Enrichment of Residual Tumor Cells in Bone Marrow Or Peripheral Blood Cells in a Neuroectodermal Tumor Model

To enrich a small population of malignant cells contaminating human bone marrow or peripheral blood, a method of immunomagnetic depletion of the normal nucleated cells was developed. In a model using neuroectodermal cell lanes, contaminations between 0.1% and 1.4% of tumor cells could be increased by a factor of 7.5 (median; range, 3.8 to 18.0; n = 10). Because incubation with antibodies is restricted to the magnetic beads in this method, th cell population after removal of the beads has not been incubated with antibodies. The risk of unspecific staining of the tumor cells during the enrichment procedure is minimal. This simple method is therefore well suited to the study of the characteristics of a minimal residual disease population.     

Enrichment of Residual Tumor Cells in Bone Marrow Or Peripheral Blood Cells in a Neuroectodermal Tumor Model

To enrich a small population of malignant cells contaminating human bone marrow or peripheral blood, a method of immunomagnetic depletion of the normal nucleated cells was developed. In a model using neuroectodermal cell lanes, contaminations between 0.1% and 1.4% of tumor cells could be increased by a factor of 7.5 (median; range, 3.8 to 18.0; n = 10). Because incubation with antibodies is restricted to the magnetic beads in this method, th cell population after removal of the beads has not been incubated with antibodies. The risk of unspecific staining of the tumor cells during the enrichment procedure is minimal. This simple method is therefore well suited to the study of the characteristics of a minimal residual disease population.     

Monoclonal Antibody MB2: a Potential Marker for Ewing's Sarcoma and Primitive Neuroectodermal Tumor

The smalt round-cell tumors of childhood present difficulties in diagnosis when differentiation is not apparent. Immunohistochemislry is helpful; however, the only antigen consistently detected in Ewing's sarcoma is vimentin, which may also he detected in the other types of small-cell neoplasms. The monoclonal antibody (MAb) MB2 is marketed as a B-lymphocyte marker that can be used on paraffin-embedded tissue. To determine its specificity, we performed immunohistochemical staining on pediatric tumors with MB2. These included 55 cases of small round-cell tumors (lymphomas, Ewing 's sarcoma, peripheral primitive neuroectodermal tumors [PNET], neuroblastomas, rhabdomyosarcomas, and nephroblastomas). MB2 positivity was detected in all B-cell lymphomas and in seven of nine cases of Ewing's sarcoma and three of three PNET. In neuroblastomas only differentiating ganglion cells were positive. In rhabdomyosarcomas only large rhabdomyoblasls were positive. Blastema of nephroblastomas was negative. Thus, in cases of poorly differentiated small round-cell tumors, MB2 was positive in all B-cell lymphomas, most Ewing's sarcomas and all cases of PNET. Lymphomas were distinguished by staining for leukocyte-common antigen and PNET by neuron-specific enolase. Therefore, the addition of MB2 to a discrete panel of antibodies may prove useful in the diagnosis of Ewing's sarcoma and PNET.     

Incidence of Bone Marrow Involvement in Ewing's Sarcoma: Value of Extensive Investigation of the Bone Marrow

Purpose: Bone marrow (BM) status is a critical matter when intensified chemotherapy with bone marrow rescue is proposed to improve the survival of patients with poor prognosis Ewing's sarcoma (ES): metastatic or relapsing disease. A systematic bone marrow investigation was performed in all the patients with newly diagnosed ES or relapsing ES to assess their BM status. Patients and Methods: From January 1985 to February 1989, 59 untreated patients and five patients at the time of relapse had a bone marrow investigation under general anesthesia: two BM biopsies and two BM aspirates until May 1986, then two BM biopsies and 10 BM aspirates. The classical method of smearing each BM aspirate was compared to cytocentrifugation of the pool of BM samples after gradient density separation. Results: The BM was involved in 13 of 59 untreated patients. BM was the single site of metastatic spread in only one patient but was involved in 52% of the patients with metastatic disease at other sites. This involvement was focal in several patients and frequent discrepancies were noted between the aspirates and biopsies at the various sites explored. The number of positive cases of BM involvement discovered by the two methods is somewhat limited. However preliminary results indicate a superior rate of positive smears with the pool technique which did however fail to detect involvement in some cases. Conclusions: The present study indicates that 1) BM involvement is a frequent event in metastatic ES (52%); 2) is often multifocal and therefore requires extensive BM investigation; and 3) further investigation of the pool technique to facilitate the BM screening is warranted. © 1995 Wi1ey-Liss, Inc.     

Primitive Neuroectodermal Tumor of the Brain Associated with Malignant Rhabdoid Tumor of the Liver: A Histologic, Immunohistochemical, and Electron Microscopic Study

A 14-day-old white male, born with a large primitive neuroectodermal tumor of the left cerebral hemisphere, was found to have a solitary rhabdoid tumor in the liver incidentally at autopsy. Cells resembling the liver rhabdoid cells were also found by histology, immunohistochemistry, and electron microscopy in the brain tumor. The concurrence of rhabdoid cells in the tumors of the brain and liver suggests a common histogenesis and further supports the previous suggestion that the rhabdoid tumor is of neuroectodermal origin. The rhabdoid tumor in the liver in this case is likely to be a metastatic tumor from the brain rather than a second primary tumor.     

So-Called Primitive Neuroectodermal Tumor in Macerated Fetuses: A Confusing Artifact

We studied 11 macerated fetuses with so-called primitive neuroectodermal tumors. Because we doubted the tumorous nature of this disorder, we produced a similar lesion in a comparable 12th fetus. Experimental compression of the skull of the macerated fetus resulted in expulsion of the nervous tissue by way of the vertebral canal and into the retroperitoneal space along the peripheral nerves, with spreading into the adjacent tissues and in blood vessels. The macroscopic and microscopic picture that was induced was essentially identical to that of the 11 spontaneous cases. This lesion, which has been called primitive neuroectodermal tumor in macerated fetuses, must therefore be considered an artifact.     

Primary Subcutaneous Primitive Neuroectodermal Tumor with Aggressive Behavior and an Unusual Karyotype: Case Report

Primitive neuroectodermal tumor/Ewing sarcoma (PNET/ES) rarely occurs in the skin and subcutaneous tissues. We present a case of a 16-year-old girl with primary cutaneous and subcutaneous PNET/ES of the abdominal wall. Despite wide local excision and chemotherapy, she rapidly developed cranial bone and brain metastases, followed by lung and skeletal metastases, and died shortly thereafter. The recurrent tumor exhibited light microscopic features of a small, round, blue cell tumor with intracytoplasmic glycogen. Immunohistochemical analysis showed positivity for CD99, CD56, S100, and glial fibrillary acid protein, and ultrastructural features included cytoplasmic glycogen and focal complex interdigitating synaptic junction-like cytoplasmic folds. Cytogenetic analysis of the relapsed tumor showed a complex karyotype: 47,XX,i(1)(q10), der(4)t(4;19) (q33q35;q13.1), + 8,t(15;17)(q24;p11.2p12),der(19)t (19;20)(q13.1;p11.2),der(22)t(20;22)(q13;q13). Cytogenetic, interphase fluorescence in situ hybridization, and molecular genetic analyses failed to show t(11:22) (q24;q12) or abnormalities of chromosome region 22q12. The clinical behavior and atypical and complex cytogenetic abnormalities exhibited by the tumor in this patient are unusual and represent the most aggressive end of the clinical spectrum of cutaneous and subcutaneous PNET/ES.     

Bone Marrow Metastasis of Neuroblastoma Analyzed by MRI and Its Influence on Prognosis

Bone marrow metastases (BM mets) of neuroblastoma were analyzed by magnetic resonance imaging (MRI). BM mets were demonstrated in 45 femurs of 23 patients with neuroblastoma by MRI. They were classified into two patterns, nodular (N) type and diffuse (D) type, on MRI, with an incidence of 16% (7/45) and 84% (38/45), respectively. No bone metastases (B mets) were accompanied by N type BM mets, whereas all of them were accompanied by D type BM mets. The rate of disappearance of N type BM mets after chemotherapy was 100% (7/7) and was significantly higher than that (26%, 10/38) of D type BM mets. The rate of disappearance (58%, 11/19) of BM mets in bones without associated B mets was also significantly higher than that (23%, 6/26) of BM mets in bones with associated B mets. BM mets present in bones with B mets tended to persist after chemotherapy. The survival rate of patients with residual BM mets after chemotherapy was significantly lower than that of patients without residual BM mets. This suggests that powerful systematic chemotherapy and additional targeted therapy to lesions remaining after chemotherapy are needed to improve the cure rate of patients with advanced neuroblastoma. © 1995 Wi1ey-Liss, Inc.     

Malignant Peripheral Nerve Sheath Tumor of Bone in Children and Adolescents

Malignant peripheral nerve sheath tumor (MPNST) of bone is a rare entity. We have examined three lesions that fit standard histopathologic criteria for MPNST of soft tissues but that arose in the skeleton of three children aged 6 to 13 years. None was affected by neurofibromatosis 1 (NF1). Histologic features typical of MPNST included spindle cells with comma-shaped nuclei, tactoid bodies, nuclear palisading, hyaline bands, and schwannoma-like and curlicue foci. Epithelioid foci were seen in two cases, and heterologous differentiation in one. Immunohistochemistry revealed positivity for S-100 (1 positive/3 tested), vimentin (3/3), glial fibrillary acidic protein (2/3), CD34 (1/1), and CD68 (1/2). Studies for CD99 (0/3), epithelial membrane antigen (0/3), cytokeratin (0/3), CD57 (0/3), and HMB-45 (0/2) were negative. Ultrastructural findings in one of two cases examined included interlacing, attenuated cytoplasmic processes, microtubules, and rare dense-core granules. We conclude that MPNST may arise as a primary bone neoplasm in children without NF1. Received February 17, 1998; accepted June 19, 1998.     

Congenital Cns Primitive Neuroectodermal Tumor: Case Report and Review of the Literature

A 30-week gestational age male fetus was found to have a congenital neoplasm involving the posterior cranial fossa, identified by fetal ultrasound examination in utero. Histological and immunohistochemical examination confirmed a diagnosis of primitive neuroectodermal tumor (PNET) of the posterior fossa, also referred to as medulloblastoma. Although PNETs have been well documented, there are relatively few reports of these occurring as congenital neoplasms. We present a case of an in utero, congenital PNET with a review of the literature and discussion of the criteria defining congenital tumors.     

Melanotic Neuroectodermal Tumor of Infancy: A Molecular Genetic Study

Melanotic neuroectodermal tumor of infancy is a rare but well-recognized entity in pediatric pathology. However, the relationship of this tumor to other pediatric small cell tumors with neuroectodermal features (such as neuroblastoma, Ewing sarcoma/peripheral primitive neuroectodermal tumor, and desmoplastic small round cell tumor) is undetermined. Molecular genetic studies of melanotic neuroectodermal tumor of infancy have not been reported. We studied three typical cases of melanotic neuroectodermal tumor of infancy in an attempt to link this tumor to other small cell tumors with well-characterized molecular genetic changes. Tests performed included: detection of MYCN gene amplification and deletion of 1p (all 3 cases), and presence of the t(11;22)(q24;q12) and the t(11;22)(p13;q12) translocations (2 of 3 cases). None of these tests yielded positive results. Thus, there is no genetic basis at present to link melanotic neuroectodermal tumor of infancy to neuroblastoma, Ewing sarcoma/peripheral primitive neuroectodermal tumor, or desmoplastic small round cell tumor. Received June 30, 1997; accepted September 19, 1997.     

Megatherapy and stem cell transplantation for Ewing's family of tumors: A critical review of current literature

Abstract:  Although a multimodal approach consisting of chemotherapy and local control with surgery and/or radiation has improved survival in children with Ewing's sarcoma family of tumors, the prognosis for patients with high-risk disease remains poor. More aggressive treatments with high-dose myeloablative chemotherapy followed by stem cell rescue have been utilized in an attempt to improve survival in these patients. Although many studies have been published, it is difficult to interpret the data since patient populations were heterogeneous with respect to disease stage, prior therapy, conditioning and stem cell source. Furthermore, there was no uniform definition of high risk, the sample sizes were small and most studies lacked appropriate control groups. Assessment of the utility of megatherapy will require prospective controlled studies.     

Quantification of Nucleated Cells,CD34-Positive Cells and CFU-GM Colonies in Single Bone Marrow Samples and Bone Marrow Harvests Derived from Healthy Children

Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10⁶ cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10⁵ cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10⁶ /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10⁵ cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.     

Peripheral Neuroepithelioma with Ganglion Cells: Report of Two Cases and Review of the Literature

Peripheral neuroepitheliomas, also known as peripheral primitive neuroectodermal tumors, are by definition primitive embryonal lesions generally composed of poorly differentiated neuroectodermal elements. We have examined two cases that paradoxically contain extensive foci of ganglionic differentiation similar to that of ganglioneuroblastoma, in addition to primitive elements. One tumor arose from the chest wall of an 8-year-old male and the other from the abdominal wall of a 15-year-old male. Differentiation into a mature ganglionic phenotype was confirmed by immunohistochemistry in one case. Rare peripheral neuroepitheliomas have a capacity for maturation that is not generally appreciated. These lesions should not be confused with ganglioneuroblastomas, which are genotypically unrelated neoplasms. Received August 27, 1997; accepted March 27, 1998.     

High‐dose therapy with stem cell rescue for pediatric solid tumors: Rationale and results

Abstract: Metastatic and recurrent pediatric solid tumors usually respond to chemotherapy but are likely to recur. Because of steep dose–response relationships, HDT requiring hematopoietic rescue may improve outcome. This strategy has recently been shown to be effective for metastatic neuroblastoma. Metastatic Ewing's sarcoma appears to be a closely analogous situation, and promising phase II studies suggest that a definitive trial of efficacy would be appropriate. Phase I or II trials remain appropriate and are needed to define further the efficacy of HDT for most other poor prognosis pediatric solid tumors.     

Molecular and Cytogenetic Analysis of a Cerebellar Primitive Neuroectodermal Tumor with Prominent Neuronal Differentiation: Detection of MYCN Amplification by Differential Polymerase Chain Reaction and Southern Blot Analysis

Oncogene amplification is uncommon in cerebellar primitive neuroectodermal tumor (medullo-blastoma) and its frequency and diversity are greater in medulloblastoma cell lines. We describe a medulloblastoma in a 10-year-old-girl with striking neuronal differentiation evident in the islands of ganglion cells intermixed with more primitive undifferentiated cells. The islands of ganglion cells showed prominent synaptophysin positivity. Karyotypic analysis revealed hypo -and hyperdiploidy with multiple random rearrangements and double minute chromosomes. Differential polymerase chain reaction and Southern blot analysis revealed up to 25-fold MYCN amplification.     

儿童骨髓间充质干细胞体外培养与富集效率的研究

目的比较密度梯度离心法和全骨髓培养法分离培养儿童骨髓间充质干细胞(BMSCs)获得的细胞纯度与克隆形成效率。方法取等量抗凝的儿童骨髓分别以密度梯度离心法和全骨髓培养法分离培养BMSCs,传代至第3代分别计数2种方法所得BMSCs的细胞总数,流式细胞仪检测表面标记物及其纯度,对第5代BMSCs分别绘制生长曲线,全骨髓培养法培养至第6代流式细胞仪再检测表面标记物及其纯度。结果2种方法均能有效培养出BMSCs,BMSCs强表达CD105及CD13,低表达CD34;3mL骨髓样本用全骨髓培养法扩增至第3代平均可获得3.15×107个细胞,而密度梯度离心法3mL骨髓只获得1.08×107个细胞,二者有显著性差异(P<0.05);密度梯度离心法第3代BMSCs纯度达95%以上,全骨髓培养法第3代BMSCs纯度约85%,多次换液传至第6代纯度可达到95%以上;2种方法第5代BMSCs生长曲线近似,接种后0～3d为生长潜伏期,3～6d为对数生长期,之后进入平台期,无显著性差异(P>0.05)。结论密度梯度离心法分离培养BMSCs,可在短时间内获取纯度较高的BMSCs;全骨髓培养法分离培养BMSCs,可从少量标本获得最大数量的BMSCs,多次换液传代也可获得纯度较高的BMSCs。     

Bone scintigraphy in nonsurgically treated Ewing's sarcoma at diagnosis and follow-up: Prognostic information of the primary tumor site

In order to detect skeletal metastases in patients with Ewing's sarcoma, bone scanning is commonly used. However, little information is available about the scintigraphic aspects of the primary Ewing's sarcoma during non-surgical treatment and follow-up. We studied retrospectively the significance of bone scintigraphic findings at the primary tumor site of 58 patients with a Ewing's sarcoma. These patients had chemotherapy and radiotherapy. At presentation 53/58 patients showed an increased tracer uptake at the primary tumor site while 5 patients with a pelvic or sacral bone localization had a normal scan. Bone scans made during treatment and more than 2 years thereafter in the 32 eligible patients demonstrated three patterns. In 16 patients the hot spot disappeared and no local tumor recurrence was encountered. In the other 16 patients the high uptake at the primary tumor site either persisted or diminished first to a normal uptake after a median period of 18 months (range 12-36 months) and returned again to a high uptake within 6-12 months. In these patients local Ewing's sarcoma was still present in 13, while in the other 3 cases a benign disorder (fracture, ectopic bone formation) was the underlying cause. These findings suggest that in non-surgically treated Ewing's sarcoma persisting increased tracer uptake or its recurrence is highly suspicious for the presence of Ewing's sarcoma, while bone scans becoming negative and remaining so for more than 12 months suggest the absence of local tumor. © 1994 Wiley-Liss, Inc.