Ultrastructrual Pathology Staff

The Pediatric Tumor Biology and Ultrastructural Pathology Section provides traditional and specialized diagnostic services on pediatric tumors to the Pediatric Branch, and diagnostic electron microscopic services to a diverse group of Clinical Center physicians at the NIH.

The services to the Pediatric Branch include quick review of pathology sections for patients considered for admission in clinical trials, frozen section diagnosis of surgical excisions, and final review and sign out of pediatric tumor cases. In the most recent years, the test of the reverse polymerase chain reaction (RT-PCR) has also been routinely introduced in the evaluation of solid pediatric sarcomas harboring specific translocations that result in the disruption of known genes. We have evaluated 40 such cases and are preparing a manuscript about the use of this method in the diagnosis of difficult cases. We anticipate an increase in the number of pediatric sarcomas that will be evaluated for the presence of fusion transcripts, since the Pediatric Branch has established a protocol by which selected patients with high-risk sarcomas expressing the fusion products will be treated with a vaccine-driven expansion of tumor effectors after chemotherapy. Pediatric solid tumors are a unique group of tumors with specific chromosomal translocations that have been implicated in their tumorigenesis. The establishment of treatment protocols that deal with the specific hybrid transcripts, such as the current one in the Pediatric Branch, will give us the opportunity to study a fair number of these tumors in relation to the presence or absence of fusion transcripts. We will also evaluate the use of the RT-PCR technique augmented by fluorescent in situ hybridization (FISH) in the accurate diagnosis of these tumors.

The section's diagnostic role on ultrastructural pathology relies on the use of electron microscopy as an adjunct diagnostic method and on correlation of its findings with those of the light microscopy and immunocytochemistry. In this capacity, the section interacts with the other sections in the Laboratory of Pathology and with NIH clinicians. The predominant tissue types submitted for electron microscopic evaluation include poorly differentiated tumors difficult to diagnose by conventional light microscopy, storage and connective tissue diseases, liver biopsies, and occasionally kidney tissue or tissue from motile cilia syndrome. The section is also responsible for training residents in diagnostic electron microscopy and pediatric tumor pathology. This is accomplished by individual teaching on a case-to-case basis and by organized lectures. Metastatic Ewing's sarcoma/peripheral primitive neuroectodermal tumor (PNET) and high-stage neuroblastoma are known to show resistance to the available chemotherapeutic agents and are usually associated with high treatment failures. This is in contrast to the original high chemotherapeutic response of Ewing's sarcoma/PNET. We have hypothesized that the chemotherapeutic failures may be due to defects in the pathway of programmed cell death (apoptosis) in this group of tumors and that such defects could be either clonal (Ewing's sarcoma) or diffuse (neuroblastoma). Apoptosis is mainly triggered upon ligation of the Fas surface receptor with its own ligand or a specific monoclonal antibody. We have studied Fas mRNA levels in neuroblastoma (NB) and peripheral primitive neuroectodermal tumor (PNET) cell lines by RT-PCR, Western analysis, and fluorescence, and found consistently high levels in PNET and variable levels in NB. Furthermore, the anti-Fas antibody induced apoptosis in most PNET cell lines, whereas all studied NB cell lines were Fas-resistant. In order to exclude the possibility that low Fas levels were the reason for the failure of NB cells to undergo Fas-induced apoptosis, we treated the cells with interferon-g that resulted in upregulation of the Fas receptor but did not induce Fas-dependent apoptosis. The possibility of an abnormal Fas protein was also excluded by parallel sequencing of the Fas cDNA from both Ewing's/PNET and neuroblastoma cell lines. Since administration of the Fas antibody resulted in downregulation of the bcl2 gene in apoptosis-sensitive Ewing's/PNET and apoptosis-resistant NB cell lines alike, we hypothesized that at least the original steps of the pathway were intact in NB. We then set out to investigate the functionality of the apoptotic machinery in NB using ceramide, which is known to induce Fas-dependent or Fas-independent apoptosis in other cell systems and to play an important apoptotic role in neural cells. We found that ceramide induced apoptosis in NB in a serum-dependent manner. These data suggested that the apoptotic machinery is functional in this tumor under certain conditions. We then made the hypothesis that inhibitory proteins may interfere with the transduction of the apoptotic signal at any level downstream of the downregulation of the bcl2 protein. An inhibitory protein of Fas-induced apoptosis has been described as FAP (Fas-associated protein), exerting its function by binding with the last 15 amino acids of the Fas molecule. We studied levels of this protein by Northern analysis but found it to be higher in PNET (responding) than in NB (nonresponding) cell lines. Therefore, this does not appear to be the inhibitory protein in the Fas pathway. However, treatment of NB cells with cycloheximide and Fas antibody resulted in induction of apoptosis that was more pronounced in conditions associated with the highest inhibition of protein synthesis, shown by protein radiolabeling. These data further confirm our suspicion that failure of NB to undergo apoptosis may be due to the existence of inhibitory proteins.

Triggering of the Fas receptor in vivo is accomplished with the Fas ligand (FasL). The expression of the FasL is conserved in normal tissues and is traditionally present in activated T lymphocytes. Chemotherapeutic agents induce apoptosis by a mechanism that has not been fully elucidated. We investigated the influence of the known chemotherapeutic agents cis-platin and adriamycin in the levels of Fas and FasL in NB and Ewing's sarcoma/PNET cell lines. We found that both Fas and FasL were upregulated in NB and Ewing's/PNET cell lines, but did not result in apoptosis in NB. We therefore concluded that the Fas pathway may be activated in chemotherapy-induced apoptosis, but in tumors with deficient Fas-pathway this does not result in apoptosis. It remains to be seen if biologic modulators will be able to reverse this defect. Understanding the defects in the apoptotic machinery in tumors will help identify new therapeutic strategies.

Recent Publications
Keleti, J; Quezado, MM; Abaza, MM, et al. Am J Pathol 1996; 149:143-51.
Wexler, LH; Delaney, TF; Tsokos, M, et al. Cancer 1996; 78:901-11.
Quezado, M; Benjamin, DR; Tsokos, M, et al. Hum Pathol 1997; in press.
Katz, RL; Quezado, M; Senderowicz, AM, et al. Hum Pathol 1997; 28:502-9.

Collaborators
Lee J. Helman, M.D.; Zhengping Zhuang, M.D.; and Jane B. Trepel, Ph.D., NIH
Paul Meltzer, NHGRI

Clinical Trials

• A pilot study of autologous T cell transplantation with vaccine-driven expansion of antitumor effectors after intensive chemotherapy for high-risk pediatric sarcomas

• New treatment approaches for patients with Ewing's sarcoma family of tumors, high-risk rhabdomyosarcoma and neuroblastoma

• Alpha-galactosidase, a replacement therapy in Fabry disease. Specimens submitted for electron microscopic examination

• A pilot study of tumor-specific peptide vaccination and IL-2 with or without autologous T cell transplantation in recurrent pediatric sarcomas

• A phase I study of SU101 in pediatric patients with refractory malignancy

• Natural history and etiology of mucolipidosis type IV and other neurometabolic disorders