Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors.

Intratumoral heterogeneous MYCN amplification (hetMNA) is an unusual event in neuroblastoma with unascertained biological and clinical implications. Diagnosis is based on the detection of MYCN amplification surrounded by non-amplified tumor cells by fluorescence in situ hybridization (FISH). To better define the genetic features of hetMNA tumors, we studied the Spanish cohort of neuroblastic tumors by FISH and single nucleotide polymorphism arrays. We compared hetMNA tumors with homogeneous MNA (homMNA) and nonMNA tumors with 11q deletion (nonMNA w11q-). Of 1091 primary tumors, 28 were hetMNA by FISH. Intratumoral heterogeneity of 1p, 2p, 11q and 17q was closely associated with hetMNA tumors when analyzing different pieces for each case. For chromosome 2, 16 cases showed 2p intact, 4 focal gain at 2p24.3 and 8 MNA. The lengths of the smallest regions of overlap (SROs) for 2p gains and 1p deletions were between the SRO lengths observed in homMNA and nonMNA w11q- tumors. Co-occurrence of 11q- and +17q was frequently found with the largest SROs for both aberrations. The evidence for and frequency of different genetic subpopulations representing a hallmark of the hetMNA subgroup of NB indicates, on one hand, the presence of a considerable genetic instability with different SRO of either gains and losses compared with those of the other NB groups and highlights and, on the other hand, the need for multiple sampling from distant and macroscopically and microscopically distinct tumor areas. Narrowing down the different SRO for both deletions and gains in NB groups would be crucial to pinpointing the candidate gene(s) and the critical gene dosage with prognostic and therapeutic significance. This complexity of segmental chromosomal aberration patterns reinforces the necessity for a larger cohort study using FISH and pangenomic techniques to develop a suitable therapeutic strategy for these patients.

Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee.

Neuroblastoma serves as a paradigm for utilising tumour genomic data for determining patient prognosis and treatment allocation. However, before the establishment of the International Neuroblastoma Risk Group (INRG) Task Force in 2004, international consensus on markers, methodology, and data interpretation did not exist, compromising the reliability of decisive genetic markers and inhibiting translational research efforts. The objectives of the INRG Biology Committee were to identify highly prognostic genetic aberrations to be included in the new INRG risk classification schema and to develop precise definitions, decisive biomarkers, and technique standardisation. The review of the INRG database (n=8800 patients) by the INRG Task Force finally enabled the identification of the most significant neuroblastoma biomarkers. In addition, the Biology Committee compared the standard operating procedures of different cooperative groups to arrive at international consensus for methodology, nomenclature, and future directions. Consensus was reached to include MYCN status, 11q23 allelic status, and ploidy in the INRG classification system on the basis of an evidence-based review of the INRG database. Standardised operating procedures for analysing these genetic factors were adopted, and criteria for proper nomenclature were developed. Neuroblastoma treatment planning is highly dependant on tumour cell genomic features, and it is likely that a comprehensive panel of DNA-based biomarkers will be used in future risk assignment algorithms applying genome-wide techniques. Consensus on methodology and interpretation is essential for uniform INRG classification and will greatly facilitate international and cooperative clinical and translational research studies.

Induction of senescence in MYCN amplified neuroblastoma cell lines by hydroxyurea.

Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei-inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cell-associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associated-beta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

DEC1 expression in 1p-aberrant oligodendroglial neoplasms.

BACKGROUND: Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). MATERIALS AND METHODS: 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. RESULTS: DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. CONCLUSION: DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Pathology and biology guidelines for resectable and unresectable neuroblastic tumors and bone marrow examination guidelines.

The recommendations concerning tumor and bone marrow handling for the evaluation of molecular-biologic and molecular-genetic and immunologic markers presented in this paper were developed by the SIOP Europe Neuroblastoma Pathology and Biology and Bone Marrow Group. Although the Guidelines were developed for neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma), they are applicable to all other tumor entities as well. The paper is subdivided in three main parts. The Pathology Guidelines give an overview about the handling, sectioning and securing of tumor material in case of resectable and non-resectable neuroblastic tumors. The Guidelines encompass open biopsies, tru cut biopsies, fine needle aspirations, and bone marrow aspiration. The importance of the pathologic evaluation for the interpretation of the molecular-genetic and molecular-biologic results, which also includes the exact determination of the tumor cell content is stressed. Besides this, recommendations concerning tumor material obtained after cytotoxic therapy, immunohistologic and immuno-cytologic issues and lymph node examination are addressed. In the Biology Guidelines, the different methods for MYCN, chromosome 1p36 investigations and DNA content measurements are discussed and DNA probes are recommended. Furthermore, specified definitions and a common terminology already used in the SIOP Europe Neuroblastoma Group are presented. In the Bone Marrow Guidelines, recommendations concerning the methods to be employed are given and the most important pitfalls are demonstrated. Both the use of standardized methods and the application of a common language will, it is hoped, contribute to the quality and reliability of collected data and thus to a better comparability between and among research reports. These improvements should prove to be of great value for the affected patients.

Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow.

PURPOSE: To improve the detection of disseminated tumor cells in bone marrow (BM) and peripheral blood samples of solid tumor patients, a novel computer-assisted scanning system for automatic search, image analysis, and repositioning of these cells was developed. This system allows precise identification and quantification of tumor cells by sequential immunological and molecular cytogenetic analysis. In this study, we attempt to demonstrate the practical use of this approach by analyzing BM samples from neuroblastoma patients. EXPERIMENTAL DESIGN: The disialo-ganglioside (GD2) molecule was used as the immunological target. The GD2 molecule was described as being specific for neuroblastoma cells, although false positive reactions had been suspected. To verify or disprove the neoplastic nature of the immunologically positive cells, sequential fluorescence in situ hybridization was performed on these cells to search for those genetic aberrations found in the corresponding primary tumors. A total of 115 samples from 40 newly diagnosed patients were evaluated for the presence of GD2(+) cells in the BM. RESULTS: GD2 positivity was detected in 95.2% of stage 4 patients, in 100% of stage 4s patients, and in 38.5% of patients with localized/regional disease. In stage 4 and 4s BM samples, the GD2(+) cells were unequivocally identified as tumor cells based on the molecular cytogenetic aberrations found by fluorescence in situ hybridization. However, in BM samples from patients with localized/regional disease, all GD2(+) cells were concluded to represent false positivity due to the absence of genetic aberrations. CONCLUSIONS: Automatic search and sequential molecular cytogenetic analysis of the immunologically positive cells provide precise information on both the number and cytogenetic profile of disseminated tumor cells.

Intratumoural heterogeneity of 1p deletions and MYCN amplification in neuroblastomas.

BACKGROUND: At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression. PROCEDURE: Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening. CONCLUSION: The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases.

Neuroblastoma cells provoke Schwann cell proliferation in vitro.

BACKGROUND: A subset of human neuroblastomas (NBs) has the capacity to mature completely, imitating sympathetic ganglia. Previously, we showed that the neuronal population in spontaneously maturing NBs usually has a near-triploid DNA content without 1p deletions, and we concluded that the constantly diploid Schwann cells (SCs) do not belong to the neoplastic component of these tumours. We therefore hypothesised that NB cells are able to stimulate SC proliferation, and that SCs trigger NB differentiation. PROCEDURE: We performed in vitro experiments to test this model and to test whether SCs can also influence the growth of aggressive NBs. Human SCs were co-cultivated with NB tumours and cell lines, and were harvested after defined time intervals. Proliferative activity of the SCs and the NB cells was determined by visualisation of 5-bromo-2'-deoxyuridine (BrdU) incorporation or Ki-67 staining. Neurite outgrowth and neurofilament (NF) expression were analysed immunocytochemically and apoptotic rate was determined by a terminal deoxynucleotidyl transferase-mediated dUTP-X fluorescein nick end labelling (TUNEL) assay. RESULTS: Human NB tumours or cell lines unequivocally increased the proliferation of SCs in vitro. In cocultivated NB cells, the proliferative activity was not altered in the first days of cocultivation, although neurite outgrowth and NF expression were enhanced. However, after 10 days, the mitotic rate of neuroblastic cells decreased and the apoptotic rate showed a marked increase. CONCLUSIONS: The results of the cocultivation experiments provide an experimental hint that the in vivo growth of SCs in NBs is caused by the neoplastic neuroblasts, and they also indicate that cells from peripheral nerves can influence the growth of aggressive NB cells if cocultivated.

Automatic detection and genetic profiling of disseminated neuroblastoma cells.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

Absence of somatostatin receptor expression in vivo is correlated to di- or tetraploid 1p36-deleted neuroblastomas.

BACKGROUND: Poor prognosis in childhood neuroblastoma is associated with deletions of chromosome region 1p36 and di/tetraploid DNA content. PROCEDURE: Forty-six patients with histopathologically proven neuroblastoma were investigated for in vivo expression of somatostatin receptors (SR) by 111In-pentetreotide scintigraphy. All tumors were analyzed for cytometric DNA content and chromosome 1p36 integrity. RESULTS: SR expression was detected in 28 tumors (61%) and correlated with young age, localized clinical stage, and favorable outcome. Fourteen tumors showed deletion at chromosome 1p36, thirteen of which did not show SR expression (P< 0.001). A triploid DNA content was correlated with the presence of SR (23 of 25, P< 0.001). No tumor with deletion of chromosome 1p36 and di/tetra DNA content showed SR expression (chi2 = 29.88, d.o.f. = 2, P < 0.001). CONCLUSIONS: We conclude that SR expression is related to genetic features of prognostic significance. This may be assessed with a minimally invasive scintigraphic method.

Prognostic significance of DNA di-tetraploidy in neuroblastoma.

BACKGROUND: Identification of biological factors may provide tools to discriminate poor risk neuroblastoma patients of diagnosis, to ultimately offer risk adapted treatment intensity. PROCEDURES: Tumour cell DNA content, MYCN amplification (NMA), deletion of the short arm of chromosome 1 (del 1p) as well as three serological markers were assessed in 179 children with neuroblastoma. RESULTS: Localised regional disease (stage 1 to 3) was diagnosed in 98 patients, and disseminated disease in 81 patients (65 with stage 4, 16 with stage 4s). Median age at diagnosis was 12 months and the median observation time 4 years. Sixty-seven of 179 patients had near di-tetraploid tumours (37%), with a significantly worse prognosis of 44% overall survival at 4 years in comparison with 88% in near triploid tumours (P < .001). The near di-tetraploid group showed a significant correlation with additional adverse biological factors (NMA, del 1p: P < 0.001), age over 1 year (P< 0.001), clinical stage 4 (P< 0.001), elevated ferritin (P = 0.023), and elevated LDH (P< 0.001). Multivariate analysis based on the overall (OS) and event free survival (EFS) estimations revealed that near di-tetraploidy was the most powerful biological factor, with a P-value of <0.001 for EFS and OS, followed by NMA (P = 0.015) for OS and del 1p (P= 0.047) for EFS. CONCLUSIONS: This analysis underlines the important influence of near di-tetraploidy on prognosis, and suggests that more efforts should be undertaken to implement this factor in future studies.

Combined (111)In-pentetreotide scintigraphy and (123)I-mIBG scintigraphy in neuroblastoma provides prognostic information.

BACKGROUND: High-affinity somatostatin receptors (SRs) have been characterized in neuroblastomas and may be used as target structures for in vivo detection of SR. PROCEDURE: Eighty-eight children with histologically proven neuroblastoma were investigated at diagnosis or relapse by (123)I-mIBG and (111)In-pentetreotide scintigraphy. All tumors were investigated for MYCN copy number, chromosome 1p36 status, and 68/88 also for DNA content, followed for a median follow-up of 35 months (range 1-88 months). RESULTS: SR expression was detected in 56/88 tumors and (123)I-mIBG showed positivity in 83/88. (111)In-pentetreotide was less sensitive in detecting tumor tissue than was (123)I-mIBG (64% vs. 94%, P = 0.005). Survival (SUR) and event-free survival probability (EFS) according to Kaplan-Meier was significantly better for children with positive SR scintigraphy than for the children with a negative SR scan (SUR: 90% vs. 48% at 4 years log rank P < 0.003, EFS: 83% vs. 39% at 4 years, log rank P < 0.0002). CONCLUSIONS: (123)I-mIBG scintigraphy remains the best scintigraphic method for detecting neuroblastoma tumor tissue, whereas additional SR scintigraphy is able to provide significant prognostic information with a minimum of invasiveness.

Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors.

Ewing's sarcoma, peripheral primitive neuroectodermal tumors, and Askin tumors are referred to as Ewing tumors (ETs), and are characterized by high MIC2 expression and a t(11;22)(q24;q12) or other rearrangements involving 22q12. In addition to these constant aberrations, facultative numerical and structural aberrations have been reported: gains of chromosomes 8 and 12, the unbalanced translocation t(1;16), and deletions at the short arm of chromosome 1. To evaluate the frequency and to study the biological impact of these facultative aberrations, we analyzed tumor specimens from 58 ET patients by classical cytogenetics and/or in situ hybridization techniques and compared these data with clinical parameters. Gains of chromosomes 8 and 12 were detected in 55% (32/58) and 24% (14/58) of the cases, respectively. Loss of chromosome 16 or der (16)t(1;16) chromosomes were found in 20% (10/51); deletions at 1p36 were observed in 18% (9/51) of the cases evaluated. The presence of these aberrations did not correlate with age and sex of the patients, with the location of the primary tumor or with the extent of disease at diagnosis by chi-square analysis and Fisher's exact test. Patients with tumors harboring gains of chromosome 8 showed a slightly better clinical outcome (n = 14/30, P = 0.17), whereas gains of chromosome 12 did not influence the clinical outcome (n = 7/30, P = 0.63). However, Kaplan and Meier analysis revealed that deletions at the short arm of chromosome 1 were associated with an unfavorable outcome in patients with localized disease (n = 6/22; P = 0.004).

Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee.

BACKGROUND: As part of the international cooperative effort to develop a complete set of International Neuroblastoma Risk Groups, the International Neuroblastoma Pathology Committee (INPC) initiated activities in 1994 to devise a morphologic classification of neuroblastic tumors (NTs; neuroblastoma, ganglioneuroblastoma, and ganglioneuroma). METHODS: Six member pathologists (H.S., I.M.A., L.P.D., J.H., V.V.J., and B.R.) discussed and defined morphologically based classifications (Shimada classification; risk group and modified risk group proposed by Joshi et al.) on the basis of a review of 227 cases, using various pathologic characteristics of the NTs. The classification-grading system was evaluated for prognostic significance and biologic relevance. RESULTS: The INPC has adopted a prognostic system modeled on one proposed by Shimada et al. It is an age-linked classification dependent on the differentiation grade of the neuroblasts, their cellular turnover index, and the presence or absence of Schwannian stromal development. Based on morphologic criteria defined in this article, NTs were classified into four categories and their subtypes: 1) neuroblastoma (Schwannian stroma-poor), undifferentiated, poorly differentiated, and differentiating; 2) ganglioneuroblastoma, intermixed (Schwannian stroma-rich); 3) ganglioneuroma (Schwannian stroma-dominant), maturing and mature; and 4) ganglioneuroblastoma, nodular (composite Schwannian stroma-richlstroma-dominant and stroma-poor). Specific features, such as the mitosis-karyorrhexis index, the mitotic rate, and calcification, were also included to allow the prognostic significance of the classification to be tested. Recommendations are made regarding the surgical materials to use for an optimal pathobiologic assessment and the practical handling of samples. CONCLUSIONS: The current article covers the essentials and important points regarding the histopathologic evaluation of NTs. Using the morphologic criteria described herein, the INPC is proposing the International Neuroblastoma Pathology Classification. It is reported in a companion article in this issue (Cancer 1999;86:363-71).

The International Neuroblastoma Pathology Classification (the Shimada system).

BACKGROUND: The International Neuroblastoma Pathology Committee, which is comprised of six member pathologists, was convened with the objective of proposing a prognostically significant and biologically relevant classification based on morphologic features of neuroblastic tumors (NTs) (i.e., neuroblastoma, ganglioneuroblastoma, and ganglioneuroma). METHODS: A total of 227 cases were reviewed. Consensus diagnoses from morphologic features (criteria described separately) based on five of six or six of six agreements by the reviewer pathologists were used for prognostic analysis. Prognostic effects of morphology, both individual and in combination, taken in conjunction with age (Shimada classification, histologic grade, and risk group), were analyzed. RESULTS: Approximately 99% of cases (224 of 227) had consensus diagnoses for categorization: neuroblastoma (Schwannian stroma-poor), 190 cases; ganglioneuroblastoma, intermixed (Schwannian stroma-rich), 5 cases; ganglioneuroma (Schwannian stroma-dominant) maturing, 1 case; ganglioneuroblastoma, nodular (composite Schwannian stroma-rich/stroma-dominant and stroma-poor), 19 cases; and NT-unclassifiable, 9 cases. For the NTs, subtype (93% consensus: undifferentiated, 6 cases; poorly differentiated, 155 cases; and differentiated, 15 cases), mitosis-karyorrhexis index (90% consensus: low, 94 cases; intermediate, 40 cases; and high, 37 cases), mitotic rate (75% consensus: low, 89 cases; high, 50 cases; and not determined, 4 cases), and calcification (100% consensus: yes, 110 cases and no, 80 cases) were recorded. Statistical analysis demonstrated that the Shimada classification system (90% consensus; 3-year event free survival: 85% for the group with favorable histology and 41% for the group with unfavorable histology; P = 0.31 x 10(-9)) had a significantly stronger prognostic effect than individual features and other combinations. CONCLUSIONS: The International Neuroblastoma Pathology Classification, a system based on a framework of the Shimada classification with minor modifications, is proposed for international use in assessing NTs.

Neuroblastoma screening in infants postponed after the sixth month of age: a trial to reduce "overdiagnosis" and to detect cases with "unfavorable" biologic features.

BACKGROUND: Encouraged by Japanese reports of the benefits of screening 6 month-old infants for neuroblastoma, a neuroblastoma screening program was introduced in Austria in 1991. However, because of concerns related to "overdiagnosis" by screening at this age, the screening test was performed at a later age. METHODS: From March 1991 to February 1995 neuroblastoma screening was performed on filter paper urine specimens in 100,043 Austrian infants (median age 8.5 months). Primary analysis of urine catecholamines (vanillylmandelic acid and homovanillic acid was performed by use of an E1A method. Questionable or positive results were confirmed by high performance liquid chromatography (HPLC). A double retest was requested following a positive HPLC result. RESULTS: Twenty-one infants were admitted to a hospital following repeatedly elevated values of vanillymandelic acid (VMA) and/or homovanillic acid (HVA). Eleven infants were found to have neuroblastoma (three stage 1, four stage 2B, four stage 3). Treatment consisted of surgery alone with total or subtotal resection in eight cases, surgery and chemotherapy in two cases, and chemotherapy alone in one case. Biologic features were assessed in all tumors excluding ploidy in one case. The majority of the tumors analyzed were near-triploid (9/10), however, two tumors revealed N-myc amplification. CONCLUSIONS: Our results demonstrate that stage distribution and biologic features of neuroblastomas diagnosed by screening at 8.5 months are different from the results of screening at 6 months. Furthermore, the detection of one neuroblastoma among 9,100 screened infants is significantly lower than the incidence of the Japanese screening program. Our results suggest that screening at an age of 7 to 10 months reduces overdiagnosis and may be of more benefit than earlier screening.