Inge M. Ambros

MIC2 is a specific marker for ewing's sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of ewing's sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration

This study reports on the specific expression of the MIC2 gene, a pseudoautosomal gene located on the short arms of the X and Y chromosomes, on Ewings sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET) cells. The gene product, a cell membrane protein, is recognized by the newly established monoclonal antibody (MoAb) HBA‐71 and the previously described MoAb 12E7 and RFB‐1. Furthermore, the reaction pattern of the MIC2 antibodies, especially HBA‐71, with normal tissues and a great number of benign and malignant tumors (70 different tumors, 199 tumor samples), as well as the correlation between the specific chromosomal aberrations, i.e., the t(11;22) and the del(22) and the expression of this antigen, are demonstrated. Both ES and pPNET cells express the MIC2 gene in very high amounts, which represents a highly selective and almost unique feature of these cells, making an assignment of these tumors in one entity even more likely. The MIC2 antibodies are of great value for clinical and research purposes.

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.

Immunohistochemical analysis of INI1 protein in malignant pediatric CNS tumors: Lack of INI1 in atypical teratoid/rhabdoid tumors and in a fraction of primitive neuroectodermal tumors without rhabdoid phenotype.

Immunohistochemical lack of nuclear INI1 protein expression has been recently described as characteristic finding in atypical teratoid/rhabdoid tumors (AT/RTs), and has been suggested as useful marker to distinguish AT/RTs from other malignant pediatric central nervous system (CNS) tumors. In this study, we examined a large series of malignant pediatric CNS tumors to determine the immunohistochemical expression of INI1 protein in different malignant pediatric tumor entities. Archival paraffin-embedded biopsy specimens of 289 malignant pediatric CNS tumors including medulloblastomas, supratentorial primitive neuroectodermal tumors, glioblastomas, anaplastic astrocytomas, anaplastic ependymomas, choroid plexus carcinomas, germ cell tumors, and AT/RTs were analyzed immunohistochemically for expression of nuclear INI1 protein. Positive INI1 staining was observed in 263 tumors. Lack of INI1 protein was detectable in 26 tumors. Seventeen of the 26 tumors showed morphologically characteristic features of AT/RTs, whereas 9 embryonal tumors did not display rhabdoid features. Tumors without rhabdoid phenotype but lack of INI1 showed an aggressive clinical course and poor response to conventional treatment regimens. In summary, immunohistochemical expression of INI1 protein is lacking in tumors displaying characteristic morphologic features of AT/RT. Furthermore, a certain number of embryonal tumors without rhabdoid features but lack of INI1 protein and aggressive biologic behavior can be detected. We conclude that INI1 protein analysis should be routinely performed in all malignant pediatric embryonal CNS tumors to detect cases with lack of INI1 protein, because patients with these tumors are likely to benefit from intensified treatment.

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.

Vascular Patterns in Glioblastoma Influence Clinical Outcome and Associate with Variable Expression of Angiogenic Proteins: Evidence for Distinct Angiogenic Subtypes

No data exist on angiogenic patterns and their prognostic impact in human glioblastoma. Such data are relevant for translation of antiangiogenic therapies into clinical applications. Using immunohistochemistry for CD34, we assessed vascular patterns in 114 primary glioblastomas. Vascular patterns comprised unevenly distributed glomeruloid/garland‐like/clustered bizarre vascular formations and evenly distributed delicate capillary‐like microvessels (“classic” vascular pattern). The combination of low content of bizarre vascular formations and prominent classic vascular pattern (n=29) was an independent factor for longer survival (p= 0.006, Cox regression), as well as postoperative high Karnofsky performance status (p=0.005). In patients with a prominent classic vascular pattern, there was no difference of MIB1 labeling index whereas microvessel density and apoptotic index (TUNEL) were significantly higher as compared to all other patients (p<0.05). In addition, diffuse expression of hypoxia‐inducible factor (HIF)‐1α and strong expression of vascular endothelial growth factor were more common (p<0.05, Chi‐square test). FISH revealed loss of chromosomes 1p and 19q only in 1/7 long‐time survivors with classic pattern. We conclude that vascular patterns in primary glioblastoma influence clinical outcome and associate with variable expression of angiogenic proteins. Our findings denote for the first time distinct angiogenic subtypes of human glioblastoma which may prove relevant for anti‐angiogenic therapy approaches.

Regression and progression in neuroblastoma. Does genetics predict tumour behaviour

Neuroblastoma (NB) is a heterogeneous disease. The clinical course may range from spontaneous regression and maturation to very aggressive behaviour. Stage 4s is a unique subcategory of NB, generally associated with good prognosis, despite skin and/or liver involvement and the frequent presence of tumour cells in the bone marrow. Another type of NB is the locally invasive tumour without bone and bone marrow involvement which can also have a good prognosis, irrespective of lymph node involvement. Unfortunately, there is only limited biological information on such tumours which have not been treated with cytotoxic therapy despite lymph node involvement, residual tumour mass after surgery and/or bone marrow infiltration. In order to find specific genetic changes common to NBs with a benign clinical course, we studied the genetic abnormalities of these tumours and compared them with highly aggressive tumours. We analysed a series of 54 localised and stage 4s tumours by means of in situ hybridisation performed on fresh cells or on paraffin embedded tissues. In addition, we performed classical cytogenetics, Southern blotting and PCR analysis on fresh tumour tissue. The majority of patients had been treated with surgery alone, and in a number of patients tumour resection was incomplete. Deletions at 1p36 and amplifications of the MYCN oncogene were absent, and diploidy or tetraploidy were not seen in any case, with residual localised tumours possessing a favourable outcome. Unexpectedly, one patient with a tetraploid 4s tumour without any genetic structural changes not receiving any cytotoxic treatment, did well. Interestingly, this genetic spectrum contrasted with that of progressing tumours, in which most had genetic aberrations, the deletion at 1p36 being the most common event. These data, although limited, suggest that an intact 1p36 (recognised by D1Z2), the absence of MYCN amplification and near-triploidy (at least in localised tumours), represent prerequisites for spontaneous regression and/or maturation.

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

Ewings 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 Fishers 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). Genes Chromosomes Cancer 24:243–254, 1999.

Diagnostic and prognostic impact of urinary catecholamines in neuroblastoma patients

Urinary catecholamine metabolites are well‐known to be elevated in patients with neuroblastoma. Some investigators have described different patterns in favorable and unfavorable cases. However, extended studies have not been published.

Segmental chromosomal alterations have prognostic impact in neuroblastoma: a report from the INRG project

Background:In the INRG dataset, the hypothesis that any segmental chromosomal alteration might be of prognostic impact in neuroblastoma without MYCN amplification (MNA) was tested.Methods:The presence of any segmental chromosomal alteration (chromosome 1p deletion, 11q deletion and/or chromosome 17q gain) defined a segmental genomic profile. Only tumours with a confirmed unaltered status for all three chromosome arms were considered as having no segmental chromosomal alterations.Results:Among the 8800 patients in the INRG database, a genomic type could be attributed for 505 patients without MNA: 397 cases had a segmental genomic type, whereas 108 cases had an absence of any segmental alteration. A segmental genomic type was more frequent in patients >18 months and in stage 4 disease (P<0.0001). In univariate analysis, 11q deletion, 17q gain and a segmental genomic type were associated with a poorer event-free survival (EFS) (P<0.0001, P=0.0002 and P<0.0001, respectively). In multivariate analysis modelling EFS, the parameters age, stage and a segmental genomic type were retained in the model, whereas the individual genetic markers were not (P<0.0001 and RR=2.56; P=0.0002 and RR=1.8; P=0.01 and RR=1.7, respectively).Conclusion:A segmental genomic profile, rather than the single genetic markers, adds prognostic information to the clinical markers age and stage in neuroblastoma patients without MNA, underlining the importance of pangenomic studies.

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.