]. In these tumors, undifferentiated small cells and ganglion cells were associated with foci of neuropil. Most of these tumors occurred in children or adolescents [8,9], but 2 tumors were discovered in adults [9]. Rarely tumors came from bones [9]. The most common location in soft tissue tumors was chest [8]. Only 2 patients died of the effects of the tumor [8]. On immunohistochemistry, NB, however, seldom, if ever, displays CD99 immunopositivity in a membrane pattern [1]. In our tumor, the CD99 remained positive only in the undifferentiated cells and not in ganglion cells. If CD56 is frequently expressed in NB [3], it could be found in ES and was associated in good prognosis [10]. In our case, the positivity of CD56 was only found in NB-like foci of the tumor. The FLI-1 immunoreactivity, in adjunction to FISH results, was critical in deciphering the 2 intermingled malignant components and, therefore, to render the diagnosis of PNET/ES with NB-like foci in this unique tumor. Cytogenetic or molecular analysis identification of mixed phenotype PNET/ES and NB has been rarely described in children. All such cases demonstrated postchemotherapy neural differentiation. Maeda et al [11] first reported in 1998 a case of a 12-year-old girl with ES of the radius bone, which after chemotherapy differentiated largely into largely ganglion cell tumor but was associated with EWS/FLI1 detected by RT-PCR. In the same year, an ES case from the forearm of a 10-year-old girl, which expressed EWS/FLI1 fusion transcripts, was reported. The tumor was treated with surgery, chemotherapy, and local radiation, but residual tumor was detected within a year lacking detectable EWS/ FLI1 expression and showed both neural and ganglion cells [12]. Collini et al [13] presented findings in a 17-year-old adolescent girl with iliac bone PNET that postchemotherapy had features of NB. Her tumor presented a EWS-FLI1 fusion transcript. Lastly, Weissferdt et al [14] analyzed the retroperitoneal tumor of an 11-year-old girl, which, on prechemotherapy biopsy, was identified as PNET/ES with t(11,22) fusion, but then after treatment and ultimately after excision of the mass, it had the morphological appearance of a NB but maintained its PNET/ES t(11,22) fusion, thus still confirming the diagnosis of PNET/ES. Interestingly, in our case, the specific EWS-FLI1 translocation was found in both morphologically different malignant components, revealing the same cell origin. We can speculate that additional genetic or epigenetic modifications led to the creation of a separate clone of neuroblastoma-like malignant cells within the Ewing sarcoma primary mass. Based on our FISH studies, these additional genetic modifications are not the ones most frequently found in NB (absence of MYCN amplification and loss of 11q23 and of 1p36). Moreover, the negative RT-PCR analysis for t(11,22) and t(21,22) was possibly due to a break point different from that typically found. FISH has been shown to be a more sensitive and reliable method than RTPCR for the diagnosis of EWS in solid tissue [15]. It is important to note that the treatments of localized neuroblastoma and of localized PNET/ES are entirely
1776 different. Historically, localized ES treated with surgery or radiotherapy alone have shown high incidence of metastatic relapses (≥70%). As a consequence, all protocols of treatment for Ewing sarcoma are currently based on heavy weighted chemotherapy regimens including high cumulative doses of alkylating agents, anthracyclins, and etoposide. These treatments put patients at risk for secondary cancer, poor health, and sterility. In contrast, localized neuroblastoma, without any other biologic factor of poor prognosis, is usually cured by surgery alone [1]. PNET/ES and NB may have overlapping histopathologic features both prior to and after chemotherapy. Therefore, immunohistochemical and molecular biology studies are essential in determining the correct diagnosis. In our case, despite the negative results of RT-PCR studies, FISH analysis on both separate malignant components revealed the presence of EWS/FLI1 translocation t(11:22). The latter, which was corroborated with FLI-1 nuclear immunoreactivity in both malignant components, was critical in the making the diagnosis of PNET/ES with foci of NB-like differentiation. The correct diagnosis is essential to provide the appropriate chemotherapy and corresponding clinical follow-up.
References [1] Weiss SW, Goldblum JR. Ewing's sarcoma/PNET tumor family and related lesions. In: Weiss SW, Goldblum JR, editors. Enzinger and Weiss's Soft Tissue Tumors. 5th ed. St Louis, MO: Mosby; 2008. p. 945-88. [2] Ambros IM, Ambros PF, Strehl S, et al. MIC2 is a specific marker for Ewing's sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of Ewing's sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer 1991;67:1886-93.
K. Vali et al. [3] Mechtersheimer G, Staudter M, Moller P. Expression of the natural killer cell-associated antigens CD56 and CD57 in human neural and striated muscle cells and in their tumors. Cancer Res 1991;51: 1300-7. [4] Aurias A, Rimbaut C, Buffe D, et al. Chromosomal translocation in Ewing's sarcoma. N Engl J Med 1983;309:496-8. [5] Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 2003;3:203-16. [6] Cavazzana AO, Miser JS, Jefferson J, Triche TJ. Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 1987;127:507-18. [7] Suvà ML, Riggi N, Stehle JC, et al. Identification of cancer stem cells in Ewing's sarcoma. Cancer Res 2009;69:1776-81. [8] Schmidt D, Harms D, Burdach S. Malignant peripheral neuroectodermal tumours of childhood and adolescence. Virchows Arch A Pathol Anat Histopathol 1985;406:351-65. [9] Ushigome S, Shimoda T, Nikaido T, et al. Primitive neuroectodermal tumors of bone and soft tissue with reference to histologic differentiation in primary or metastatic foci. Acta Pathol Jpn 1992; 42:483-93. [10] Ash S, Luria D, Cohen IJ, et al. Excellent prognosis in a subset of patients with Ewing sarcoma identified at diagnosis by CD56 using flow cytometry. Clin Cancer Res 2011;17:2900-7. [11] Maeda G, Masui F, Yokoyama R, et al. Ganglion cells in Ewing's sarcoma following chemotherapy: a case report. Pathol Int 1998;48: 475-80. [12] Knezevich SR, Hendson G, Mathers JA, et al. Absence of detectable EWS/FLI1 expression after therapy-induced neural differentiation in Ewing sarcoma. HUM PATHOL 1998;29:289-94. [13] Collini P, Mezzelani A, Modena P, et al. Evidence of neural differentiation in a case of post-therapy primitive neuroectodermal tumor/Ewing sarcoma of bone. Am J Surg Pathol 2003;27: 1161-6. [14] Weissferdt A, Neuling K, English M, et al. Peripheral primitive neuroectodermal tumor with postchemotherapy neuroblastoma-like differentiation. Pediatr Dev Pathol 2006;9:229-33. [15] Bridge RS, Rajaram V, Dehner LP, et al. Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT-PCR in malignant round cell tumors. Mod Pathol 2006;19:1-8.
www.elsevier.com/locate/humpath
Case study
Extraosseous Ewing sarcoma with foci of neuroblastoma-like differentiation associated with EWSR1(Ewing sarcoma breakpoint region 1)/FLI1 translocation without prior chemotherapy K. Vali MD a , V. Kokta MD b , M. Beaunoyer MD a , R. Fetni PhD b , P. Teira MD c , Hervé Sartelet MD, PhD b,⁎ a
Department of Surgery, Hôpital Sainte-Justine, Montreal, Quebec, Canada Department of Pathology and Cytogenetic, Hôpital Sainte-Justine, Montreal, Quebec, Canada c Department of Pediatric Oncology, Hôpital Sainte-Justine, Montreal, Quebec, Canada b
Received 11 November 2011; revised 28 February 2012; accepted 2 March 2012
Keywords: Ewing sarcoma; Neuroblastoma
Summary Peripheral primitive neuroectodermal tumor/Ewing sarcoma and neuroblastoma are distinct malignant tumors belonging to the group of undifferentiated solid pediatric tumors. We report a case of a 14year-old adolescent girl who presented with a right lower quadrant mass. At surgery, a mobile retroperitoneal mass was entirely removed. Histologic evaluation revealed 2 distinct components; the first, consisting of sheets of undifferentiated cells, was CD99+ and CD56−, whereas the second, consisting of multiple foci of neuropil and maturing neuroblasts, was CD99− and CD56+. Fluorescence in situ hybridization analysis revealed the presence of EWSR1/FLI1 translocation in both histologic distinct components. MYCN (myelocytomatosis viral related oncogene, neuroblastoma derived) was not amplified. The tests for t(11;22) and t(21;22) performed by reverse transcription–polymerase chain reaction were negative. The final diagnosis corresponds to an extraosseous Ewing sarcoma with foci of neuroblastoma-like differentiation. This is the first case, documented by molecular studies, in which neuroblastoma-like differentiation has been noted in primitive neuroectodermal tumor/Ewing sarcoma without prior chemotherapy. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Peripheral primitive neuroectodermal tumor/Ewing sarcoma (PNET/ES) and neuroblastomas (NBs) belong to the group of pediatric small round cell tumors and are 2 clearly distinct malignant tumors [1]. However, the presence of morphological ⁎ Corresponding author. Department of Pathology, CHU Sainte-Justine, 3175, Côte Sainte-Catherine Montréal (QC), Canada H3T 1C5. E-mail address: [email protected] (H. Sartelet). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2012.03.006
and immunophenotypic neural differentiation suggests a link between the two. Clinically, ES is a frequent highly malignant tumor that usually affects children and young adults. In the same age group, extraskeletal PNET/ES is most frequently developed in extremities, paravertebral regions, and the retroperitoneum. Conversely, NB occurs in early childhood and is distributed along sympathetic ganglia in addition to the adrenal medulla. Approximately 80% to 90% of patients with NB have elevated levels of catecholamine metabolites (vanillylmandelic acid and homovanillic acid) in their urine [1]. PNET/ES are nonsecreting
Ewing sarcoma and neuroblastoma tumors. Pathologically, the gross appearance of both tumors varies, but in general, they are multilobulated, soft, fleshy, gray, and partially hemorrhagic tumors. Microcalcifications are rare in PNET/ES but are frequent in NB. Microscopically, NB tumors have a broad spectrum of differentiation varying between undifferentiated tumors composed only of small rounded blue cells and ganglioneuroma composed uniquely of ganglia and Schwann stromal cells [1]. NB with varying degrees of differentiation containing Schwann and ganglion cells and/or neuropil is easily distinguished from PNET/ES. In contrast, undifferentiated NB tumors and ES have overlapping histopathologic morphology. Moreover, Homer-Wright rosettes, containing a central solid core of neurofibrillary material surrounded by neuroblasts, can be found in both NB and in PNET. Immunohistochemically, the MIC2 (CD99) antigen demonstrates a membranous expression in a large majority of PNET/ES [2]. NB cells are characterized by an intense expression of the 140-kd neural cell adhesion molecule, neural cell adhesion molecule (N-CAM) (CD56) [3]. Both tumors stain
1773 positive for neural markers, including neuron-specific enolase, Leu-7, and synaptophysin. Cytogenetic and molecular analyses have revealed that 90% to 95% of PNET/ES are characterized by rearrangement of the EWSR1 (Ewing sarcoma breakpoint region 1) gene on 22q12 [4]. The most frequent translocation is t(11;22)(q24; q12), resulting in fusion of the FLI gene on 11q24. The fusion gene encodes an oncoprotein (EWS-FLI fusion protein) domain of FLI1 that generates aberrantly active transcription factors capable of DNA binding and malignant transformation [4]. Many cytogenetic markers have been described in NB: MYCN amplification is the most important prognostic factor in this tumor, whereas 11q23 deletion is associated with aggressive tumors and a poor prognosis [5]. The treatment varies greatly between the localized stages of these 2 distinct tumors. The treatment of low-risk nonmetastatic NB is uniquely surgical removal of the primary tumor. In contrast, patients with localized PNET/ES systematically receive chemotherapy before or after surgery. The chemotherapy
Fig. 1 A, Most of the tumor consisted of sheets of malignant small, uniform, round blue cells without stroma (hematoxylin-eosin-saffron, original magnification ×200). B, The second pattern consisted of poorly differentiated NB with a rich neuropil stroma, and some tumor cells had cytomorphologic features of differentiation toward ganglion cells (hematoxylin-eosin-saffron, original magnification ×400). C, In the small, round, blue cell areas, there was membranous positivity with CD99 (CD99, original magnification ×400). D, In the neuropil-rich areas, malignant cells had a strong membranous expression of CD56 and an absent CD99 expression (CD56, original magnification ×400).
1774 consists of a combination of several drugs including anthracyclines and, frequently, doxorubicin. We present the first case documented with molecular studies of an extraosseous ES with foci of NB-like differentiation without prior chemotherapy, discovered in a 14-year-old adolescent girl who presented with a retroperitoneal mass.
2. Case report 2.1. Clinical course A 14-year-old adolescent girl presented with a painless, mobile, right lower quadrant abdominal mass. No significant medical or family history was noted. The patient was well in appearance and afebrile. Physical examination was otherwise unremarkable. Hemoglobin level, white blood cells, and platelets were normal. Tumoral markers such as alpha-1fetoprotein (AFP) and human chorionic gonadotropin (βHCG) were negative. Ca 125 was mildly elevated at 67.9 U/mL (upper limit of normal, 35 U/mL). Ultrasound studies revealed a noncalcified right lower quadrant mass, in continuity with right ovary, measuring 9.6 × 5 × 6.9 cm. Centrally, it was heterogeneous with hemorrhagic elements and more solid appearing on the outer rim. No enlarged lymph nodes could be observed, and no metastatic disease was seen on the bone scan, computed tomographic scan of the chest, or bilateral bone marrow aspirate/biopsy. Based on these findings, the patient was scheduled for surgery within a week after presentation. After entering the abdominal cavity, both ovaries were seen, and both were found to be normal. Upon exploration of the abdomen, a mobile retroperitoneal mass with a short mesentery, originating at the iliac bifurcation, was identified. This entire mass was removed without any complications or spillage. The abdominal cavity was inspected, and no tumor implants or other retroperitoneal masses were found. Because of this retroperitoneal mass, she underwent whole-body positron emission tomography (PET)/computed tomographic scan, which was negative. At tumor board, the decision was made to treat the patient according to the protocol AEWS 0031 of the Children Oncology Group with 14 cycles of chemotherapy done, alternating every 2 weeks VDC cycle (vincristine/doxorubicine/cyclophosphamide) and IE cycle (ifosfamide/etoposide). She is currently 6 months posttreatment, and she does not have any sign of relapse.
K. Vali et al. of malignant small, uniform, round blue cells without any intervening stroma (Fig. 1A). Nuclei contained fine powdery chromatin and 1 or 2 small nucleoli. The cytoplasm was scanty and pale staining. Mitotic figure were rare. The second pattern, representing approximately 10% of the tumor, consisted of poorly differentiated NB with a rich neuropil (Fig. 1A and B). Less than 5% of the tumor cells in these foci had cytomorphologic features of differentiation toward ganglion cells (Fig. 1B). Indeed, some cells presented an enlarged, eccentric nucleus associated with a single prominent nucleolus. The cytoplasm was more abundant and eosinophilic in the latter. The 2 histologic patterns were always intermingled. Immunohistochemistry was performed on both of these areas. In the small, round, blue cell areas, there was membranous positivity with CD99 (dilution, 1:50, Mouse Monoclonal; Dako, Carpinteria, CA) (Fig. 1C). Synaptophysin was negative, and Ki-67 was evaluated at 50%. In the neuropil-rich areas, there was no CD99 staining
2.2. Pathologic and immunohistochemical findings The surgical resection measured 9 × 6.5 × 5 cm and was mostly solid with a few hemorrhagic cysts. Representative tissue specimen was selected for cytogenetics and molecular biology analysis, frozen samples and imprints were made, and the rest was processed for routine histologic examination. Histologic evaluation revealed 2 different morphological patterns. Firstly, most of the tumor bulk consisted of sheets
Fig. 2 Interphase FISH analysis was realized in fresh imprints on small undifferentiated cells (A, FISH, counterstain 4′,6′-diamidino2-phénylindole [DAPI], original magnification ×1000) and on paraffin section in neuroblastoma-like foci (B, FISH, original magnification ×100 and, inset, DAPI, original magnification ×200) using FLI1/EWSR1 probes. We found a FLI1/EWSR1 t(11:22) translocation in both areas.
Ewing sarcoma and neuroblastoma (Fig. 1C), but cells demonstrated a membranous expression of CD56 (dilution, 1:20, mouse Monoclonal; Vector Laboratories, Burlingame, CA) (Fig. 1D). Weak cytoplasmic staining was seen with chromogranin, and the neuropil background was highlighted with synaptophysin and neuron-specific enolase (NSE). Ki-67 index was low (b10%). Immunohistochemical studies of Friend leikemia virus integration 1 (FLI-1) demonstrated positive nuclear staining in both malignant components.
2.3. Molecular cytogenetic analysis A fresh tumor fragment and imprints were sent for cytogenetic investigation. Interphase fluorescence in situ hybridization (FISH) analysis using the 2 probes near EWSR1 and FLI1 genes (FLI1/EWSR1 TRANSLOCATION LPS007, Aquarius solid tumor probes; Cytocell, Cambridge, United Kingdom) revealed an abnormal signal pattern in 92% of the undifferentiated malignant cells that consisted of a FLI1/EWSR1 t(11:22) translocation (Fig. 2A). A second interphase FISH analysis was performed on paraffin section in the neuroblastoma-like foci and also revealed the presence of FLI1/EWSR t(11:22) (Fig. 2B). FISH analysis did not demonstrate specific cytogenetic abnormalities of NB (absence of MYCN amplification and loss of 11q23 and of 1p36). These finding were consistent with a diagnosis of PNET/ES. The tests for t(11;22)( EWS/FLI1) and t(21;22)(EWS/ERG) performed by reverse transcription– polymerase chain reaction (RT-PCR) were negative. The clinicopathologic results were, therefore, consistent with a primary ES with foci of NB-like differentiation.
3. Discussion This fascinating tumor in an adolescent girl demonstrates the association in a same tumor of an ES associated with poorly differentiated NB. This case also demonstrates that the differences between the 2 tumor types are sometimes overlapping. NB are derived from primordial neural crest cells that migrate from the mantle layer of the developing spinal cord and populate the primordia of the sympathetic ganglia and adrenal medulla [1]. The histogenesis of PNET/ ES is still controversial. For a long time, a neural crest origin was also evoked for PNET/ES based on in vitro, immunohistochemical, and morphological studies [6]. A more recent hypothesis is that it might arise from mesenchymal stem cells [7]. These cells have the ability to differentiate into multiple distinct lineages including bone, cartilage, and neural cells. Based on the 2 latter hypotheses, either neural crest cell or mesenchymal stem cell origins, PNET/ES has a very close origin similar to that of NB, which may explain a mixed tumor like those we describe in our case. Homer-Wright rosettes can be seen in both NB differentiated and PNET/ES. Moreover, several publications described
1775 PNET/ES associated with the presence of ganglion cells [8,9]. In these tumors, undifferentiated small cells and ganglion cells were associated with foci of neuropil. Most of these tumors occurred in children or adolescents [8,9], but 2 tumors were discovered in adults [9]. Rarely tumors came from bones [9]. The most common location in soft tissue tumors was chest [8]. Only 2 patients died of the effects of the tumor [8]. On immunohistochemistry, NB, however, seldom, if ever, displays CD99 immunopositivity in a membrane pattern [1]. In our tumor, the CD99 remained positive only in the undifferentiated cells and not in ganglion cells. If CD56 is frequently expressed in NB [3], it could be found in ES and was associated in good prognosis [10]. In our case, the positivity of CD56 was only found in NB-like foci of the tumor. The FLI-1 immunoreactivity, in adjunction to FISH results, was critical in deciphering the 2 intermingled malignant components and, therefore, to render the diagnosis of PNET/ES with NB-like foci in this unique tumor. Cytogenetic or molecular analysis identification of mixed phenotype PNET/ES and NB has been rarely described in children. All such cases demonstrated postchemotherapy neural differentiation. Maeda et al [11] first reported in 1998 a case of a 12-year-old girl with ES of the radius bone, which after chemotherapy differentiated largely into largely ganglion cell tumor but was associated with EWS/FLI1 detected by RT-PCR. In the same year, an ES case from the forearm of a 10-year-old girl, which expressed EWS/FLI1 fusion transcripts, was reported. The tumor was treated with surgery, chemotherapy, and local radiation, but residual tumor was detected within a year lacking detectable EWS/ FLI1 expression and showed both neural and ganglion cells [12]. Collini et al [13] presented findings in a 17-year-old adolescent girl with iliac bone PNET that postchemotherapy had features of NB. Her tumor presented a EWS-FLI1 fusion transcript. Lastly, Weissferdt et al [14] analyzed the retroperitoneal tumor of an 11-year-old girl, which, on prechemotherapy biopsy, was identified as PNET/ES with t(11,22) fusion, but then after treatment and ultimately after excision of the mass, it had the morphological appearance of a NB but maintained its PNET/ES t(11,22) fusion, thus still confirming the diagnosis of PNET/ES. Interestingly, in our case, the specific EWS-FLI1 translocation was found in both morphologically different malignant components, revealing the same cell origin. We can speculate that additional genetic or epigenetic modifications led to the creation of a separate clone of neuroblastoma-like malignant cells within the Ewing sarcoma primary mass. Based on our FISH studies, these additional genetic modifications are not the ones most frequently found in NB (absence of MYCN amplification and loss of 11q23 and of 1p36). Moreover, the negative RT-PCR analysis for t(11,22) and t(21,22) was possibly due to a break point different from that typically found. FISH has been shown to be a more sensitive and reliable method than RTPCR for the diagnosis of EWS in solid tissue [15]. It is important to note that the treatments of localized neuroblastoma and of localized PNET/ES are entirely
1776 different. Historically, localized ES treated with surgery or radiotherapy alone have shown high incidence of metastatic relapses (≥70%). As a consequence, all protocols of treatment for Ewing sarcoma are currently based on heavy weighted chemotherapy regimens including high cumulative doses of alkylating agents, anthracyclins, and etoposide. These treatments put patients at risk for secondary cancer, poor health, and sterility. In contrast, localized neuroblastoma, without any other biologic factor of poor prognosis, is usually cured by surgery alone [1]. PNET/ES and NB may have overlapping histopathologic features both prior to and after chemotherapy. Therefore, immunohistochemical and molecular biology studies are essential in determining the correct diagnosis. In our case, despite the negative results of RT-PCR studies, FISH analysis on both separate malignant components revealed the presence of EWS/FLI1 translocation t(11:22). The latter, which was corroborated with FLI-1 nuclear immunoreactivity in both malignant components, was critical in the making the diagnosis of PNET/ES with foci of NB-like differentiation. The correct diagnosis is essential to provide the appropriate chemotherapy and corresponding clinical follow-up.
References [1] Weiss SW, Goldblum JR. Ewing's sarcoma/PNET tumor family and related lesions. In: Weiss SW, Goldblum JR, editors. Enzinger and Weiss's Soft Tissue Tumors. 5th ed. St Louis, MO: Mosby; 2008. p. 945-88. [2] Ambros IM, Ambros PF, Strehl S, et al. MIC2 is a specific marker for Ewing's sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of Ewing's sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer 1991;67:1886-93.
K. Vali et al. [3] Mechtersheimer G, Staudter M, Moller P. Expression of the natural killer cell-associated antigens CD56 and CD57 in human neural and striated muscle cells and in their tumors. Cancer Res 1991;51: 1300-7. [4] Aurias A, Rimbaut C, Buffe D, et al. Chromosomal translocation in Ewing's sarcoma. N Engl J Med 1983;309:496-8. [5] Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 2003;3:203-16. [6] Cavazzana AO, Miser JS, Jefferson J, Triche TJ. Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 1987;127:507-18. [7] Suvà ML, Riggi N, Stehle JC, et al. Identification of cancer stem cells in Ewing's sarcoma. Cancer Res 2009;69:1776-81. [8] Schmidt D, Harms D, Burdach S. Malignant peripheral neuroectodermal tumours of childhood and adolescence. Virchows Arch A Pathol Anat Histopathol 1985;406:351-65. [9] Ushigome S, Shimoda T, Nikaido T, et al. Primitive neuroectodermal tumors of bone and soft tissue with reference to histologic differentiation in primary or metastatic foci. Acta Pathol Jpn 1992; 42:483-93. [10] Ash S, Luria D, Cohen IJ, et al. Excellent prognosis in a subset of patients with Ewing sarcoma identified at diagnosis by CD56 using flow cytometry. Clin Cancer Res 2011;17:2900-7. [11] Maeda G, Masui F, Yokoyama R, et al. Ganglion cells in Ewing's sarcoma following chemotherapy: a case report. Pathol Int 1998;48: 475-80. [12] Knezevich SR, Hendson G, Mathers JA, et al. Absence of detectable EWS/FLI1 expression after therapy-induced neural differentiation in Ewing sarcoma. HUM PATHOL 1998;29:289-94. [13] Collini P, Mezzelani A, Modena P, et al. Evidence of neural differentiation in a case of post-therapy primitive neuroectodermal tumor/Ewing sarcoma of bone. Am J Surg Pathol 2003;27: 1161-6. [14] Weissferdt A, Neuling K, English M, et al. Peripheral primitive neuroectodermal tumor with postchemotherapy neuroblastoma-like differentiation. Pediatr Dev Pathol 2006;9:229-33. [15] Bridge RS, Rajaram V, Dehner LP, et al. Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT-PCR in malignant round cell tumors. Mod Pathol 2006;19:1-8.