Plasticity of neuroblastoma tumor cells to differentiate along a fetal adrenal ganglionic lineage predicts for improved patient survival.

We have recently presented a model of human adrenal medullary histogenesis that incorporates all neural crest-derived lineages (chromaffin, sustentacular, and ganglionic) known to compose this tissue. To determine if neuroblastomas correspond to the arrested maturation of embryonal adrenal medullary cells, we evaluated the expression of adrenal medullary developmental markers in 81 neuroblastoma tumors. We found that patterns of chromaffin-related gene expression in these tumors correlated exactly with the patterns observed during maturation of adrenal medullary cells (P2 < 10(-5). In a multivariate Cox proportional hazards analysis of developmental marker expression and other well-recognized prognostic variables, evidence of maturation along a fetal ganglionic lineage, as monitored by HNK-1 immunoreactivity (relative risk of 6.42, P2 = 0.0001), and age at diagnosis (relative risk of 5.05, P2 = 0.0042) were independent and significant prognostic indicators of patient survival. These studies demonstrate that neuroblastomas correspond to embryonal adrenal medullary cells arrested at recognizable stages during development, and that evidence of maturation along a fetal ganglionic lineage appears to have major importance in predicting patient survival.

Insulin-like growth factor II-mediated proliferation of human neuroblastoma.

Neuroblastoma is an embryonal tumor that typically arises in cells of the developing adrenal medulla. IGF-II mRNA is expressed at high levels in the adrenal cortex before birth but it is not detectable until after birth in the adrenal medulla. Neuroblastoma cell lines corresponding to early adrenal medullary precursors did not express IGF-II, although all three cell lines we tested were growth stimulated by IGF-II. Cell lines corresponding to more mature adrenal medullary cells expressed IGF-II, and one, SK-N-AS, grows by an IGF-II autocrine mechanism (J. Clin. Invest. 84:829-839) El-Badry, Romanus, Helman, Cooper, Rechler, and Israel. 1989. An examination of human neuroblastoma tumor tissues for IGF-II gene expression using in situ hybridization histochemistry revealed that IGF-II is expressed by tumor cells in only 5 of 21 neuroblastomas, but is detectable in cells of nonmalignant tissues including adrenal cortical cells, stromal fibroblasts, and eosinophils in all 21 tumors. These findings indicate that IGF-II may function as an autocrine growth factor for some neuroblastomas and as a paracrine growth factor for others. They suggest that the growth regulatory pathways utilized by neuroblastoma mimic those used in the precursor cell type from which individual tumors arise.

Identification of subsets of neuroblastomas by combined histopathologic and N-myc analysis.

BACKGROUND: Neuroblastomas show different histopathologic phenotypes, and the tumor cells can carry normal or multiple copies of the N-myc proto-oncogene (MYCN). Studies of the N-myc gene and histopathology of untreated primary neuroblastomas have demonstrated that both these factors are important in risk assessment. PURPOSE: Our purpose was to determine if there are any associations between N-myc gene copy number, histopathologic features, clinical stage, and progression-free survival (PFS) and if joint analyses of histopathology and N-myc gene copy number improve risk assessment. METHODS: The histopathologic phenotype and N-myc gene copy number were determined for 232 biopsy/surgery specimens obtained from untreated primary neuroblastoma patients. Tumors were classified as having favorable or unfavorable histology on the basis of Schwannian stroma (rich versus poor), neuroblastic differentiation (differentiating versus undifferentiated), and mitosis-karyorrhexis (fragmenting nucleus) index (MKI; high, intermediate, or low) in the context of age at diagnosis (Shimada classification). N-myc gene amplification was considered significant when the gene copy number was at least 10-fold higher than normal as determined by Southern blot analysis. Otherwise, tumors were classified as nonamplified for N-myc. RESULTS: Among 19 stroma-rich tumors, 11 had grossly visible neuroblastic nodules, and two of these had N-myc amplification. Of 213 stroma-poor tumors, 51 had N-myc amplification, all of which were undifferentiated, and 45 (88% of 51) had high MKI. This histologic phenotype was present in less than 10% of tumors with nonamplified N-myc. Of 162 stroma-poor tumors that showed nonamplified N-myc, 45 (28%) were differentiating and 121 (75%) had low MKI. Neuroblastomas of clinical stages I, II, and IV-S nearly always had favorable histology and no amplification of N-myc. Stage III (regional) and particularly stage IV (metastatic) tumors, however, frequently had unfavorable histologic features with or without N-myc amplification. The estimated PFS at the end of 4 years after diagnosis was 83% for patients whose tumors had favorable histology and no N-myc amplification. The estimated PFS for the patients whose neuroblastomas had unfavorable histology, however, was 29% without and 13% with N-myc amplification, respectively. Subsets of patients with stage II, III, or IV disease were identified by both histologic evaluation and N-myc analysis. Multivariate Cox regression analysis indicated that both the histologic and N-myc-based stratifications provided prognostic information that was independent of staging. CONCLUSIONS: Neuroblastomas with N-myc amplification have a characteristic histopathologic phenotype and an aggressive clinical course. In contrast, neuroblastomas without N-myc amplification exhibit a wide range of histologic features that can define prognostic subsets.

Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas.

Histopathologic prognostic factors of 295 pretreatment tumors of a total 641 neuroblastomas and ganglioneuroblastomas were studied with the use of the following proposed tumor classification. The tumors were divided into 2 groups: stroma-poor (235 cases) and stroma-rich (60 cases) according to their organizational pattern (stromal development). The stroma-poor group was classified further into 2 subgroups: favorable stroma-poor (84% survival) and unfavorable stroma-poor (4.5% survival) according to the patient's age at diagnosis, degree of maturation, and nuclear pathology [mitosis-karyorrhexis index (MKI)] of the neuroblastic cells. The stroma-rich group was further classified into 3 subgroups: well differentiated (100% survival), intermixed (92% survival), and nodular (18% survival) on the basis of morphology of the immature element in the tumor tissue without regard to patient's age or quantitative maturation. Favorable stroma-poor and well-differentiated and intermixed stroma-rich groups seem to make good prognosis groups (87% survival), which show gradual progression along a maturational sequence according to the age of the patient. Unfavorable stroma-poor and nodular stroma-rich groups form poor prognosis groups (7% survival) and show morphological evidence of malignant or aggressive behavior, such as inappropriate immaturity for age, higher MKI, and gross nodule formation by immature neuroblasts.

HLA class I and beta 2-microglobulin expression in frozen and formaldehyde-fixed paraffin sections of neuroblastoma tumors.

Ten solid neuroblastoma tumors were examined for beta 2-microglobulin (beta 2-m) and HLA-class I expression in an immunocytochemical assay. Blood vessel endothelium and a small population of cells (less than 6% of small round cells) were consistently stained in frozen sections of each tumor. No beta 2-m or HLA-class I reactivity was detected in the remainder (greater than 94%) of the small round cells in each tumor. The localization of most of the positive cells near stromal tissue and blood vessels suggests that most of these cells may be of non-tumor origin. The patients from which the tumors were taken varied in degree of disease involvement, yet no differences with respect to beta 2-m and HLA-class I expression were found among the tumors. A procedure for visualizing beta 2-m and HLA-class I molecules in formaldehyde-fixed, paraffin-embedded tissue is described. This confirmed the results in frozen sections. These findings extend previous reports of weak beta 2-m and HLA-class I levels in cells of neuronal origin. The clinical implications are discussed.

Lipoblastoma/lipoblastomatosis: a clinicopathologic study of 25 tumors.

Lipoblastoma/lipoblastomatosis is an uncommon benign adipose tissue tumor of children. Since 1958, 25 of these tumors from 24 patients have been reviewed in the Department of Pathology at The Children's Hospital of Philadelphia. Tumors were resected from 19 boys (79%) and five girls, and 20 patients (84%) were < or =5 years of age at diagnosis. Twenty-three tumors presented as painless superficial soft-tissue masses; one tumor was retroperitoneal and was discovered because of vomiting; one hand tumor was present at birth. Tumors occurred in an extremity (n = 11 patients), the head and neck (n = 5), groin (n = 2), axilla (n = 2), back (n = 1), chest (n = 1), flank (n = 1), labia (n = 1), and retroperitoneum (n = 1). Thirteen tumors occurred on the left side, and five occurred on the right. Lesions measured 1.0-21.0 cm in greatest dimension; 15 of 25 (60%) measured < or =5.0 cm. The largest (retroperitoneal) tumor weighed 450 g. Eleven tumors were discrete lipoblastoma, and 14 had irregular margins (lipoblastomatosis). Microscopically, the tumors displayed adipocytes in different stages of maturation; lobules bordered by septae that were cellular in 11 cases; prominent blood vessels in 19 cases; and myxoid foci in 13 cases. Chart review of 22 patients showed that one tumor recurred 4 years after resection; one tumor recurred after 7 years as fibrolipoma; and one incompletely resected tumor enlarged and at second resection was lipoma. There were no metastases. Three patients also had hemangioma. Juvenile aponeurotic fibroma occurred in one patient near the site of resection of a lipoblastoma 4 years earlier. We conclude that lipoblastoma/lipoblastomatosis behaves benignly, occurs in both superficial and deep sites, occasionally attains large size, may mature, can recur, and may be associated with other benign soft-tissue lesions. Complete surgical excision is the treatment of choice.

Monoclonal antibody NB84 in the differential diagnosis of neuroblastoma and other small round cell tumors.

Two hundred fifty-five well-characterized formaldehyde-fixed and paraffin-embedded small round cell tumors mainly from children and young adults including 105 neuroblastomas were immunohistochemically analyzed with the NB84 monoclonal antibody raised to neuroblastoma cells. Most of the undifferentiated neuroblastomas (21 of 22) and all 83 differentiated neuroblastomas reacted with NB84, but none of these tumors were CD99 positive. Compared with synaptophysin, NB84 was more sensitive, although less specific, in the identification of neuroblastoma in formaldehyde-fixed tissue. In addition to neuroblastoma, skeletal and extraskeletal Ewing's sarcoma and medulloblastoma showed NB84 reactivity in approximately 20% of cases, and 50% of desmoplastic small round cell tumors showed positive cells, usually in smaller numbers than the neuroblastomas. The NB84 reactivity was seen slightly more commonly in morphologically defined (rosette-positive) cases of peripheral primitive neuroectodermal tumors than in Ewing's sarcoma. However, the NB84 positivity did not correlate with the expression of other neural markers (neurofilament proteins, CD57, and synaptophysin) in these tumors. All other small round cell tumors including rhabdomyosarcomas, Wilms' tumors, and lymphomas were NB84 negative. In the case of NB84-positive tumors other than neuroblastoma, their specific reactivity for other markers was useful (Ewing's sarcoma CD99 positive, desmoplastic small round cell tumor desmin and keratin positive). The NB84 monoclonal antibody is a useful reagent to separate neuroblastoma from other small round cell tumors. In problem cases it is best used in a panel together with other markers that address the significant differential diagnostic alternatives.

Cancer in neonates: the experience at the Children's Hospital of Philadelphia.

Among 22 neonates treated at the Children's Cancer Research Center of Philadelphia, 11 had neuroblastoma, which in two cases was widely metastatic. There were three infants with teratoma, three with sarcoma, three with leukemia, one with Wilms' tumor, and one with parotid carcinoma. Nine of eleven patients (82%) are long-term survivors following complete surgical excision of tumor, whereas only one of eight (13%) has survived following incomplete surgical excision. All three neonates with leukemia died. The overall two-year actuarial survival is 45% (10/22). The problems associated with treating neonates with chemotherapy, radiation therapy, or both are especially difficult because of the immaturity of the organs and structures. Surgical excision alone has been the treatment of choice for solid tumors. Chemotherapy or radiation therapy, when indicated, require careful monitoring for both acute toxicities and potential long-term morbidities.

Genetic disorders and major extracardiac anomalies associated with the hypoplastic left heart syndrome.

All pediatric autopsies of patients with hypoplastic left heart syndrome seen during an 11-year interval were reviewed to determine the frequency of underlying chromosomal and single-gene defects and idiopathic major extracardiac anomalies associated with this common, lethal congenital heart abnormality. Of 83 patients identified, nine had underlying chromosomal abnormalities, four had single-gene defects, ten had one or more major extracardiac anomalies without an identifiable chromosomal or mendelian disorder, and two were infants of insulin-dependent diabetic mothers. Overall, 23 patients (28%) had a genetic disorder and/or major extracardiac anomaly. The substantial prevalence of genetic causes of and major extracardiac anomalies associated with hypoplastic left heart syndrome underscores the need for a detailed genetic evaluation for all patients with hypoplastic left heart syndrome.

Prognostic value of histopathology in advanced neuroblastoma: a report from the Childrens Cancer Study Group.

We report the histopathologic findings in 420 patients with stage III and IV neuroblastoma enrolled in Childrens Cancer Study Group trials conducted from 1980 to 1983. A prospective study of individual cytohistologic features showed that outcome was related in a statistically significant manner to mitotic rate, multi-nuclearity, foam cells, ganglion cells, necrosis, and calcification, but only the latter was consistent for both stages. A similar test of four selected published classifications indicated the greatest prognostic value for the system developed by Shimada et al to distinguish favorable from unfavorable tumors. This classification proved significant in both stages and on examination of both primary and metastatic sites. Concordance in histologic assignment of prognosis by two observers was 83%. We conclude that the Shimada classification is valid and reproducible, and that it may be useful in planning therapy in advanced neuroblastoma. Selected cytohistologic parameters and the other classifications were less strongly predictive of outcome, but are worthy of continued study.

Coarctation resection in children with Turner's syndrome: a note of caution.

Eight children were recognized to have Turner's syndrome, among 353 patients over 1 year of age who had undergone surgical treatment for coarctation of the aorta. Of these eight children, three developed a significant perioperative hemorrhage from aortic rupture, resulting in one death and one instance of paraparesis related to a period of prolonged hypotension. In two of the other five patients with Turner's syndrome, a decision was made to perform an angioplasty rather than a resection of the coarctation because of apparent friability of the aortic wall. In contrast, only one of the 345 patients without Turner's syndrome died as a result of surgical treatment, and none developed spontaneous perioperative aortic rupture or neurologic deficit. This experience suggests that the operative risk for coarctation of the aorta in this subgroup of patients is considerably greater than that in patients without Turner's syndrome (p < 0.001). Special precautions should include use of rubber-jaw vascular clamps, choice of technique to avoid tension at the anastomotic suture line, and careful control of systemic blood pressure intraoperatively and postoperatively. Indications for surgical treatment of coarctation as well as the type of operative procedure must be individualized cautiously in patients with Turner's syndrome.

Immunohistochemical evaluation of myeloid leukemia infiltrates (granulocytic sarcomas) in formaldehyde-fixed, paraffin-embedded tissue.

Paraffin sections of granulocytic sarcomas (GS) (n = 30) were immunohistochemically evaluated for CD3, CD15 (LeuM1), CD20 (L26), CD31, CD34, CD43, CD45, CD68 (KP1), lysozyme, myeloperoxidase (BM1), CD45RO (UCHL1), and LN5 with an avidin-biotin amplification system and a peroxidase-based color development system with DAB as a chromogen. CD45 positivity was present in all lymphomas and 24 of 25 granulocytic sarcomas. Lysozyme and CD43 labeled 26 of 29 granulocytic sarcomas, showing intense cytoplasmic staining. LN5 (membrane-staining) and CD68 (subtle cytoplasmic caplike staining) were found in 20 of 30 cases, often only focally. BM1 and CD15 mainly labeled maturing granulocytes and mostly were negative in primitive myeloid cells. Myeloid progenitor cell antigens CD31 and CD34 were seen in 7 and 12 of 30 cases, respectively. They seemed to recognize different subsets of myeloid leukemia infiltrates (16 cases positive for at least one); the use of CD31 and CD34 for defining these subsets should be evaluated further. Features suggesting a dual phenotype--T-cell and myeloid (positive for CD3, CD68, and lysozyme)--were documented in two cases. In contrast, lymphoblastic lymphomas (n = 4) were positive for CD3 and CD43 but negative for CD68, lysozyme, CD31, CD34, LN5, and myeloperoxidase. Lymphocytic lymphomas (n = 10) were positive for CD20 and CD43 but negative for all other markers. Small, round-cell tumors (n = 15) were negative for all markers. If T-cell and B-cell differentiation can be excluded with other markers, CD43+ is a sensitive marker for myeloid differentiation. Our results show that several markers are useful in the identification of myeloid leukemia infiltrates and in distinguishing them from lymphoblastic and lymphocytic lymphomas and small round-cell tumors in formaldehyde-fixed, paraffin-embedded tissue.

Report of a case of gross thymic hyperplasia in a child.

A large thoracic mass and a mediastinal lymph node were excised from an infant with a peripheral blood and bone marrow lymphocytosis. The 224-mass was composed of histologically normal thymus, and the lymph node architecture was partially effaced. Hypogammaglobulinemia was detected two years after thymectomy. The enormous thumus in this case fits the classic gross pathologic definition of hyperplasia. The possibility of associated thymic hyperfunction in this case is discussed.

Detection of gene fusions in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction assay of archival samples.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue tumor that is often difficult to distinguish from other small round-cell tumors. The PAX3-FKHR and PAX7-FKHR gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival material, we used reverse transcriptase-polymerase chain reaction and oligonucleotide hybridization methodology to develop an assay capable of identifying PAX3-FKHR and PAX7-FKHR fusion transcripts in formalin-fixed, paraffin-embedded tissue. Use of a control assay for wild-type FKHR mRNA indicated that RNA was successfully isolated, reverse-transcribed, and amplified in 15 of 16 archival cases. Comparison of assay results for the PAX3-FKHR and PAX7-FKHR fusions with standard molecular assays of paired frozen material revealed that all eight cases of known fusion-positive rhabdomyosarcoma were correctly identified and distinguished as PAX3-FKHR or PAX7-FKHR. The seven cases of known fusion-negative rhabdomyosarcoma showed no evidence of either product. These results indicate that we have developed a molecular assay that accurately identifies the fusion transcripts characteristic of alveolar rhabdomyosarcoma in archival samples. This assay will be useful for diagnosis and for retrospective clinicopathologic correlative studies.

Pulmonary manifestations of juvenile rheumatoid arthritis. A report of eight cases and review.

Pleuropulmonary disease was seen in 4 per cent of patients with juvenile rheumatoid arthritis. Roentgenographic abnormalities seen in association with juvenile rheumatoid arthritis include: transient pneumonitis, interstitial reticular and nodular infiltrates, pleural and pericardial effusions, and patchy pleural infiltrates. Pathologic abnormalities seen in association with juvenile rheumatoid arthritis include pulmonary hemosiderosis, lymphoid follicular bronchiolitis, and lymphocytic interstitial pneumonitis. Patients with juvenile rheumatoid arthritis and pleural disease recover fully. In children with parenchymal disease, residual abnormalities include roentgenographic evidence of interstitial fibrosis and minimal abnormalities of pulmonary function.

Acute renal failure secondary to acute pyelonephritis.

A six year-old girl presented with acute oliguric renal failure, secondary to acute, non-obstructive pyelonephritis. Evidence for pyelonephritis as the cause of renal failure included: the evolution of typical changes on serial intravenous pyelograms, an acute interstitial inflammatory exudate on percutaneous renal biopsy, and gram-positive cocci on gram stain of the biopsy tissue. Although a specific causative organism was not conclusively identified, enterococcus was isolated from the initial catheterized urine specimen. The patient recovered from the acute illness but was left with impaired renal function, hypertension, and cortical scarring. Acute, non-obstructive pyelonephritis can produce acute renal failure in children and must be considered in the differential diagnosis of this syndrome.

Hydropic cell variant (clear cell variant) of Wilms' tumor.

We report an unusual clear cell tumor of the kidney in infancy. The presence in our patient of a discrete mass with foci of primitive nephroblastic cells indicates the neoplastic nature of the clear cells. We discuss its distinction from clear cell sarcomas and carcinomas, and we suggest that this lesion may be inadequately separated from pediatric renal carcinomas in the literature. We recommend the term hydropic cell variant as a less confusing and more accurate descriptive designation.

Nodular renal blastema in trisomy 13.

The autopsy records and material of 14 cases of karyotype-proved trisomy 13 were reviewed. There were one or more foci of nodular renal blastema in six cases. Patients with nodular renal blastema had lived up to five days; patients in whom foci were not found at autopsy had lived six to 37 days. The presence of nodular renal blastema did not substantially correlate with other renal anomalies or the number or severity of extrarenal anomalies. The presence of nodular renal blastema in trisomy 13 expands the spectrum of syndromes associated with nodular renal blastema and supports its relationship with disorders of growth regulation. The presence of the lesions only in very young infants suggests that most, or all, foci of nodular renal blastema regress with age.

The role of surgery in localized neuroblastoma.

A 30-year experience with 83 patients, median age two years, with Children's Cancer Study Group (CCSG) Stages I, II, and III localized neuroblastoma was studied to determine factors that influence outcome. In addition, histology was reclassified in all patients based on the Shimada system, which is divided into five subtypes according to age and cytohistologic criteria. A multivariant survival analysis was carried out and patients were considered to have failed if they relapsed or died from any cause. Initial analysis determined that CCSG stage, Shimada histologic classification, and presence of disease in lymph nodes were statistically significant predictors of failure. Histology was the most important factor with Shimada subtypes 1, 2, and 4 having good outcome and 3 and 5 poor outcome. The latter three variables were combined to create four prognostic groups that had distinctly different rates of survival. Further analysis showed that after controlling for prognostic groups, extent of surgery was a statistically significant predictor. Patients who had more complete surgical resection had better disease-free survival.

Acute pancreatitis and increased intracranial pressure.

A retrospective review of 41 patients with post-mortem evidence of acute pancreatitis revealed that 19 patients (46%) showed concomitant evidence of increased intracranial pressure (ICP). A prospective analysis of five patients with increased ICP demonstrated two patients with evidence of acute pancreatitis. The pathogenesis of acute pancreatitis in these patients appears to be dependent on the following risk factors: increased intracranial pressure, steroids, hypovolemia, morphine infusion and hypothermia. These factors increase vagal stimulation, predispose to pancreatic duct obstruction and enhance cellular hypoperfusion. Acute pancreatitis occurring in Reye's Syndrome is probably a consequence of increased ICP and the therapy instituted.