Circulating T-regulatory cells in PNET: a prospective study.

PURPOSE: Bone marrow regulatory T-cells (Tregs) have been evaluated in patients with peripheral neuroectodermal tumor (PNET); data on peripheral blood circulating Tregs are lacking. The objective of our study was to determine baseline Tregs (both Treg frequency and absolute number) in patients with PNET and correlate with patient characteristics, and observe their change with treatment and relapse. METHODS: Five milliliters blood was evaluated in de novo patients with PNET at diagnosis, post-neoadjuvant chemotherapy and at relapse/progression, along with nine healthy controls using flow-cytometric analysis for Treg cells (CD4+ CD25+ FoxP3+). RESULTS: Thirty-seven patients with median age 17 years; male/female ratio 5.1:1 had significantly higher baseline absolute Tregs than controls (mean 338.95 ± 264.63/mm(3) vs. 34.83 ± 24.90/mm(3); P < 0.001). Patients with fever had a significantly higher mean Treg frequency than those without fever (11.27 ± 8.36% vs. 8.40 ± 2.58%; P = 0.014). There was significant reduction in the circulating Tregs after neoadjuvant chemotherapy (mean 339.78 ± 294.31/mm(3) vs. 82.09 ± 91.25/mm(3), P < 0.001) and rise at progression (n = 13) as compared to values post-neoadjuvant chemotherapy (mean 240.92 ± 191.90/mm(3) vs. 57.67 ± 39.01/mm(3), P = 0.012). There was no significant difference in the event-free survival (EFS) or overall survival (OS) between the high and low Treg cell groups (2-year EFS 51.6% vs. 52.1%; P = 0.689 and OS 61.3% vs. 59.2%; P = 0.891). CONCLUSION: This study on circulating Tregs in PNET demonstrated that peripheral blood Tregs are higher in patients than healthy controls. There was significant reduction in Tregs with chemotherapy and rise at progression.

Ewing's sarcoma of the lesser sac masquerading as a pancreatic tumor.

Extraosseous Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) is an uncommon, aggressive, and malignant tumor with a poor patient outcome. Its occurrence in the lesser sac is a rare event and to the best of our knowledge, has not been previously described. The present case was clinically and radiologically misdiagnosed as a pancreatic tumor/gastrointestinal stromal tumor. Histopathology revealed a tumor with "small round cells" that were positive for CD99, confirming the diagnosis of ES/PNET. This report highlights the importance of considering Ewing's sarcoma in the differential diagnosis of intraabdominal, extraintestinal masses.

Feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous, cytotoxic T lymphocytes in patients with solid tumor.

BACKGROUND AIMS: Adoptive T-cell therapy with tumor-specific T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specific cytotoxic T lymphocytes (CTL) generated by stimulation of patients' peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cells. Methods. Six patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo. RESULTS: Patients received a median of nine CTL infusions (range 2-19). The median number of CTL administered per infusion was 11 × 10(8) (range 1-55 × 10(8)). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values. CONCLUSIONS: Our study demonstrates that autologous ex vivo-generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical efficacy of this immunotherapeutic approach in phase I/II studies.

Perigastric extraskeletal Ewing's sarcoma: a case report.

Ewing's sarcoma (ES) is a neoplasm of undifferentiated small round cells, which occurs in the bones and deep soft tissues of children and adolescents. We present a rare case of a 44-year-old woman with gastric ES presenting with epigastric pain and weight loss. Ultrasound and computed tomography scans indicated a solid/cystic mass in the pancreatic tail. At laparotomy, the tumor was found attached to the posterior surface of the stomach, completely free from the pancreas, with no lymphadenopathy or local metastases. The polynodal, partly pseudocystic, dark-red soft tumor was excised. Histopathology revealed an anaplastic small-round-cell tumor with strong membranous CD99 immunoexpression. Additionally, there was patchy immunostaining for S-100 protein, vimentin, protein gene product (PGP) 9.5 and neuron-specific enolase, and weak focal CD117 cytoplasmic immunoreactivity. The patient had no adjuvant chemotherapy; her postoperative recovery was uneventful, and she remains symptom-free, and without any sign of recurrence at 20 mo. To the best of our knowledge, this is only the third ever case of gastric ES.

[Application of the in situ hybridization with EWS dual-color break-apart fluorescence probe and anti-CD99 and anti-FLI-1 antibodies in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor].

OBJECTIVE: To investigate the value of using EWS dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique, CD99 and FLI-1 antibodies immunohistochemistry in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET). METHODS: Thirty-five cases of EWS/PNET and 24 cases of non-EWS/PNET small round cell tumor were analyzed by FISH and immunohistochemically detected with FLI-1 and CD99 antibodies. Comparison between FISH and immunohistochemical results was carried out and their diagnostic value was evaluated. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of FISH in EWS/PNET were 93.8%(30/32), 81.8%(18/22), 88.2%(30/34) and 90%(18/20); those of CD99 were 100%(35/35), 58.3% (14/24), 77.8%(35/45) and 100%(14/14); those of FLI-1 were 71.4%(25/35), 62.5%(15/24), 73.5%(25/34), 60%(15/25), respectively. The sensitivity, specificity, positive predictive value and negative predictive value of combined use of CD99 and FLI-1 were 71.4%(25/35), 75%(18/24), 80.6%(25/31) and 64.3%(18/28), those of combined use of FLI-1 and FISH were 68.7%(22/32), 86.4%(19/22), 88%(22/25) and 65.5%(19/29), those of combined use of CD99 and FISH were 93.7%(30/32), 95.5%(21/22), 96.8%(30/31) and 91.3%(21/23), respectively. CONCLUSION: EWS dual-color, break-apart rearrangement probe FISH is a highly sensitive and specific technique in the diagnosis of EWS/PNET. The combination of CD99 and FISH is the method of choice for the diagnosis of EWS/PNET. The combination of CD99 and FLI-1 can improve the specificity in EWS/PNET without the data of FISH.

Primitive neuroectodermal tumor of the lung with pericardial extension: a case report.

A 16-year-old student presented with a 4-week history of progressive shortness of breath, loss of appetite, and occasional blood-tinged sputum. The chest X-ray revealed massive right-sided pleural effusion with cardiomegaly. An echocardiogram revealed a large pericardial mass with massive pericardial effusion. Subsequent computed tomography of the thorax revealed a large heterogeneous mass in the right lung with extension into the pericardium. Lung biopsy revealed primitive neuroectodermal tumor (PNET) with small round blue cells, Homer-Wright rosettes, and CD99 positivity. We discuss pericardial metastases of PNET and its implication in this patient.

Ewing's sarcoma and peripheral primitive neuroectodermal tumor cells produce large quantities of bioactive tumor necrosis factor-alpha (TNF-alpha) after radiation exposure.

PURPOSE: In the present study, we examined human Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET) cell lines that are able to produce TNF-alpha as part of the response to irradiation. Radiation-induced tumor cell production of TNF-alpha may enhance irradiation efficacy and improve the effect of local tumor irradiation. On the other hand, radiation-induced tumor cell production of TNF-alpha may adversely affect the normal tissue. METHODS AND MATERIALS: Twelve different ES/pPNET cell lines were investigated in vitro and (after establishment as tumor xenografts in athymic nude mice) in vivo for their TNF-alpha mRNA expression (real-time quantitative reverse transcriptase polymerase chain reaction) and TNF-alpha protein production (in vitro: enhanced amplified sensitivity immunoassay; in vivo: immunohistochemistry) after exposure to different irradiation doses (2, 5, 10, 20, 30, or 40 Gy) and after different time intervals (1, 3, 6, 12, 24, 48, or 72 h after irradiation). The bioactivity of the TNF-alpha protein was evaluated in chromogenic cytotoxicity and neutralization assays. RESULTS: Nine out of 12 ES/pPNET cell lines express constitutively significant quantities of bioactive TNF-alpha in vitro. ES/pPNET cells originating from primary tumors secreted higher TNF-alpha levels than cells derived from metastatic lesions. In 5 of the 9 TNF-alpha-producing cell lines, TNF-alpha mRNA and protein levels were upregulated after irradiation exposure in a time- and dose-dependent manner. After establishment of the ES/pPNET cell lines in athymic nude mice, the radiation-induced TNF-alpha release could be demonstrated also in the xenograft tumors in vivo (analogous to the in vitro experiments). Using the same methods for quantitative analysis, it was determined that the TNF-alpha expression of the radiation-responsive tumor cells was up to 2000-fold higher compared to the maximal radiation-induced TNF-alpha release in normal lung tissue measured during the pneumonic phase. CONCLUSION: Certain ES/pPNET cell lines produce extremely large quantities of bioactive TNF-alpha after radiation exposure in a time- and dose-dependent manner. Radiation-induced TNF-alpha production of tumor cells may be of paramount importance in respect to not only tumor behavior, but also to potential damage to normal tissue and the clinical status of the host.

Primary primitive neuroectodermal tumor of the lung: report of two cases.

Two cases of primitive neuroectodermal tumor of the lung are reported. The first case is a 41-year-old man with a tumor in the left upper lung, and the second case is a 30-year-old woman with a tumor in the right lower lung. In both cases, the tumors originated in the lung but not in the chest wall. No distant metastasis was detected. In case 1, transcutaneous fine-needle biopsy (TCNB) revealed small round cell proliferation, although bronchoscopic examination showed no abnormal findings. Both the expression of Mic2 protein and t(11;22)(q24;q12) translocation were proved in the tumor cells. The tumor cells were positive for periodic acid-Schiff (PAS), neuron-specific enolase (NSE), and vimentin, but negative for Leu7, chromogranin A, and pro-gastrin-releasing peptide (ProGRP). In case 2, bronchoscopic examination showed only compressive change in right lower lobe bronchi. TCNB revealed small round tumor cells expressing Mic2 protein. The tumor cells were negative for leukocyte common antigen, S100 protein, pankeratin, chromogranin A, and desmin, but weakly positive for NSE and moderately positive for Ki-67 (MIB1). Both patients were successfully treated by the combination of surgical resection and chemotherapy, and are alive with no sign of recurrence for approximately 22 months in case 1 and 16 months in case 2.

Neuroblastoma and peripheral neuroectodermal tumors.

Peripheral neuroblastic tumors in childhood present unique problems in terms of diagnosis, classification, and histologic determinants of prognosis. The proper specimen handling of these neoplasms requires integration of gross and microscopic pathologic examination along with cytogenetic, immunohistochemical, and electron microscopy studies. The appropriate gross examination and processing of these tumors are described with particular emphasis on ancillary studies. Important special studies such as immunohistochemistry, flow cytometry, and genetic and molecular oncologic investigation are stressed, and the appropriate methods of processing materials for these studies are discussed. The handling of small biopsy specimens, fine-needle aspirates, or both is also addressed. The staging of neuroblastoma varies according to Pediatric Oncology Group and Children's Cancer Study Group systems, and the importance of obtaining appropriate information to satisfy either system is noted. Ancillary information for classification of neuroblastoma and related neoplasms is also presented.

Molecular features in a biphenotypic small cell sarcoma with neuroectodermal and muscle differentiation.

We report a case of a 13-year-old girl with soft tissue sarcoma of the hand, which showed muscle and neuroectodermal immunophenotypes. Molecular studies were performed on RNA collected from fine-needle aspiration (FNA) cytology and peripheral blood samples by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis. This biphenotypic tumor showed simultaneous expression of EWS-FLI1 and PAX3-FKHR transcripts, specific of Ewing family tumors and alveolar rhabdomyosarcoma, respectively. Although childhood sarcomas with simultaneous muscle and neural differentiation have been described to have EWS-FLI1 transcripts, there are no reports of tumors with both transcripts. Cytological specimens are a good source of RNA for molecular studies.

Appraisal of the comparative utility of immunohistochemistry and electron microscopy in the diagnosis of childhood round cell tumors.

To provide an objective assessment of the comparative utility of fluorescence- and peroxidase-based immunohistochemistry and electron microscopy, an observer blinded study was conducted under realistic study conditions utilizing a large sampling of poorly differentiated pediatric round cell tumors. Working independently, using a single ancillary technique of particular expertise, each of three investigators attempted to render a specific diagnosis with regard to 50 diagnostically challenging tumors. The results were compared against the subsequent "file diagnosis" established by consensus with all relevant information made available. A grading scheme was applied wherein points were awarded based on the accuracy and confidence of diagnosis. A comparative efficiency rating, expressed as a percentage, was formulated by dividing the number of points awarded each technique by the total number of points theoretically available. Electron microscopy proved superior overall, with an efficiency rating of 89%. Immunoperoxidase and immunofluorescence studies yielded efficiency ratings of 71 and 61%, respectively. Used in combination, the techniques achieved an efficiency rating of 95%. Application of these ancillary techniques resulted in a revision of the provisional diagnosis in 11 of 50 cases, and left only two cases without a firm specific diagnosis.

Immunohistology, cytogenetics, and molecular studies of small round cell tumors of childhood. A review.

Malignancies of childhood include a well-defined spectrum of hematolymphoid, organ specific (adrenal, kidney, liver), soft tissue, bone, and nervous system (central and peripheral) neoplasms with variable biology. Small round cell neoplasms, a subset of childhood malignancies, are histologically similar but differ markedly in their histogenesis, therapy, and prognosis. Traditionally, clinical information and light microscopy, with the aid of histochemistry and ultrastructural evaluation, establish a diagnosis or at least narrow the differential diagnosis. Additionally, immunohistology, cytogenetics, and molecular studies have become important in diagnosis and in defining phenotype/genotype, patient treatment modalities, and prognosis in specific cases. The 11;22 chromosomal translocation typifies Ewing's sarcoma, primitive neuroectodermal tumor, and Askin's tumor, as does the resultant chimeric transcript, while expression and amplification of N-myc oncogene are predictive of the prognosis in neuroblastoma. Furthermore, studies of genes and gene products are elucidating mechanisms of oncogenesis and tumor progression.

[Embryonal neuroepithelial tumors of the cerebral hemispheres]. / Embrional'nye neiroépitelial'nye opukholi bol'shikh polusharii golovnogo mozga.

33 embryonal neuroepithelial tumours of the cerebral hemispheres were examined light- and electron-microscopically, immunohistochemically. 4 types of tumours were distinguished: neuroblastoma, neuroepithelioma, ependymoblastoma and choroid carcinoma. Each type was characterised by its own pathohistological, immunohistochemical and ultrastructural features. Our results and literature data prove immunophenotypic and ultrastructural heterogeneity of embryonal neuroepithelial tumors of the cerebral hemispheres, in spite of some similarities in their pathohistological features.

Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing's sarcoma and peripheral neuroepithelioma.

Ewing's sarcoma (ES) and peripheral neuroepithelioma (PN) are closely related tumors, and it can be difficult to distinguish them from other small-round-cell tumors (SRCTs). The glycoprotein p30/32MIC2 is highly, but not exclusively, expressed in both ES and PN. Although the monoclonal antibody (Mab) HBA71, which reacts with P30/32MIC2, has been reported to be relatively specific and highly sensitive for both neoplasms, it is not readily available. Yet, Mab O13 is commercially available, and it purportedly displays the same immunostaining characteristics as HBA71. Because O13 has not been studied extensively, we immunostained 21 ES/PNs and 147 other tumors or lesions that might show SRCT-like features with O13. The results were similar to those reported for HBA71. We found O13 to be 100% sensitive for ES/PN; and, no immunostaining was noted on the SRCTs often included in the differential diagnosis of ES/PN (i.e., conventional neuroblastoma, rhabdomyosarcoma, and non-lymphoblastic lymphomas). But, O13 immunoreacted with lymphoblastic lymphomas and some other tumors and normal tissues. Nonetheless, this nonspecific reactivity should not cause diagnostic problems, if an antibody panel containing anti-desmin and anti-leukocyte common antigen is used in conjunction with O13. We conclude that, within the proper diagnostic context, strong immunoreactivity of a SRCT tumor for O13 should be considered good evidence that the tumor is ES/PN.

Induction of MHC class I antigen expression following infection of a human esthesioneuroblastoma cell line with cytomegalovirus and human immunodeficiency virus.

Productive infections with cytomegalovirus (CMV) and human immunodeficiency virus (HIV) were established in the Tp41ON cell line derived from a human esthesioneuroblastoma. HIV antigen expression was highest in cultures coinfected with CMV and HIV. Viral infection caused increased MHC class I antigen expression while class II and CD4 antigens remained undetectable using immunofluorescence methods. Uninfected cultures showed 10% and coinfected cultures 80% class I antigen positive cells. In coinfected cultures, CMV and HIV antigens were detected in 4% and 8% of the cells, respectively. The detection of CMV antigens in some multinucleated cells suggests coinfection with both viruses in these cells, as multinucleated cells were not found in cultures infected with CMV only. The study shows that a cell line showing neuronal differentiation in vitro can be infected with CMV and HIV and that this infection increases MHC class I antigen expression.

Two malignant peripheral primitive neuroepithelial tumor cell lines established from consecutive samples of one patient: characterization and cytogenetic analysis.

A 6-year-old girl presented with a tumor of the right shoulder involving bone, adjacent soft tissue, and regional lymph nodes. The conventional histologic diagnosis was ambiguous, initially suggesting lymphoma. After relapse on lymphoma therapy, reevaluation with additional multiple diagnostic techniques performed on the biopsy tissue and on two cell lines derived from the biopsies established the diagnosis of a primitive neuroepithelial tumor of bone and soft tissue. This was strongly supported by 1) focal rosette formation by the tumor cells and positive immunostaining for neuron-specific enolase and synaptophysin, with absent staining for leukocyte common antigen; 2) at the ultrastructural level, formation of cellular processes containing microtubules, a paucity of neurosecretory granules, absence of synaptic junctions, formation of long "intermediate" junctions between cells, and, in culture, widespread development of rosettes; 3) marked surface positivity to W 6/32 and negativity to HSAN 1.2 antibodies; and 4) elevated expression of MYC and lack of overexpression of MYCN oncogenes. Numerical and structural abnormalities were present in the karyotype, but the expected t(11;22)(q24;q12) was not present in the tumor-involved marrow or in either of the established tumor cell lines, although there was an interstitial deletion of 11q involving breakpoints in q21 and q23.

Effects of target antigen density on the efficacy of immunomagnetic cell separation.

Immunomagnetic cell separation uses binding of an antibody to its epitope to identify the target cell, which is then removed by attachment to an anti-immunoglobulin-coated paramagnetic bead, and passage through a magnetic field. This method has previously been shown to be less sensitive to the effects of low target antigen density than are other cell elimination methods, such as complement-mediated lysis. In this paper we demonstrate that, with certain antibody/target cell combinations, the efficiency of immunomagnetic depletion can be adversely affected by high expression of the target antigen. This can occur by two non-mutually exclusive mechanisms. These are (i) steric hindrance of bead binding due to crowding of monoclonal antibodies on the cell surface; and (ii) binding of the monoclonal antibody molecule in a configuration that is poorly-accessible to the anti-immunoglobulin immobilized on the microspheres. The predominant effect operating in any system can be determined by analysis of the cells remaining after the separation procedure. In both cases pre-attachment of the monoclonal to the beads results in improved separation efficiency. These results emphasize the necessity of optimizing experimental conditions in each system that is investigated.

Expression of class I histocompatibility antigens in neuroectodermal tumors is independent of the expression of a transfected neuroblastoma myc gene.

We have evaluated the relationship between the neuronal myc gene (NMYC) and class I major histocompatibility complex (MHC) expression in human neuroblastoma (NB) tumor cell lines. Class I MHC surface Ag expression in NB cell lines varied from nearly undetectable to levels nearly as high as in a lymphoblastoid cell line. Class I MHC mRNA levels in NMYC-amplified NB cell lines were lower than levels observed in single copy NMYC NB cell lines. However, considerable variation in class I MHC surface Ag and mRNA expression was evident in NMYC-amplified cell lines. To determine directly whether NMYC might modulate class I MHC expression in NB, we transfected a plasmid containing a recombinant NMYC gene into two tumor cell lines derived from a NB and a related neuroepithelioma tumor. Constitutive overexpression of the recombinant NMYC gene produced no consistent change in class I MHC surface Ag or mRNA levels. To determine whether class I MHC expression might be developmentally regulated in adrenal medullary cells, the precursor cells of adrenal NB tumors, beta 2-microglobulin expression was measured in fetal and adult adrenal glands. beta 2-Microglobulin expression was not evident in the neuroblasts of a 24-wk-old fetal adrenal gland, whereas beta 2-microglobulin expression was present in the adult adrenal medulla. These data suggest that variation in class I MHC expression among NB cells may reflect the developmental stage at which neuroblasts were arrested during tumorigenesis.