Molecular Genetic Analysis of a SmallBowel Primitive Neuroectodermal Tumor

DOUGLAS K. GRAHAM,1* LINDA C. STORK,1 QI WEI,2 J. DAVID INGRAM,3

FREDERICK M. KARRER,4 GARY W. MIERAU,2 AND MARK A. LOVELL2

1Department of Hematology, Oncology, and Bone Marrow Transplant, The Children’s Hospital, University ofColorado Health Sciences Center, 1056 East 19th Avenue, Denver, CO 80218, USA2Department of Pathology, The Children’s Hospital, University of Colorado Health Sciences Center, 1056 East19th Avenue, Denver, CO 80218, USA3Department of Radiology, The Children’s Hospital, University of Colorado Health Sciences Center, 1056 East19th Avenue, Denver, CO 80218, USA4Department of Surgery, The Children’s Hospital, University of Colorado Health Sciences Center, 1056 East 19thAvenue, Denver, CO 80218, USA

Received September 13, 2001; accepted September 28, 2001.

ABSTRACTWe present a pediatric peripheral primitive neuroectoder-mal tumor (pPNET) localized exclusively to the smallbowel. The tumor presented in an adolescent male and thediagnosis was confirmed by electron microscopy, CD99immunopositivity, and molecular genetic analysis thatdemonstrated an EWS-FLI1 type 2 fusion transcript. Thiscase report and a review of the literature underscore theconsiderable phenotypic overlap in EWS-related tumors inthis site and the necessity for molecular genetic analysis topermit accurate classification.

Key words: peripheral primitive neuroectodermal tu-mor, pPNET, Ewing’s sarcoma, EWS-FLI1, pediatric on-cology, small bowel tumor

INTRODUCTIONThe peripheral primitive neuroectodermal tumorsof childhood include the spectrum of Ewing’s sar-coma (ES), the second most common malignantbone tumor of children and young adults, and pe-ripheral primitive neuroectodermal tumors(pPNETs). Whereas classical ES is a poorly differ-entiated small round blue cell tumor, pPNETs

show discernable differentiation with definite neu-ral features. In addition, ES typically arises in boneand pPNETs more commonly are derived from softtissues. Despite these differences, these tumortypes have many immunohistochemical character-istics and typically have a translocation involvingchromosome 22 (EWS gene) with either chromo-some 11 (FLI1 gene) or chromosome 21 (ERGgene); they are sometimes referred to as Ewing’sfamily of tumors (EFT) [1–3].

The translocation t(11;22)(q24;q12) occurs in�80%–95% of cases of ES [1,4]. The recombinedgenes retain the DNA-binding portion of the ETS-like oncogene FLI1 while coming under the controlof the EWS promoter. EWS-FLI1 translocationsconsist of 18 different theoretical transcript com-binations and many of these fusion products havebeen detected in patient samples [4,5]. The twomost common fusions unite EWS exon 7 with ei-ther FLI1 exon 6 (type 1 fusion) or FLI1 exon 5(type 2 fusion). The type 1 and type 2 fusion prod-ucts have been reported to occur in 72% and 24%of cases, respectively [4]. The EWS-FLI1 type fu-sion may have a favorable prognostic significance*Corresponding author

Pediatric and Developmental Pathology 5, 86–90, 2002

DOI: 10.1007/s10024-001-0192-1

© 2002 Society for Pediatric Pathology

independent of other factors such as patient age,tumor location, size, and stage [4].

Soft tissue pPNETs often arise near bones andmay be difficult to differentiate from primary bonetumors. Approximately one-fourth of patients havemetastases to either the lung, cortical bone, or bonemarrow. Other sites of metastases, including thesmall intestine, are rare. We report a case of a pedi-atric primary pPNET arising from the small bowelwall. The electron microscopic and immunohisto-chemical findings supported the pPNET diagnosis,and the diagnosis was confirmed by the moleculardemonstration of the EWS-FLI1 transcript.

CLINICAL PRESENTATIONThe patient is a 14-year-old previously healthymale who presented with a 2-month history ofprogressive fatigue and weakness and a 2-weekhistory of nausea and dizziness. The patient waspale and tachycardic but otherwise had a normalphysical examination. A complete blood count(CBC) revealed a hemoglobin (Hgb) of 6.0 g/dl andthe patient was referred for further evaluation. Hehad no recent history of weight loss, fever, consti-pation, diarrhea, abdominal pain, fever, blood inthe stool, or dark-colored urine. Diet was appropri-ate for age. There was no family history of anemia,bleeding disorder, or cancer in relatives less thanage 40. He had no risk factors for HIV exposure.

Further evaluation included a hemoccult-pos-itive stool analysis. His sedimentation rate wasnormal. A repeat CBC showed a white blood cellcount of 6100/�l with a normal differential, Hgb6.1 g/dl, and platelet count of 666,000/�l. The redcell distribution width (RDW) was increased to17.0% and the mean corpuscular volume (MCV) of54 fl was significantly lower than normal. His re-ticulocyte count was 4.1%. The peripheral smearrevealed marked hypochromia, microcytosis, andanisocytosis without targets, spherocytes, or frag-ments. The peripheral smear and red blood cellindices were consistent with iron deficiency ane-mia. This diagnosis was confirmed by iron studiesof total iron 11 �g/dl (56–175 normal range), totaliron-binding capacity 503 �g/dl (205–400 normalrange), and ferritin �1 ng/ml (7–140 normalrange). The lactate dehyrogenase was normal.

An abdominal computed tomography (CT)scan to determine the source of occult blood loss

causing the severe iron deficiency showed a loop ofbowel wall focally thickened by a soft tissue massmeasuring 7 � 3 � 3 cm (Fig. 1). The liver, spleen,and kidneys were normal and no adenopathy wasappreciated. Laparoscopic examination revealed awell-circumscribed mass arising from the wall ofthe distal ileum. Hypervascularity of the adjacentbowel wall was noted. No other intestinal, mesen-teric, or omental abnormalities were found by ex-tensive visual inspection. The mass, a wedge ofmesentery, and a 15-cm segment of distal ileumwere then completely resected through a low mid-line abdominal incision.

Additional metastatic work-up included anormal chest CT and bone scan. Bilateral bonemarrow aspirates and biopsies showed no evidenceof tumor. HIV serology was negative. Followingthe diagnostic work-up and surgical resection, thepatient was treated with 39 weeks of chemother-apy, which included Adriamycin (cumulative dose375 mg/m2) as well as 7 doses of Cytoxan (1200–1800 mg/m2/dose) and 7 doses of Ifosfamide/VP-16. He is currently 13 months off chemotherapyand remains in complete remission.

PATHOLOGIC FINDINGSA smooth, gray-white, ovoid mass measuring 6.0 �

3.5 � 3.0 cm arose from the bowel wall and causedcompression of the lumen. Internally, the lesion ex-

Figure 1. Abdominal computed tomography scan. Alobulated mass (thick arrow) narrows the contrast-filledlumen (thin arrow) of a loop of bowel on the left side ofthe pelvis.

PRIMARY SMALL BOWEL PPNET 87

tended into the mucosa. On microscopic examina-tion, the surgical margins were free of tumor and themass consisted of nests and sheets of mitotically ac-tive small round blue cells that filled the mucosa andsubmucosa, with lymphovascular invasion, and ex-tended into the muscularis propria, subserosa, andmesentery. Histologically, the pattern of tumor infil-tration suggested a lymphoma, but flow cytometricanalysis showed lack of expression of CD2, CD3,CD4, CD7, CD8, CD19, CD20, kappa, and lambda,arguing against this diagnosis. Focally, the tumordisplayed pseudorosettes (Fig. 2A), and dense fibro-sis that divided the tumor into nests resembling des-moplastic small round cell tumor (DSRCT) were alsoseen (Fig. 2B). Cytologically, the tumor cells hadvariable amounts of featureless eosinophilic cyto-plasm with round-to-oval nuclei displaying an openchromatin pattern often with a single prominent nu-cleolus. Immunohistochemical stains were stronglypositive for CD99 in a membranous pattern and neu-ron-specific enolase (NSE), with focal cells stronglypositive for cytokeratin (AE1/AE3) and negative forCD45, CEA, synaptophysin, epithelial membrane an-tigen, S-100, smooth muscle actin, desmin, and vi-

mentin (Fig. 2C,D). Electron microscopy demon-strated rudimentary intercellular junctions and largefocal aggregates of cytoplasmic glycogen. In theirtotality, these findings supported the diagnosis ofpPNET.

Routine cytogenetics performed on the tumorwere normal, presumably because of fibroblastovergrowth. However, reverse transcriptase poly-merase chain reaction (RT-PCR) demonstrated thepresence of a non–type 1 fusion transcript of theEWS gene and the FLI1 gene (Fig. 3). Sequenceanalysis has confirmed that the transcript was atype 2 EWS-FLI1 transcript derived from fusion ofexon 7 of the EWS gene with exon 5 of FLI1.

DISCUSSIONLymphoma, usually of B-cell lineage, is the mostcommon pediatric malignancy localized to the in-

Figure 2. Histopathology of tumor. A: Focally, pseudo-rosettes are present, typical of a peripheral neuroepithe-lial tumor. H&E; original magnification, �20. B: Otherregions show dense stromal tissue with nests of tumorcells, in a pattern typical for desmoplastic small roundcell tumor. H&E; original magnification, �20. C: The in-filtrating tumor cells display crisp cytoplasmic membraneimmunopositivity. CD99 immunostain; original magnifi-cation, �20. D: The tumor cells display scattered immu-nopositivity while the glandular epithelial cells show uni-form strong positivity. Pancytokeratin immunostain;original magnification, �20.

Figure 3. A: Reverse transcriptase-polymerase chain reac-tion (RT-PCR) for EWS-FLI1 gene fusion product performedon RNA extracted from snap-frozen tumor tissue. PCRproducts are run on a 3% agarose gel stained withethidium bromide. Lanes are as follows: M, molecularweight marker; lane 1, negative control; lanes 2 and 3,duplicate samples of current case; lane 4, positive controlfrom Ewing’s sarcoma with cytogenetic confirmation oft(11;22). The RT-PCR product in lanes 2 and 3 was 484 base-pairs (bp) long while the product in lane 4 was 418 bp long,with the 66 bp difference resulting from the insertion ofthe FLI1 exon 5 sequence, confirming a type 2 transcript.The lower bands in lanes 2, 3, and 4 are artifactual. Theforward primer used in this experiment was 5�TGTTGGGCT-TGCTTTTCCGCTC 3� and the reverse primer was 5� CCCAC-TAGTTACCCACCCCAAA 3�. B: Sequence analysis of the RT-PCR product from lane 2 demonstrating the EWS exon7–FLI1 exon 5 junction. Sequence analysis on the product inlane 4 confirmed a type 1 transcript (data not shown).

88 D.K. GRAHAM ET AL.

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PRIMARY SMALL BOWEL PPNET 89

testines. However, the flow cytometric and immu-nohistochemical analysis did not support this di-agnosis. Other candidate tumors such asrhabdomyosarcoma and leiomyosarcoma and epi-thelial neoplasms were also ruled out on the basisof the negative electron microscopic and immuno-histochemical findings. Given the histologic ap-pearance in some foci, DSRCT was also considereda possible diagnosis. However, DSRCT is associ-ated with the translocation t(11;22)(p13;q12) in-volving WT-1 and EWS [6].

In reviewing the literature, several similarcases (see Table 1) have been reported [7–12],which demonstrate that tumors in this site mayhave a polyphenotypic nature. Despite the poly-phenotypic histologic features of the tumor in ourpatient, the diagnosis of a pPNET tumor is stronglysupported by immunohistochemical (positiveCD99/MIC2 and NSE stains), electron micro-scopic, and molecular genetic data. Similarly, thesignificant overlap between DSRCT and EFT isevident in the case presented by Ordi et al. [10], inwhich the histologic appearance and immunohis-tochemical phenotype could be consistent with ei-ther of these tumor types. However, the presenceof CD99-MIC2 protein and EWS-ERG transcriptargue in favor of a diagnosis of pPNET. In poly-phenotypic cases, the molecular analysis is essen-tial for appropriate tumor classification.

Only four other pediatric patients [7–9] withintraabdominal pPNET have been described. Onepatient had a type 1 EWS-FLI1 fusion transcript[9], two patients had type 2 EWS-FLI1 fusion tran-scripts [7], and the fourth patient did not have theEWS-FLI1 fusion type reported [8]. The fusion ofEWS exon 7 with FLI1 exon 6 (type 1 fusion) maybe associated with a higher probability of relapse-free survival than fusion of EWS exon 7 with FL1exon 5 (type 2 fusion) or other non–type 1 EWS-FLI1 fusions [4,13]. The inclusion of exon 5 in thetype 2 fusion protein yields an additional struc-tural domain that could contribute to the transac-tivating potential of the oncogene. Despite thepresence of type 2 transcripts in our patient andtwo additional pediatric cases [7], all three patientscontinue to be in clinical remission.

In conclusion, pPNET should be consideredin the differential diagnosis of a pediatric intestinaltumor, recognizing the variable histologic pheno-

type, which can be misleading if only a small bi-opsy is obtained for diagnosis. In addition, thiscase emphasizes the necessity of the totality ofinformation available, including molecular geneticanalysis in conjunction with the more establishedmethods of histology, immunohistochemistry, andelectron microscopy, to appropriately classify anunusual pediatric malignancy.

A C K N O W L E D G M E N T S

D.K.G. was supported by the National ChildhoodCancer Foundation, the Brent Eley Foundation,and NIH/NCI training grant T32-CA82086-0141.

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Ewing’s sarcoma. An evaluation of 85 cases and remark-able consistency of t(11;22)(q24;q12). Cancer Genet Cyto-genet 1988;32:229–238.

2. Delattre O, Zucman J, Plougastel B, et al. Gene fusion withan ETS DNA-binding domain caused by chromosometranslocation in human tumors. Nature 1992;359:162–165.

3. Delattre O, Zucman J, Melot T, et al. The Ewing family oftumors. A subgroup of small round cell tumors defined byspecific chimeric transcripts. N Engl J Med 1994;331:294–299.

4. de Alava E, Kawai A, Healey JH, et al. EWS-FLI1 fusiontranscript is an independent determinant of prognosis inEwing’s sarcoma. J Clin Oncol 1998;16:1248–1255.

5. Zucman J, Melot T, Desmaze C, et al. Combinatorial gen-eration of variable fusion proteins in the Ewing family oftumours. EMBO J 1993;12:4481–4487.

6. Ladanyi M, Gerald W. Fusion of the EWS and WT1 genesin the desmoplastic small round cell tumor. Cancer Res1994;54:2837–2840.

7. Sarangarajan R, Hill DA, Humphrey PA, Hitchcock MG,Dehner LP, Pfeifer, JD. Primitive neuroectodermal tumorsof the biliary and gastrointestinal tracts: clinicopathologicand molecular diagnostic study of two cases. Pediatr DevPathol 2001;4:185–191.

8. Thorner P. Intra-abdominal polyphenotypic tumor. Pedi-atr Pathol Lab Med 1996;16:161–169.

9. Rosoff PM, Hatcher S, West DC. Biphenotypic sarcomawith characteristics of both a Ewing sarcoma and a des-moplastic small round cell tumor. Med Pediatr Oncol2000;34:407–412.

10. Ordi J, deAlava E, Torne A, Mellado B, Pardo-Mindan J,Iglesias, X, Cardesa, A. Intraabdominal desmoplasticsmall round cell tumor with EWS/ERG fusion transcript.Am J Surg Pathol 1998;22:1026–1032.

11. Katz RL, Quezado M, Senderowicz AM, Villalba L, LaskinWB, Tsokos M. An intra-abdominal small round cell neo-plasm with features of primitive neuroectodermal tumorand desmoplastic round cell tumor and an EWS/FLI-1fusion transcript. Hum Pathol 1997;28:502–509.

12. Horie Y, Kato M. Peripheral primitive neuroectodermaltumor of the small bowel mesentery: a case showing per-foration at onset. Pathol Int 2000;50:398–403.

13. Zoubek A, Dockhorn-Dworniczak B, Delattre O, et al. Doesexpression of different EWS chimeric transcripts defineclinically distinct risk groups of Ewing tumor patients?J Clin Oncol 1996;14:1245–1251.

90 D.K. GRAHAM ET AL.