Gynecologic Pathology
Moderator: Dr. Marisa Nucci

"Peripheral-type" Primitive Neuroectodermal Tumor (PNET) of the Ovary

Yoshiki Mikami M.D.
Kyoto University Hospital
Kyoto, Japan


Clinical History:
A 48-year-old G1P1 female presented with constipation and abdominal fullness, and was referred to the hospital because of an intrapelvic mass and ascites. Computed tomography revealed massive ascites and a solid and cystic mass measuring 12x19 cm anteriorly in the pelvic cavity. Laparotomy disclosed a left ovarian mass invading the retroperitoneum and small intestine. Disseminated tumors were identified in the omentum and on the peritoneum. Grossly, the tumor was necrotic and was widely spread.


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Diagnosis :
"Peripheral-type" Primitive Neuroectodermal Tumor (PNET) of the Ovary

Microscopic description:
The tumor measured 14x10x3cm, and was whitish in color. Microscopically, it was composed of uniform small round cells having vesicular nuclei and scant cvtoplasm, arranged in solid sheets with occasional abortive or Hormer-Wright rosettes. Marked nuclear pleomorphism was not identified, but extensive coagulative necrosis was observed in some areas. Mitotic figures were clearly evident. The neoplastic cells harbored diastase-sensitive PAS-positive glycogen granules in the cytoplasm. Immunohistochemically, the tumor cells were positive for CD99 (MIC2) with membranous staining, as well as positive for NCAM (CD56), vimentin, and synaptophysin. Chromogranin A, alpha-inhibin, cytokeratin (AE1/AE3), EMA, GFAP and desmin were negative.

Genetic analysis:
RT-PCR assay, using RNA extracted from formalin-fixed and paraffin-embedded tissue blocks and reverse-transcribed into cDNA, demonstrated EWS/FLI-1 chimeric gene transcripts. These products were sequenced and proven to be made up of EWS exon 7 and FII-1 exon 6.

Discussion:
Ovarian primitive neuroectodermal tumor (PNET), showing morphologic features and cytogenetic alterations similar to Ewing's sarcoma/PNET family tumors arising in bone and soft tissues, is a rare neoplasm, and only several cases have hitherto been reported. The "peripheral-type" PNET can be confused with "central-type" neuroectodermal tumor, in particular its poorly differentiated form such as primitive neuroectodermal tumor (PNET) of the cerebrum and medulloblastoma of the cerebellum. These tumors are commonly associated with neuroglial tissue in mature or immature teratoma, and are a genetically distinct group of tumors.

"Central-type" neuroectodermal tumor of the ovary is defined as a tumor closely resembling neoplasms of the central nervous system with a similar spectrum of differentiation, ranging from "primitive" and "anaplastic" tumors to differentiated gliomas [1]. The largest series included 25 cases studied and reported by Kleinman et al. in 1993, who subdivided ovarian neuroectodermal tumors into three categories: differentiated, primitive, and anaplastic [2]. The primitive subtype resembled primitive neuroectodermal tumors of the cerebrum and medulloblastoma arising in the cerebellum. They were typically highly cellular and composed of small cells with hyperchromatic and round to oval nuclei, and scant cytoplasm in a background of a varying amount of fine fibrillary matrix. The patients were 24.5 years old on average [2]. About half of the tumors were associated with teratomatous components [2]. They were therefore thought to arise from neuroglial tissue in mature or immature teratomas, explaining the histogenesis of neuroectodermal tumor in the ovary. The prognosis was poor in the presence of extraovarian spread, and approximately 50% of the patients died of the disease within seven months [2].

In 1998, Kawauchi in Japan reported a case of an ovarian tumor showing features of PNET/Ewing's sarcoma arising in bone or soft tissues with EWS/FLI-1 chimeric mRNA demonstrated by RT-PCR [3]. Thereafter, additional two cases of tumors of PNET/Ewing's sarcoma family were reported in the English literature [4, 5]. The age of the patients in these three single but well-examined cases was 29, 18, and 13 years. Interestingly, Chow demonstrated multiple chromosomal aberrations including deletions of Rb, ARHI, and FAT, as well as amplification of N-myc, FASL, GITRL, and EGFR [5]. On the other hand, medulloblastoma and supratentorial PNET show distinct genetic alterations. EWS/FLI-1 fusion gene has been considered to be essential in tumorigenesis of pPNET. Therefore, pPNET appears to be a rather minor but distinct entity in the category of ovarian PNET [6, 7]. In other words, primitive or poorly differentiated forms of neuroectodermal tumor in the ovary include at least two distinct groups of neoplasms, i.e., "central-type" (cPNET) and "peripheral-type" (pPNET). In the current WHO scheme (2003), however, these two categories are lumped together under the term neuroectodermal tumor and the distinction between them is not clear [1]. Since information on the management of ovarian neuroectodermal tumors is very limited, differences in therapeutic strategies and outcome between patients with central and peripheral types of tumors remain to be elucidated, although both tumors are considered to be aggressive. Importantly, recent studies suggest that EWS/FLI1 antagonists have been found to inhibit cell growth of the tumor of Ewing's sarcoma /PNET family in vitro [8, 9, 10]. Therefore, the correct diagnosis of pPNET with EWS/FLI1 chimeric gene may become crucial for individualized and optimal therapeutic strategies.

Although genetic analysis may contribute to establishing the diagnosis, morphologic features of pPNET are characteristic and triage for genetic study can be achieved by careful morphologic observation with the aid of immunohistochemistry. cPNET typically shows, as mentioned above, features of medulloblastoma or supratentorial PNET characterized by primitive cells with scant cytoplasm and hyperchromatic nuclei with elongated or so-called carrot-shaped morphology and hyperchromasia. On the other hand, pPNET is composed of a proliferation of cells with rather uniform morphology characterized by small round and vesicular nuclei with occasional rosette formation. MIC2 (CD99), a transmembrane glycoprotein product of the MIC2 gene, is commonly expressed in pPNET, whereas negative in cPNET [7].

The list of differential diagnoses for ovarian pPNET (PNET/Ewing's sarcoma group tumor) is long, and a variety of small round cell tumors need to be considered before establishing the diagnosis, including other ovarian neuroectodermal tumors of central-type, anaplastic carcinoma, small cell carcinomas, intra-abdominal desmoplastic small round cell tumor, endometrial stromal sarcoma, granulosa cell tumor, malignant lymphoma, and leukemia.

Among the other central type ovarian neuroectodermal tumors, ependymoma, diffuse astrocytoma, oliogodendroglioma, and neuroblastoma may be considered. Ependymoma, in particular its "cellular" form, is characterized by perivascular "nuclear-free zones" formed by radiating processes of neoplastic cells with small round nuclei (perivascular pseudorosette). A "true" ependymal rosette can be seen, and its surface is lineally delineated by antibodies to epithelial membrane antigen (EMA). Diffuse astrocytoma can be distinguished by the presence of neoplastic cells with eosinophilic cytoplasm and cell processes and GFAP-positivity. Oligodendroglioma is characterized by perinuclear halos, delicate arcuate arteries, calcospherites, and microcysts. Neuroblastoma shows a varying amount of fibrillary matrix and immature looking cells forming nests encircled by fine fibrovascular septae.

Small cell carcinoma of the ovary is divided into two distinct groups including pulmonary and hypercalcemic types. The former shows features similar to pulmonary small cell endocrine carcinoma characterized by cells with hyperchromatic nuclei with molding and DNA streaming resulting in the so-called Azzopardi effect. This tumor should be distinguished from metastatic small cell carcinoma of the lung or other organs. The coexistence of other components, such as endometrioid adenocarcinoma or squamous or mucinous differentiation, indicates the ovarian origin of the tumor. On the other hand, small cell carcinoma of hypercalcemic type is characterized by younger aged patients, and shows a variety of features including cystic spaces lined by stratified squamous epithelium and/or mucus cells, and rhabdoid features. It is notable that hypercalcemia can be absent in the case of morphologically typical hypercalcemic-type. Immunohistochemically, small cell carcinoma is distinguished from PNET by positivity for cytokeratin and negative staining for vimentin.

Intra-abdominal desmoplastic small cell tumor rarely affects the ovary of children, and few cases have been reported. This tumor is characterized by dense fibrous stroma traversing between sheets of small round cells with scant cytoplasm and hyperchromatic nuclei, and divergent differentiation such as evidenced by immunoreactivity for epithelial markers (cytokeratin, EMA), WT1, or desmin. The detection of EWS/WT1 chimeric gene transcripts, resulting from translocation t(11;22)(p13;q12), is diagnostic of this tumor.

Low-grade endometrial stromal sarcoma (ESS) is a cellular neoplasm composed of a proliferation of short spindle cells with ovoid nuclei, with arterioles resembling spiral arteries of the endometrium, imparting a resemblance to endometrial stroma of the proliferative phase. Cellular portions can look like PNET, but finding the classical features resolves the problem. In addition, CD10 immunohistochemistry is useful.

Granulosa cell tumor (GCT) can be a serious diagnostic concern. From the view of routine practice, it is a practical approach for the diagnostic pathologist to think first of rather common diagnoses. Adult-type GCT is a representative blue cell tumor of the ovary, characterized by proliferation of cells with nuclear grooves, arranged in a microfollicular, macrofollicular, trabecular, insular, or glandular pattern. Occasionally tumors show diffuse patterns, and in such cases, the characteristic nuclear morphology is crucial and may resolve the problem. Although commonly the nuclear morphology is uniform, GCT may show marked nuclear pleomorphism focally. Call-Exner bodies are fluid-filled spaces between neoplastic cells, and may form a rosette-like structure, which should be distinguished from the true rosette as seen in PNET. Juvenile-type GCT is composed of cells with rather hyperchromatic nuclei and abundant cytoplasm, arranged in sheets with or without variably-sized follicles. The nuclei are larger than those of adult-type GCT and may be very bizarre in appearance. Inhibin immunohistochemistry is an adjunctive tool for the diagnosis of GCT.

A variety of malignant lymphomas rarely involve the ovary primarily or secondarily. The types of lymphoma involving the ovary include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and Burkitt lymphoma. Granulocytic sarcoma may be manifested by an ovarian mass, showing proliferation of immature looking myeloid precursor cells mixed with a varying amount of cells with eosinophilic and somewhat granular cytoplasm. Immunostaining for CD13, CD33, and myeloperoxidase, is diagnostic.

Finally the diagnosis of small round cell tumor of the ovary is challenging and needs a constellation of findings including clinical features, gross and microscopic findings, immunophenotype, and/or genetic abnormalities, combined together. MIC2 (CD99) immunohistochemistry may be contributory in the diagnosis of PNET, but it should be kept in mind that this marker can be positive in cases of T-lymphoblastic lymphoma, rhabdomyosarcoma, desmoplastic small round cell tumor, and sex cord-stromal tumors. Therefore, a use of panel of antibodies is recommended to resolve the problems.

References
  1. Pathology and Genetics of Tumours of the Breast and Female Genital Organs. Lyon: IARCPress, 2003.

  2. M., Young, R. H., and Scully, R. E. Primary neuroectodermal tumors of the ovary. A report of 25 cases. Am J Surg Pathol, 17: 764-778, 1993.

  3. Kawauchi, S., Fukuda, T., Miyamoto, S., Yoshioka, J., Shirahama, S., Saito, T., and Tsukamoto, N. Peripheral primitive neuroectodermal tumor of the ovary confirmed by CD99 immunostaining, karyotypic analysis, and RT-PCR for EWS/FLI-1 chimeric mRNA. Am J Surg Pathol, 22: 1417-1422, 1998.

  4. Kim, K. J., Jang, B. W., Lee, S. K., Kim, B. K., and Nam, S. L. A case of peripheral primitive neuroectodermal tumor of the ovary. Int J Gynecol Cancer, 14: 370-372, 2004.

  5. Chow, S. N., Lin, M. C., Shen, J., Wang, S., Jong, Y. J., and Chien, C. H. Analysis of chromosome abnormalities by comparative genomic hybridization in malignant peripheral primitive neuroectodermal tumor of the ovary. Gynecol Oncol, 92: 752-760, 2004.

  6. Jay, V., Pienkowska, M., Becker, L., and Zielenska, M. Primitive neuroectodermal tumors of the cerebrum and cerebellum: absence of t(11;22) translocation by RT-PCR analysis. Mod Pathol, 8: 488-491, 1995.

  7. Gyure, K. A., Prayson, R. A., and Estes, M. L. Extracerebellar primitive neuroectodermal tumors: A clinicopathologic study with bcl-2 and CD99 immunohistochemistry. Ann Diagn Pathol, 3: 276-280, 1999.

  8. Kovar, H., Aryee, D. N., Jug, G., Henockl, C., Schemper, M., Delattre, O., Thomas, G., and Gadner, H. EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro. Cell Growth Differ, 7: 429-437, 1996.

  9. Toretsky, J. A., Connell, Y., Neckers, L., and Bhat, N. K. Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides. J Neurooncol, 31: 9-16, 1997.

  10. Tanaka, K., Iwakuma, T., Harimaya, K., Sato, H., and Iwamoto, Y. EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest, 99: 239-247, 1997.