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Aim of the Study: Radiolabelled meta-iodobenzylguanidine (MIBG) is an effective option in treatment of neuroblastoma (NBL) tumours. We studied feasibility, toxicity and efficacy of upfront 131 I-MIBG and induction treatment in stage 4 NBL patients., Patients and Methods: Retrospective, multi-centre (AMC and EMC) pilot regimen (1/1/2005-2011). Newly diagnosed stage 4 NBL patients, were treated with 2 courses of 131 I-MIBG, GPOH 2004 NBL protocol, myeloablative therapy (MAT) and autologous stem cell rescue (ASCT). 131 I-MIBG was administered in a fixed dose. Response rate (RR) was defined as complete remission, very good partial response and partial response., Results: Thirty-two patients, (median age [range] 2.9 [0-11.4] years), 21 received 131 I-MIBG therapy, 11 did not because of: MIBG non-avid (N = 5) and poor clinical condition (N = 6). In 95% of eligible patients 131 I-MIBG treatment was feasible within 2 weeks from diagnosis. Interval between chemotherapy courses was 25 days ( 131 I-MIBG group) versus 22 days (chemotherapy group). No stem cell support was needed after 131 I-MIBG therapy. Stem cell harvest in both groups was feasible, neutrophil recovery was comparable, but platelet recovery post MAT, ASCT was slower for 131 I-MIBG-treated patients. RR post 131 I-MIBG was 38%, post MAT + ASCT was 71% ( 131 I-MIBG group), 36% (chemotherapy group) and overall 59%., Conclusions: Induction therapy with 131 I-MIBG before the HR GPOH NB 2004 protocol is feasible, tolerable and effective in newly diagnosed stage 4 NBL patients. 131 I-MIBG upfront therapy induces early responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: The clinical importance of the detection of neuroblastoma messenger RNA (mRNA) in bone marrow (BM) of localised neuroblastoma patients at diagnosis remains unclear. In this prospective multicentre study, BM samples of a large cohort, were studied using real-time quantitative polymerase chain reaction (qPCR)., Methods: BM samples at diagnosis from 160 patients with localised neuroblastoma were prospectively collected at Dutch and German centres between 2009 and 2013. qPCR was performed using five neuroblastoma specific markers. The association with other biological factors and the prognostic impact of BM positivity and clinical response was assessed., Results: In 58 out of 160 patients neuroblastoma mRNA was detected in BM. In 47 of the 58 positive samples only one marker was found positive. BM positivity was significantly associated with MYCN amplification (p = 0.02) and deletion of chromosome 1p (p = 0.04). In total 31 patients had an event, of which only five patients had progression to stage IV. BM positivity was not associated with an unfavourable outcome. However, the detection of more than one marker was associated with an unfavourable outcome (systemic or local relapse) (event free survival 48% versus 85%; p = 0.03) in the whole cohort and in the observation group., Conclusions: BM positivity was associated with unfavourable biological factors and might represent more aggressive tumours. Patients with qPCR positive BM should not be upstaged, because of very few systemic events in the cohort. However, for patients with more than one marker positive a more careful follow-up is advisable. These results need to be verified in a very large cohort of localised patients., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Increasing age has been an adverse risk factor in children with neuroblastoma (NB) since the 1970's, with a 12-month age-at-diagnosis cut-off for treatment stratification. Over the last 30 years, treatment intensity for children >12 months with advanced-stage disease has increased; to investigate if this strategy has improved outcome and/or reduced the prognostic influence of age, we analysed the International Neuroblastoma Risk Group (INRG) database., Patients and Methods: Data from 11,037 children with NB (1974-2002) from Australia, Europe, Japan, North America. Cox modelling of event-free survival (EFS) tested if the era and prognostic significance of age-of-diagnosis, adjusted for bone marrow (BM) metastases and MYCN status, effects on outcome had changed., Results: Outcome improved over time: 3-year EFS 46% (1974-1989) and 71% (1997-2002). The risk for those >18 months against ≤12 decreased: hazard ratio (HR); 4.61 and 3.94. For age 13-18 months, EFS increased from 42% to 77%. Outcome was worse if: >18 months (HR 4.47); BM metastases (HR 4.00); and MYCN amplified (HR 3.97). For 1997-2002, the EFS for >18 months with BM involvement and MYCN amplification was 18%, but 89% for 0-12 months with neither BM involvement nor MYCN amplification., Conclusions: There is clear evidence for improving outcomes for children with NB over calendar time. The adverse influence of increasing age-at-diagnosis has declined but it remains a powerful indicator of unfavourable prognosis. These results support the age-of-diagnosis cut-off of greater than 18 months as a risk criterion in the INRG classification system., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aim/purpose: (123)I-meta-iodobenzylguanidine ((123)I-mIBG) scintigraphy is well established for staging and evaluation of response in children with high-risk neuroblastoma but its prognostic value in highly intensive first-line treatment protocols is uncertain. The presence of any (123)I-mIBG positive tumour tissue was correlated with event-free survival (EFS) and overall survival (OS)., Patients and Methods: The prognostic impact of residual (123)I-mIBG uptake into the primary tumour and metastases for predicting outcome in 113 stage 4 neuroblastoma patients >1 year of the German Neuroblastoma Trial NB97 was assessed using a univariate log-rank test and multivariate Cox regression analysis., Results: All patients had (123)I-mIBG positive disease at initial staging. After four courses of induction chemotherapy, 71% of patients were still (123)I-mIBG positive for the primary tumour and 61% for metastases. After six courses, 39% of patients had (123)I-mIBG uptake by the primary tumour and 45% residual (123)I-mIBG positive metastatic disease. The (123)I-mIBG status of the primary tumour site had no bearing on outcome. Residual (123)I-mIBG positive metastatic disease after four (3-y-EFS 25.7+/-5.3% versus 55.9+/-7.6%, p=0.009; 3-y-OS 49.8+/-6.1% versus 65.0+/-7.3%; p=0.021) and after six chemotherapy cycles (3-y-EFS 27.5+/-6.2% versus 47.4+/-6.4%, p=0.011; 3-y-OS 50.5+/-7.1% vs 60.0+/-6.4%, p=0.031) was associated with poor outcome., Conclusion: Functional imaging with (123)I-mIBG scintigraphy can identify poor responders with any persistent metastatic (123)I-mIBG uptake who are at a high risk of disease relapse. (123)I-mIBG response of the primary tumour site had no bearing on outcome.

While the role of MYCN-amplification (MNA) for risk assessment in neuroblastoma is undisputed, the phenomenon of gene copy excess below the amplification threshold is rarely described. To discuss biological characteristics and the clinical impact of the so-called MYCN-gain versus amplified or non-amplified cases, we investigated the MYCN status of 659 patients uniformly analysed by fluorescence in situ hybridisation. The number of MYCN-amplified tumours in our cohort was 18% (116/659); an additional 38 tumours (6%) displayed MYCN-gain. Both alterations were associated with an advanced stage disease, an increased patient age and further chromosomal alterations. Most of the amplified neuroblastomas displayed 1p aberrations, whereas MYCN-gain tumours correlated with 11q alterations. In contrast to the amplified cases, tumours with gain displayed no increased MYCN RNA levels. MNA versus non-amplification discriminated between good and poor outcomes, independent of stage, age and the degree of amplification. However, patients with amplified tumours showed a significantly better outcome when this was combined with non-stage 4 disease and age <1 year versus stage 4 and age < 1 year. Although MYCN-gain was associated with poor event-free-survival (EFS) in stages 1-3, 4S (P=0.005), this might be related to associated genetic aberrations and not to the MYCN-gain itself. A survival difference between neuroblastomas with gain and single copy MYCN could not be delineated. In conclusion, MNA predicts a poor outcome for neuroblastoma patients of all stages and age. MYCN-gain is also a characteristic feature of advanced stage tumours and older patients, but is not associated with higher MYCN expression and appears not to be discriminative in predicting patient outcome.

Familial neuroblastoma is of special interest in view of the oncogenesis of this tumour with its early manifestation in childhood. The inheritance seems to follow an autosomal-dominant mendelian trait with incomplete penetrance. Familial neuroblastomas and ganglioneuromas have not been reported in detail within large treatment studies. A retrospective clinicopathological survey of patients reported to the German neuroblastoma treatment studies over 24 years was performed. Among 2863 patients (2752 neuroblastomas, 111 ganglioneuromas) included in five consecutive trials, only 22 hereditary cases in ten families were observed. Neuroblastomas were found in 18 patients and ganglioneuromas in four, accounting for less than one percent of all cases. Six patients with neuroblastomas had localised disease, seven had stage 4, three had stage 4S, and stage was unknown in two patients. Two families had three affected patients. Contrary to previous reports, age distribution and number of primary tumours in patients with familial data confirm the low prevalence of familial neuroblastoma and may help in counselling the affected families.

The value of the tumour markers vanillylmandelic acid (VMA) and homovanillic acid (HVA) in urine (u) and serum (s), neurone-specific enolase (NSE), and lactate dehydrogenase (LDH) in the early prediction of relapse/progression in neuroblastoma is not known. We analysed the data of neuroblastoma patients who had successfully completed first-line treatment and had laboratory results available from their initial diagnosis and from relapse/progression (n=196). Patients' overall survival from relapse or progression was 21.5+/-4.2% (mean+/-standard deviation). At diagnosis, we found abnormal results in 75% for VMA and/or HVA (s), 92% for VMA and/or HVA (u), 90% for NSE, and 81% for LDH. We found a lower incidence of abnormal results at relapse or progression with 40% for VMA and/or HVA (s), 54% for HVA and/or VMA (u), 61% for NSE, and 48% for LDH. Sensitivity of all markers was higher for metastatic compared with local recurrence. NSE was the best, being able to detect 42% of the localised relapses, 77% of the combined local/metastatic relapses, and 69% of the metastatic recurrences. Relapse or progression in neuroblastoma cannot be detected reliably by monitoring tumour markers alone. Therefore, follow-up of neuroblastoma patients must include clinical assessment and imaging studies.

Between January 1986 and May 1996, 870,313 children were tested in European neuroblastoma (NB) screening programmes. Among these children, 82 cases of NB (age range 4-24 months, median 11 months) were detected by screening. 83% of the patients had localised NB and 17% were diagnosed with generalised NB (stage 4, 10%; stage 4s, 7%). Unfavourable biological markers (MYCN amplification, loss of heterozygosity (LOH) 1p36, DNA di/tetraploidy) were observed in 14% of 76 biologically examined cases. The median follow-up time of all the patients was 21.5 months (range 1-101 months). To date, 69 patients are in complete remission (CR) and 2 patients have died due to therapy (stage 4, 1 patient; stage 3, 1 patient with unfavourable markers). Apart from screened patients, 16 other patients with NB were found who had previously had a normal screening test, i.e. 'false negative' patients (age range 10-41 months, median 31.5 months). The median interval between screening and diagnosis was 24.5 months (range 6-35 months). 11 of the 'false negative' patients suffered from generalised NB (stage 4) and 5 had localised NB at diagnosis. Unfavourable biological markers were observed in 7/12 patients. 5 patients have died, 2 achieved partial remission and 9 CR. 9 of the 11 patients with unfavourable biological markers diagnosed due to NB screening are currently in CR. It is very likely that, among the patients without unfavourable biological markers, we detected tumours which may have regressed spontaneously. These children may have undergone 'unnecessary,' but unavoidable, diagnostic procedures and therapy. To reduce the number of 'false negative' patients, a later screening could be helpful and should be evaluated.

The association of molecular characteristics with prognosis has been reported, but not their relationship with each other and their impact in the context of known clinical risk factors. In this study, data of 1249 consecutive intent-to-treat-neuroblastoma patients with more than 1 year follow-up were examined by multivariate analysis using loglinear and Cox proportional hazard regression models on a stage-related basis (stages 1-3: 600, 4S: 116, 4: 533). In a first step, risk factors were identified from 18 selected clinical variables, and risk groups defined. The second step investigated whether molecular characteristics (MYCN, LOH 1p, del 1p, CD44, N-ras, NGF-R, bcl-2, APO-1 (CD95)) contributed additional prognostic information to the model. The loglinear model demonstrated several interactions between clinical factors. By the Cox regression model, seven independent clinical risk factors were found for stages 1-3, seven for stage 4 and two for stage 4S. By subsequent introduction of all molecular variables, MYCN amplification only added significant prognostic information to the clinical factors in localised and stage 4 neuroblastoma. The models allowed the definition of risk groups for stages 1-3 patients by age (e beta = 5.09) and MYCN (e beta = 4.26), for stage 4 by MYCN (e beta = 2.78) and number of symptoms (e beta = 2.44) and for stage 4S by platelet count (e beta = 3.91) and general condition (e beta = 2.99). Molecular factors and in particular MYCN contribute significantly to risk estimation. In conjunction with clinical factors, they are powerful tools to define risk groups in neuroblastoma.

The role of single drugs in the treatment of neuroblastoma is poorly defined. We, therefore, tested neuroblastoma cell survival after a 72 h exposure to one of 19 cytostatic drugs by monolayer proliferation assay. 6 cell lines (IMR-5, Kelly, SK-N-SH, GI-CA-N, CHP-100, CHP-134) were selected on the basis of MYCN amplification and PGY1 overexpression. ED50 drug concentrations were related to plasma levels achievable in patients during chemotherapy. More effective substances were mitoxantrone, doxorubicin, hydroxyurea, bleomycin, dactinomycin, cisplatinum, thiotepa, melphalan, carboplatinum, etoposide, vincristine, cytarabine, 6-thioguanine, cyclophosphamide, ifosfamide and zilascorb. Parental drugs (cyclophosphamide, cisplatinum) appeared more cytotoxic on a molar basis than derived drugs (ifosfamide, carboplatinum). Less effective drugs included 5-fluorouracil, 6-mercaptopurine, CCNU and procarbazine. Fractional application of a given dose was more efficient than a single dose of cyclophosphamide, ifosfamide and cisplatinum. The tested neuroblastoma cell lines showed distinct sensitivities to cytostatic drugs. Cell lines with MYCN amplification appeared more sensitive than PGY1 overexpressing cells. In conclusion, comparative in vitro testing of cytostatic drugs may provide a rationale for their clinical evaluation. Investigation of drug combinations and application of the monolayer proliferation assay to tumour biopsy material for preclinical chemosensitivity testing are clearly warranted.

Cell-cell and cell-extracellular matrix interactions mediated by cell adhesion molecules (for example CD44) play an important role in the cascade of metastasis and the progression of human malignant tumours. The most important aim of this review was, on the basis of our results and the literature, to show the correlation between the expression of CD44s and differentiation and prognosis of neuroblastoma. Surprisingly and in contrast to most other malignant tumours, neuroblastomas exhibited an inverse correlation between CD44s expression and tumour progression. It can be stated that CD44s is a prognostic marker in neuroblastoma which correlates significantly with the grade of tumour cell differentiation, but not with clinical stage. Moreover, there exists a statistically significant correlation between MYCN oncogene amplification and the lack of CD44s expression.

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

In 211 patients with neuroblastoma, serum vanillylmandelic acid (VMA) and homovanillic acid (HVA) levels were determined and correlated to stage, histological differentiation, ferritin, neuron-specific enolase, lactate dehydrogenase (LDH) and outcome. Elevated serum VMA and/or HVA levels were found 16% less frequently than elevated urine levels. The incidence of the elevated serum levels increased with stage (stages I-III 58%, IV 78%, IVS 100%). Increased VMA/HVA ratios were not associated with a higher grade of tumour differentiation. Serum ferritin and neuron-specific enolase showed no correlation, and LDH a borderline non-random correlation with the serum catecholamine metabolites. Using age-related reference values a quotient of serum VMA/HVA (P = 0.061) < 0.7 indicated a poorer event-free survival (48 +/- 10%) than ratios > or = 0.7 (event-free survival 81 +/- 6%) for children with localised neuroblastoma (P = 0.0004). No correlation with prognosis was detected for patients with stage IV and stage IVS disease. We conclude that serum VMA and HVA determinations may be useful as tumour markers for 71% of neuroblastoma patients, and aid in estimating the prognosis in children with localised disease.