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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.

Background: Neuroblastoma is a rare malignant disease and patients with high-risk neuroblastoma have a poor prognosis. Retinoic acid has been shown to inhibit growth of human neuroblastoma cells and has been considered as a potential candidate for improving the outcome. Methods: The objective was to evaluate effects of retinoic acid after consolidation with high-dose chemotherapy and bone marrow transplantation as compared to placebo or no further treatment in patients with high-risk neuroblastoma. We searched the databases CENTRAL, MEDLINE, and EMBASE from inception to 01 October 2014 and included randomized controlled trials. Results: We identified one relevant randomized controlled trial with 50 participants receiving retinoic acid and 48 participants receiving no further therapy. There was no statistically significant difference between the treatment groups in overall survival (hazard ratio 0.87, 95% confidence interval 0.46–1.63, P=0.66) and event-free survival (hazard ratio 0.86, 95% confidence interval 0.50–1.49, P=0.59). We did not identify results for other outcomes, including toxicity. Conclusion: The difference in overall and event-free survival between treatment alternatives was not statistically significantly different. Based on the currently available evidence, we are uncertain about the effects of retinoic acid after bone marrow transplantation in patients with high-risk neuroblastoma.

International audience; The Fourth Metronomic and Anti-angiogenic Therapy Meeting was held in Milan 24-25 June 2014. The meeting was a true translational meeting where researchers and clinicians shared their results, experiences, and insights in order to continue gathering useful evidence on metronomic approaches. Several speakers emphasised that exact mechanisms of action, best timing, and optimal dosage are still not well understood and that the field would learn a lot from ancillary studies performed during the clinical trials of metronomic chemotherapies. From the pre-clinical side, new research findings indicate additional possible mechanisms of actions of metronomic schedule on the immune and blood vessel compartments of the tumour micro-environment. New clinical results of metronomic chemotherapy were presented in particular in paediatric cancers [especially neuroblastoma and central nervous system (CNS) tumours], in angiosarcoma (together with beta-blockers), in hepatocellular carcinoma, in prostate cancer, and in breast cancer. The use of repurposed drugs such as metformin, celecoxib, or valproic acid in the metronomic regimen was reported and highlighted the potential of other candidate drugs to be repurposed. The clinical experiences from low- and middle-income countries with affordable regimens gave very encouraging results which will allow more patients to be effectively treated in economies where new drugs are not accessible. Looking at the impact of metronomic approaches that have been shown to be effective, it was admitted that those approaches were rarely used in clinical practice, in part because of the absence of commercial interest for companies. However, performing well-designed clinical trials of metronomic and repurposing approaches demonstrating substantial improvement, especially in populations with the greatest unmet needs, may be an easier solution than addressing the financial issue. Metronomics should always be seen as a chance to come up with new innovative affordable approaches and not as a cheap rescue strategy.

In Germany and Austria, more than 90% of pediatric cancer patients are enrolled into nationwide disease-specific first-line clinical trials or interim registries. Essential components are a pediatric cancer registry and centralized reference laboratories, imaging review, and tumor board assistance. The five-year overall survival rate in countries where such infrastructures are established has improved from20% before 1950 to80% since 1995. Today, treatment intensity is tailored to the individual patient's risk to provide the highest chances of survival while minimizing deleterious late effects. Multicenter clinical trials are internationalized and serve as platforms for further improvements by novel drugs and biologicals.

Part II demonstrates the results of MRI follow up studies of 23 children with leukemia. Differentiation between leukemic cells and normal hematopoiesis by MRI is impossible. The state of disease can only be determined together with bone marrow biopsy. Clinical remission precedes the normalization of bone marrow signal by weeks. In non-responders, however, cytological findings and MRI findings seem to correlate at the same time. During therapy, areas of osteonecrosis may develop. They do not necessarily contradict continuing complete remission. But this does not affect the prognosis and complete remission is still a possibility. Diagnosis of relapse by MRI is only possible in correlation with studies during the different states of disease especially at clinical remission.

The results of MRI studies performed on the medullary cavity in the knee region of 15 children with leukemia and 5 healthy children are reported. By the age of a few years the signal intensities and the relaxation times of bone marrow begin to resemble fat. Early leukemic infiltration can therefore be more easily recognised in the knee region than in the spine using simple T1 weighted Spin-Echo images. We observed an abnormal signal pattern in all our patients which fell into three groups: (a) diffuse uniform (b) diffuse non-uniform and (c) patchy. We have not been able to correlate these into age, sex or the risk factor determined by clinical or laboratory methods. The diffuse patterns seem to dominate in cases of ALL, the patchy forms in AML. No correlation could be found between the blast levels established by the iliac crest biopsy and the results of MRI.

(131)I-meta-iodobenzylguanidine ((131)I-MIBG) therapy has been used in neuroblastoma treatment for many years but its value in high intensive first line treatment protocols is not exactly known.Stage 4 neuroblastoma patients1 year with (123)I-MIBG positive residual disease (primary tumour and/or metastasis) after complete induction chemotherapy according to the German neuroblastoma trial NB97 were retrospectively analyzed.One-hundred-eleven patients had (123)I-MIBG positive residual disease after complete induction chemotherapy. Forty patients received (131)I-MIBG therapy using a median activity of 0.44 GBq/kg body weight. By univariate analysis, patients who underwent (131)I-MIBG therapy had a better 3-year event free survival (3-y-EFS 46 +/- 8%) and 3-year overall survival (3-y-OS 58 +/- 9%) than 71 patients without (131)I-MIBG therapy (3-y-EFS 19 +/- 5%, p = 0.003; 3-y-OS 43 +/- 6%, p = 0.037). However, subgroup analysis of 66 patients who underwent high dose chemotherapy with autologous stem cell transplantation (ASCT) during treatment found a very similar outcome with (131)I-MIBG therapy (3-y-EFS 49 +/- 9%, 3-y-OS 59 +/- 10%) and without (131)I-MIBG therapy (3-y-EFS 33 +/- 9%, p = 0.171; 3-y-OS 59 +/- 9%, p = 0.285) due to the dominating effect of ASCT. By multivariate analysis, (131)I-MIBG therapy had no impact on EFS (p = 0.494) and OS (p = 0.891). Only ASCT, external beam radiation therapy and MYCN amplification were important for EFS and OS.An independent advantage of I-131-MIBG therapy could not be proven in this retrospective analysis. The ongoing German Neuroblastoma Trial NB2004 will address the influence of (131)I-MIBG therapy with emphasis on tumour dosimetry.

Availability of statistically sufficient numbers of tumor samples and other biomaterials in high quality together with corresponding clinical data is crucial for biomedical research. Tumor repositories from individual scientists are mostly not sufficient to satisfy these criteria, especially since pediatric tumors are rare. In 2000 three centralized tumor repositories (neuroblastoma in Cologne, nephroblastoma in Würzburg, hepatoblastoma, brain tumors in Bonn) have been established by the "German Competence Net Pediatric Oncology und Hematology". The aim was to collect biomaterial including tumor samples, normal tissue, and blood in high quality for research and diagnostic purposes at a central institution. Informed consent of the parents or patients is a prerequisite for scientific use of the samples and is requested by the therapy trial. The samples are collected according to accepted standards and shipped in the specially designed Tumorbox. The tumor repository organizes the distribution of the samples to the cooperating diagnostic laboratories. The number of collected tumor samples has increased over the years. In 2000, samples from 200 patients were collected while the patient number increased to 321 in 2005. Over the years the tumor repositories collected more than 7,150 samples (fresh frozen tumor, fresh frozen normal tissue, and blood). Through links with clinical trial databases the samples can be connected with clinical data. 12 of 14 applications for tumor material to be used in specific scientific projects have been approved by an independent supervisory board. The establishment of central tumor repositories represents a major step for biomedical research activities and quality control in pediatric oncology.

The sonic hedgehog (Shh) and the Wnt signalling pathways are involved in the development of medulloblastomas (MBs), the most frequent malignant brain tumours in children. Components of these two developmental and cancer-associated pathways, including (Patched) PTCH, SMOH, adenomatous polyposis coli (APC), beta-catenin and AXIN1 show somatic mutations in sporadic MBs. In this study we analysed SUFU (human Suppressor of Fused), which acts as a negative regulator of both the Shh and Wnt signalling pathways and therefore represents a putative tumour suppressor gene, to find out if it is also involved in the pathogenesis of sporadic MBs. We screened 145 primitive neuroectodermal tumours (PNETs) including 90 classic MBs, 42 of the desmoplastic variant and two medullomyoblastomas as well as 11 MB cell lines for mutations using single-strand conformational polymorphism (SSCP) and sequencing analysis. 18% of the MBs exhibited allelic losses on chromosome 10q. In contrast to a previous report, in which truncating mutations of SUFU have been identified in 9% of MBs, we were not able to identify somatic mutations of SUFU in our large tumour panel. We uncovered single nucleotide polymorphisms (SNPs) in exon 4, 8, 11 and in intron 2 in the SUFU gene. Expression analysis by competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed no difference in SUFU mRNA levels of both MB subtypes and normal foetal or adult cerebellar tissues. Our results indicate that genetic alterations of the SUFU gene, do not contribute significantly to the molecular pathogenesis of MBs.

Medulloblastoma (MB) represents the most frequent malignant brain tumor in children. Most MBs appear sporadically; however, their incidence is highly elevated in two inherited tumor predisposition syndromes, Gorlin's and Turcot's syndrome. The genetic defects responsible for these diseases have been identified. Whereas Gorlin's syndrome patients carry germ-line mutations in the patched (PTCH) gene, Turcot's syndrome patients with MBs carry germ-line mutations of the adenomatous polyposis coli (APC) gene. The APC gene product is a component of a multiprotein complex controlling beta-catenin degradation. In this complex, Axin plays a major role as scaffold protein. Whereas APC mutations are rare in sporadic MBs, a hot-spot region of beta-catenin (CTNNB1) mutations was identified in a subset of MBs. To find out if Axin is also involved in the pathogenesis of sporadic MBs, we analyzed 86 MBs and 11 MB cell lines for mutations in the AXIN1 gene. Using single-strand conformation polymorphism analysis, screening for large deletions by reverse transcription-PCR, and sequencing analysis, a single somatic point mutation in exon 1 (Pro255Ser) and seven large deletions (12%) of AXIN1 were detected. This indicates that AXIN1 may function as a tumor suppressor gene in MBs.

In the multicenter trial AML-BFM 93 daunorubicin or idarubicin was randomly applied in all patients during induction in combination with cytarabine and etoposide. After induction all patients were stratified to the standard or high risk group. To improve outcome in high risk patients high dose cytarabine and mitoxantrone (HAM) was introduced. The placing of HAM as either the 2nd or 3rd therapy block was randomized to evaluate the efficacy and toxicity accordingly.471 children with de novo AML entered the trial AML-BFM 93 (161 standard risk, 310 high risk).Overall, 387 of 471 (82 %) patients achieved remission, 5-year survival, event free survival (EFS), and disease free survival were 60 % SE 3 %, 51 % SE 2 % and 62 % SE 3 %, respectively. Idarubicin-based induction resulted in a significantly better blast cell reduction in the bone marrow on day 15 (25 of 144=17 % patients with5 % blasts compared to 46 of 149=31 % patients after daunorubicin, pchi(2)=0.01). This was, however, mainly seen in high risk patients treated with idarubicin (19 % vs. 38 %, pchi(2)=0.007). Cardiotoxicity, WHO grade 1 - 3 shortening fraction reduction after induction occurred in 6 % patients in both arms. In the total group of patients probabilities of five years event-free survival and disease-free survival were similar for patients treated with daunorubicin or idarubicin. However, in patients presenting with more than 5 % blasts on day 15 there was a trend for a better outcome after treatment with idarubicin (p logrank 0.06). Outcome in high risk patients was superior in study 93 compared to study 87 (remission rate and 5-year pEFS in study AML-BFM 93 vs. study 87: 78 % vs. 68 %, p=0.007, and 44 % vs. 31 %, p logrank=0.01). The placing of HAM as the 2nd or 3rd therapy block was of minor importance. However, patients who received the daunorubicin treatment during induction benefited from early HAM.Compared to study AML-BFM 87 treatment results in study AML 93 improved significantly in high risk patients. This can partly be contributed to the better response on day 15 after idarubicin induction but is mainly due to the introduction of HAM.

To evaluate the impact of megatherapy on the outcome of high risk neuroblastoma, 39 patients with stage IV neuroblastoma, who underwent megatherapy, were analyzed retrospectively and compared to 49 patients, who continued chemotherapy according to the German cooperative trial NB85. The groups were comparable concerning age, primary localisation, pattern of metastases, clinical risk groups and response to induction therapy. Overall survival of the megatherapy group was 0.23 +/- 0.07 compared to 0.14 +/- 0.05 of the chemotherapy group (p = 0.0149). In particular patients with partial response to induction chemotherapy appeared to gain from megatherapy, while patients in complete remission did not benefit. Subset analysis confirmed the superiority of megatherapy in patients with bone marrow infiltration at diagnosis, in patients with or without bone metastases at diagnosis and in patients older than 2 years. On the background of the poor prognosis of stage 4 neuroblastoma and lack of other effective treatment modalities these data support the utilization of megatherapy in treatment strategies for stage IV neuroblastoma.

Inactivating mutations in the PTCH gene, a human homologue of the Drosophila segment polarity gene patched, have been identified recently in patients with nevoid basal cell carcinoma syndrome. These patients are predisposed to various neoplasias including basal cell carcinomas and medulloblastomas (MBs). To determine the involvement of PTCH in sporadic MBs, which represent the most frequent malignant brain tumors in children, we screened for PTCH alterations in an unselected panel of 64 biopsy samples from 62 patients and four continuous MB cell lines, all derived from patients with sporadic MBs. Using single-strand conformational polymorphism analysis, we screened exons 2-22 and detected nonconservative PTCH mutations in 3 of 11 samples from sporadic cases of the desmoplastic variant of MB but none in 57 MBs with classical (nondesmoplastic) histology. In two of the tumors with mutations and in two additional desmoplastic cases, loss of heterozygosity was found at 9q22. These findings suggest that PTCH represents a tumor suppressor gene involved in the development of the desmoplastic variant of MB.

The prognostic significance of the immunohistochemically assessed growth fraction in neuroblastomas was determined in relation to tumor grade and tumor stage. A total of 101 cases of neuroblastoma were examined with the monoclonal antibodies PC10 against proliferating cell nuclear antigen (PCNA) and Ki-S5 against the Ki-67 protein. Patients were followed for a mean time of 4.8 years. Expression of both PC10 and Ki-S5 was found to be significantly linked to tumor grade and tumor stage. Prognostically favorable stage IVs was associated with low PCNA and Ki-S5 levels. For ganglioneuroblastoma, significant differences were found between the diffuse and the composite type. In univariate analysis of stage III and IV tumors, Ki-S5 and PCNA scores were significantly correlated with disease-free survival (P < 0.0015), allowing definition of a subset of cases with favorable outcome. As to Shimada's group with poor prognosis, significant differences in the clinical course were found for low and high Ki-S5 scores (P = 0.036) but not for PCNA. In multivariate analysis, only patient age, Shimada's grade, and Ki-S5 scores achieved prognostic significance. We conclude that proliferation marker Ki-S5 may provide substantial prognostic information and might become a useful adjunct for predicting the clinical courses of neuroblastoma.

Current therapy strategies for localized neuroblastomas are mainly based on surgical procedures. In our analysis of 657 patients with localized neuroblastomas (mean follow-up: 6.2 years), the extent of surgical removal predicted outcome (complete resection: 0.91 +/- 0.02; partial resection: 0.79 +/- 0.04; biopsy 0.49 +/- 0.11). But for stage 2/II (0.96 +/- 0.02 vs. 0.91 +/- 0.06) and stage 3/III tumors (0.82 +/- 0.04 vs. 0.78 +/- 0.04) no difference in survival was seen between complete and partial resection of the primary tumor. Outcome was worse in stage 3/III patients who underwent biopsy only (0.48 +/- 0.11). Complications were seen after 20% of 853 surgical procedures, sometimes requiring second operations (n = 29) or leading to death (n = 11). Infants were especially at risk from complications: eight infants died of complications. Looking at these results, extended surgical procedures should be avoided to minimize the risk for the patient.

Prognosis of neuroblastoma is primarily dependent on the stage of disease. While stages 1 to 3 show a survival rate of 94 to 66%, stage 4 has a survival rate below 20%. The most important prognostic factor is the extent of disease. Age, LDH, resectability of primary tumor, cytologic and molecular parameters are defined to have a prognostic impact as well. A stage- and risk-adapted therapy of neuroblastoma needs a thorough assessment of all these factors.

182 untreated neuroblastomas were examined for the expression of the adhesion molecule CD44s by immunohistochemistry; all tumors were also tested for amplification of the oncogene N-myc by conventional Southern blot analyses. Positive CD44s immunoreactivity correlated not with a more favorable prognosis in contrast to an absence of CD44s expression in stage dependent or independent evaluations. All patients with stage 4S disease (n = 16) expressed CD44s on a high level and had an event-free survival probability of 82%. In undifferentiated neuroblastoma subgroups could be defined, depending on the expression of CD44s (p0.01). In 93% of N-myc non-amplified tumors CD44s expression was detected, whereas only 62% of N-myc amplified tumors were positive for CD44s expression. These results point to CD44s expression as a new histological marker in neuroblastoma, having prognostic impact in tumors of undifferentiated morphology.

A multitude of risk factors has been described for patients with neuroblastoma. Little is known about the mutual interrelationship of these factors and their impact on patients with localized disease only.We investigated the possible influence of 37 variables univariately on event-free survival (EFS) in 308 consecutive patients with neuroblastoma stages I-III using Kaplan-Meier estimates. The chi 2 test was applied to detect nonrandom correlations, and the Cox's regression model was used for the multivate evaluation of identified factors.Seventeen factors appeared to influence EFS in stage I-III patients (p0.05, log-rank3.84), whereas 10 factors were found in the subgroup of stage III patients with midline crossing tumors (= stage III*, n = 128). The majority of univariately identified risk factors showed a nonrandom correlation to several others (p0.05). The multivariate analysis according to Cox selected for the patients with stages I-III the factors lactate dehydrogenase (LDH) (p = 0.0011), resectability (p = 0.0167), weight loss (p = 0.0185), tumor extension beyond midline (p = 0.0207), and age (p = 0.0233). For stage III* patients the model identified the factors LDH (p = 0.0089), weight loss (p = 0.0135), resectability (p = 0.0408), and age (p = 0.0700). The identification of these independent risk factors permitted the description of risk groups with EFS ratios after6 years between 22% and 96%.Risk estimation of high discriminating power is possible for patients with localized neuroblastoma using simple, readily available clinical data.

Despite an increasing interest in the clinical application of positron emission tomography (PET) in tumors of the adult patient as a diagnostic and prognostic tool, only a few studies have been concerned with the usefulness of PET with [18F]2-deoxy-2-fluoro-D-glucose (FDG) in childhood tumors.Fifteen children and young adults (0.5-26.0 years of age) with histologically confirmed brain tumors were studied with PET and FDG. Seven children with medulloblastoma (n = 5) or primitive neuroectodermal tumor (PNET) (n = 2) underwent repeated PET studies during their therapy. The other eight children with medulloblastoma (n = 5) or astrocytoma (n = 3) were studied only once before initiation of treatment. A close clinical follow-up was performed in every case.Comparison of local glucose metabolic rates obtained in the various tumor lesions revealed that the mean rates found in medulloblastomas (mean glucose metabolic rate, 42.8 +/- 14.03 mumol/100 g/min) were twice as high as the rates measured in either PNET (17.3 +/- 4.5 mumol/100g/min) or infratentorial gliomas (21.8 +/- 4.2 mumol/100g/min). The high metabolism of medulloblastomas enabled the observer to identify the tumor more easily and to clearly separate it from the surrounding unaffected brain tissue. In the seven patients with follow-up PET studies during therapy, decreasing or increasing ratios of tumor-to-white matter metabolic rate were not only commensurate with neuroradiologically defined tumor reduction or growth, but also corresponded to the duration of initial clinical improvement.These preliminary results suggest that PET with FDG may be a useful tool to evaluate metabolic activity of pediatric brain tumors over time and to assess response to treatment.

From January 1, 1982 to November 1, 1992 24 patients suffering from neuroblastoma were operated in the course of a neuroblastoma study at the Department of Surgery, Cologne University. 23 patients were in advanced stages 3, 4 and 4s. In 13 cases the initial operation was prior to applying chemotherapy, whereas in 10 cases chemotherapeutical treatment had taken place before. As a result of preoperative chemotherapy operation time and blood loss could be reduced, respectively, and despite delayed operative treatment a better demarcation of the tumor and an easier preparation were seen. Due to the preoperative treatment, tumors could be operated on more radically without increasing the rate of complications. Utilizing all possible surgical means, secondary and tertiary operative procedures and chemotherapeutical treatment. all patients in stage 2, 3 and 4s survived with safe remission. Nine of the fifteen patients in stage 4 died due to side effects from bone marrow transplantation or from recurrent neuroblastomas and not because of the surgical procedures. In the light of existing current multicenter neuroblastoma studies stage oriented surgical and chemotherapeutical concepts were developed, that are presented in this paper.

Children with malignant disease have an increased risk for bacterial infections. We investigated a possible correlation between septic episodes and decreased IgG subclass levels in 63 patients. At diagnosis 13 of 50 children showed decreased IgG subclass levels: 10x IgG4, 2x IgG1, and 1x IgG3 + IgG4 were reduced. Bone marrow infiltration by tumor cells did not increase the frequency of subclass reductions (4/25 with, 9/25 without bone marrow infiltration). The time course of subclass levels was followed during 37 febrile episodes (mainly fever of unknown origin, septicemia, pneumonia) of 23 children under cytostatic therapy. 6 patients showed transient low IgG subclasses: 2x IgG4, 1x IgG1, 1x IgG3, 1x IgG2 + IgG4, and 1x IgG1 + IgG3 + IgG4. Children with decreased IgG subclass levels appeared to occur more independently of leucopenia. In general, febrile episodes in children with subclass decreases did not last a longer period and did not occur more frequently than in children without IgG subclass deficiencies. In conclusion, the determination of IgG subclasses in cancer children at diagnosis or during chemotherapy did not add substantial information of prognostic or therapeutic relevance.

The proof for the general evolution of metastatic neuroblastoma from localized disease by a low-speed process is lacking. The epidemiological approach would require the decrease of neuroblastoma related mortality by reduction of the number of metastatic cases per population unit as a result of a screening program. During 1983 through 1990 the neuroblastoma incidence in the FRG was 0.85-1.09 cases per 100,000 children less than 15 years of age. The mortality rate ranged between 0.25 and 0.49 deaths per 100,000 children. The project "Niedersachsen/Nordrhein-Westfalen" started April 1st, 1992 and is investigating urinary catecholamine metabolite excretion in infants at the age of 10-12 months and for a second time in toddlers at 17-19 months. The target population includes 24.7 million inhabitants with 267,000 births per year. The urine sampled on filter paper is analyzed using thin layer chromatography. Positive and questionable positive results are confirmed by HPLC and GC-MS. The aim of the study is to demonstrate the feasibility of the chosen approach. Furthermore first epidemiological data will be obtained. Until a population-based study on neuroblastoma mortality in screened versus controlled populations has proven the usefulness of the screening it cannot be recommended as a general service to the public.

In patients undergoing high-dose ifosfamide treatment tubular nephrotoxicity with hypophosphatemia has been described. Long-lasting hypophosphatemia may be associated with bone disease including rickets. Serum osteocalcin is considered a sensitive marker for reduced osteoblast activity and bone formation. In our study we determined osteocalcin serum levels in 11 children with cancer and chemotherapy, comparing them with fractional reabsorption of phosphate, aminoaciduria and a1-microglobulin excretion. A decrease of serum levels of osteocalcin and serum phosphate during chemotherapy was found. Progressive hyperaminoaciduria, a1-microglobulin loss and a low fractional reabsorption of phosphate were also detected during chemotherapy. All parameters tended to become normal after treatment. We conclude that serum osteocalcin levels may decrease soon after the initiation of chemotherapy, indicating low osteoblast activity, probably as a result of phosphate loss. Patients with decreased osteocalcin levels may therefore be at risk for osteoporosis.

Serum neuron specific enolase (NSE) was determined in 159 patients with neuroblastoma at diagnosis and in 183 children of various age groups. We found an age dependence of reference intervals for NSE and defined the 95th percentiles as upper normal limits. The specificity was 91.3% and the sensitivity 73.0%. The incidence of abnormal NSE levels increased with stage. The NSE serum levels were not influenced by histologic differentiation. Neuron specific enolase proved to be a reliable tumor-marker for monitoring the disease. Moreover, abnormal NSE values at diagnosis were of prognostic significance for patients with localized neuroblastoma (stages I-III) and for children with metastatic disease (stage IV), but not for infants with stage IV S. In comparison to catecholamine metabolite determination neuron specific enolase appeared to be a slightly less specific, equally sensitive tumor marker but with prognostic information for children with neuroblastoma.

Genomic configuration and expression of the N-myc gene was investigated by Southern and Northern blot analyses in 18 neuroblastomas of different clinical stages. We observed a 4-100-fold amplification of this oncogene in one of six stage III, two of four stage IV as well as one of five stage IVS tumours. Remarkably, an 80-fold N-myc amplification was demonstrated in a patient with stage IV neuroblastoma being in remission for more than 2 years; moreover, a 100-fold amplification could be detected in a stage IVS tumour from a newborn. These data are discussed in view of the recently postulated close association of N-myc amplification with rapid progression of neuroblastomas.

In therapy study ALL-BFM 83 a total of 630 patients with acute lymphoblastic leukemia (ALL) have prospectively been evaluated for initial response on therapy with corticosteroids. It was the aim to qualify the in vivo cytoreduction as a new predictor for therapy failure. All patients were exposed for 7 days to prednisone before combination chemotherapy at day 8 has been started. At day 0 one additional dose of Methotrexate was given intrathecally. Therapy for all patients with non-B-ALL has been stratified according to initial tumor burden (risk factor) providing four therapy branches: standard risk low (SR-L), standard risk high (SR-H), medium risk (MR), high risk (HR). After a median duration of study of 21 months, event-free survival (EFS) is for all 630 patients 73%, 81% for SR-L, 76% for SR-H, 69% for MR, and 35% for HR patients (date of evaluation Jan. 1, 1987). In this prospective study, a small subgroup of patients (n = 48; 7.6% of total group) is characterized by greater than 1000 leukemic blasts/mm3 peripheral blood at day 8 after exposure to prednisone. In this subgroup the EFS is only 43% in contrast to 76% in the complementary group of 582 patients with less than 1000 leukemic blasts/mm3 peripheral blood at day 8. Patients of that risk group are derived from therapy branches SR-H, M and HR, the latter contributing relatively most patients. In this negatively selected group all patients with an initial high white blood count, CNS disease at diagnosis, immune subtypes as prae-T-ALL (n = 6), T-ALL (n = 18), null-ALL (n = 5), and males clearly dominate. Of 48 patients with greater than 1000 blasts/mm3 at day 8 4 subsequently failed to enter remission and 8 were qualified as lateresponders. 18 patients relapsed, most of them earlier compared to those of the complementary group. The initial in vivo response on corticosteroid therapy is considered a supplementary prognostic predictor for early failure. It will be utilized in trial ALL/NHL-BFM 86 to qualify patients at the highest risk for relapse. This group of patients is supplemented in addition by non- and lateresponders and children with acute undifferentiated leukemia (AUL). The in vivo corticosteroid test is simple, generates early and reliable results and can be obtained almost always. Thus it may be recommended for use in a multicenter trial.

Within 1 year 74 children with neuroblastoma were registered, 30 patients with stage I-III (= 41%) and 44 with stage IV-metastatic disease (= 59%). An aggressive chemotherapy regimen employing Adriamycine, Cyclophosphamide, Vincristine, and Dacarbazine yielded 10/24 partial and 9/24 complete remissions after 9 weeks. 5/24 children were treated less than 9 weeks so far. At the end of the chemotherapy protocol (week 33) 6 recurrences were observed; 3 of these children died. 5 patients remained in complete remission, 1 in partial remission. 12/24 of patients were not evaluable because of treatment less than 33 weeks so far. The one year run of the study is too short to evaluate the benefit of Interferon (randomized trial). The toxicity of the regimen is tolerable, including bone marrow depression, vomiting and hyperpyrexia. Breaking off therapy was only necessary in one patient.

The selective uptake and accumulation of 131J-Metaiodobenzylguanidine in neuroblastoma cells in vivo may be utilized for targeted irradiation. The experience with 32 neuroblastoma patients refractory to conventional high dose chemotherapy is reported. At diagnosis 8 patients had Evans stage III and 22 stage IV. 11/32 experienced recurrences after complete tumor disappearance and before mIBG treatment, 16/32 progressed from residual or nonresponding tumor and in 3/32 insufficient tumor regression by chemotherapy was observed. 2 children received one mIBG course each with no evidence of disease. Mean applied activity was 128 mCi per course (35-300 mCi), 360 mCi per patient (80-1033 mCi) and 19.2 mCi/kg per patient (3.2-37.9 mCi/kg), respectively. A total of 84 courses was given (mean 2.6 per patient). Pain relief was noticed in 14/14 patients with bone pain. Complete or very good partial remission was achieved in 5/32, partial remission in 11/32 and stable disease in 6/32 patients. In 8 children progression occurred and 2 patients were not evoluable. 20 children died, 12 are still alive (6 patients with initial stage IV, 6 with stage III disease). Main side effect was transient thrombocytopenia, which became more severe with increasing number of courses. We conclude that mIBG treatment is effective in some patients with refractory neuroblastoma and may be utilized in the future as front line therapy for patients achieving only incomplete regressions after high dose chemotherapy.

In the years 1980–1985 72 children with acute lymphoblastic leukemia were diagnosed and treated by intensive combination chemotherapy (BFM protocols 79, 81, 83). Of these children 33 acquired a Hepatitis B-virus-carrier state with 1983 as the peak year of incidence. Both groups of patients, the infected and the uninfected ones, were comparable as to prognostic factors. All except 8 patients are off chemotherapy after a total duration of treatment of 1 1/2 or 2 years. Probability for event-free survival (life table analysis, maximum observation time 82 months, minimum 12 months) is equal (0.77 vs. 0.75) in both groups. With 3 exceptions, all HBV-infected patients still carry the HBs-antigen in the serum; 22 of the 30 living patients in the infected group developed anti-HBc.

An intensive 7-day combination chemotherapy protocol was designed to reinduce children with early bone marrow relapse of acute lymphoblastic leukemia (less than 6 months after the end of or during preceding treatment). This aggressive approach seemed to be justified for a group of patients who were at the highest risk for ultimate treatment failure. In all, 38 children were enrolled for study. The ratio of male (median age, 10 years) to female (median age, 13 years) subjects was 27:11. Thirty patients were treated for their first relapse and eight for their second or subsequent relapse. Isolated bone marrow involvement was present in 24 cases. All patients had received heavy pretreatment including anthracyclines with cumulative doses of between 120 and 240 mg/m2. 22 of these patients, achieved complete remission, ten did not respond to therapy, and six died from the toxicity of the protocol. Cardiac failure was the cause of death in one child (after additional radiotherapy for a mediastinal mass). No further clinical manifestation of cardiomyopathy could be observed. The other five patients died from hemorrhages or infectious complications. The main side effects were fever, gastrointestinal problems, stomatitis, and severe bone marrow aplasia lasting for about 2 weeks with nadirs of platelets and white blood count around days 10-14. The remission rate of 60% was acceptable, though not satisfactory. Only four children survived disease-free for 13+, 14+, 20+, and 22+ months after diagnosis of relapse.

Two children with acute myeloid leukemia (FAB M1 and M2) experienced bone marrow relapse during maintenance chemotherapy 7 and 10 months after diagnosis. Low-dose ARA-C monotherapy (2 X 10 mg/m2 per day s.c. for 14 days) was then initiated, as suggested by others reporting induction of differentiation and achievement of remission without toxic side effects. In contrast to these reports, remission induction was not observed in the two children after low-dose ARA-C but was achieved by subsequent high-dose chemotherapy. However, blast cell characteristics revealed some alterations. Blast count and chromosome pattern remained unchanged. Cytochemistry revealed the appearance of esterase- (0----11%), 0----21%) and PAS- (0----74%, 0----45%) positive cells in the patients and a remarkable increase (patient 1: 0----71%) and decrease (patient 2: 90----12%) in acid phosphatase positivity. Expression of myeloid marker VIM D5 decreased distinctly (70----4%, 77----11%). However, the biologic relevance of these alterations remains in question. The failure to respond clinically to low-dose ARA-C in both children is discouraging.

Five children treated for acute myeloid leukemia according to the BFM protocol AML 83 experienced first bone marrow relapse after 7, 10, 14, 18, and 30 months and were retreated for second remission induction. The chemotherapy consisted of mAMSA (100 mg/m2 per day i.v., days 1-3), ARA-C (100 mg/m2, twice daily, days 1-6), and VP 16 (150 mg/m2 per day, days 4-6). Four of the children achieved a complete second remission after one course of chemotherapy, and the fifth child died of pneumonia during bone marrow aplasia. All surviving children received an identical second course within 4-5 weeks, followed by maintenance chemotherapy. Remission duration was 0, 3, 4, 5, and 5 months. Toxicity was confined to heavy bone marrow depression with thrombocytopenia (nadir 2-7000, days 7-13) and leukocytopenia (nadir 0-400, days 8-14). Bleeding episodes could be prevented by substitution with platelets. Four patients experienced infections (pneumonia, septicemia). We conclude that combination chemotherapy using mAMSA, ARA-C, and VP 16 is effective in inducing a second remission in patients with early bone marrow relapse. The main side effect was considerable bone marrow toxicity.

During induction therapy of acute lymphoblastic leukemia a 10 year old boy developed a hyperuremic nephropathy and subsequently a staphylococcal septicemia at the beginning of the 3. week. Specific treatment was started leading to severe hypomagnesemia and generalized seizures with coma for 30 hours, which finally responded to magnesium replacement. The possible additive effect of nephropathy, gentamicin, and furosemide due to urinary loss of magnesium is discussed and should encourage further observations.

22 children with metastatic neuroblastoma received myeloablative chemoradiotherapy followed by bone marrow transplantation (BMT). The duration of preceding chemotherapy was 4-30 months and included treatment of recurrences in 10 children. At BMT 12 patients were in CR, 9 in PR and one had tumor progression. 10/15 of autologous bone marrows were purged using immunomagnetic bead method of Kemshead and 2/15 using 4 hydroperoxycyclophosphamide. Myeloablative therapy consisted of melphalan and total body irradiation (TBI) in 13 patients (three each supplemented by vincristine or adriamycin/etoposide), in one child of melphalan and mIBG and in 3 children of melphalan alone. 3 children received double autograft and 2 cyclophosphamide (and TBI). 10 patients survived 0-32 months from BMT and 5-48 months from diagnosis, respectively. 12 patients died including 7/12 of tumor progression and 5/12 of toxicity (venoocclusive disease, gut toxicity, septicemia, pneumonia). We conclude that at this point BMT after conventional high dose chemotherapy may provide the only real chance of survival for a significant number of children with metastatic neuroblastoma.

Case reports are presented of two related patients suffering from a hereditary megaloblastoid hemolytic anemia which at the moment cannot be categorized into one of the well-known entities. The main characteristics of the disease consisted of constant jaundice, macrocytic normochromic anemia, marked hemolysis without a substantial decrease in osmotic resistance, increased iron turnover and hepatic hemosiderosis at a relatively young age. One patient had to undergo splenectomy due to hemolytic crises, the other one cholecystectomy due to gallstones. In contrast to their uncharacteristic morphology in smear, red cells displayed highly variable forms ("lumpy", "Y", "U", drumstick forms) when examined in transmission and scanning electron microscopes. These changes corresponded well with reduced filtrability and aggregability of erythrocytes. The apparent relative blood viscosity was unchanged. The protein pattern of ghosts in SDS gel-electrophoresis revealed neither defects nor additional bands. Changes in the lipid composition of the membrane were indirectly deduced from electron spin-resonance studies, which showed an additional signal at g = 2.192. Similarly, the lipid related membrane mobility agent A2C failed to exert the usual stabilizing effect against osmotic stress. The negative surface potential, estimated by free flow electrophoresis, was only altered in the splenectomized patient. It is concluded that the primary abnormal physical properties of the enlarged red cell contribute at least in part to the marked hemolysis. The similar findings in the two related patients and the fact that the disorder was obviously congenital suggest a special subtype of a megaloblastoid hemolytic anemia.