Off-label drug use in paediatric haemato-oncology patients: financial implications and proposed solutions for Belgian patients.

Treatment of children with cancer requires access to and reimbursement of effective drugs. Children with haemato-oncological diseases are often treated according to established treatment recommendations or in the framework of late-phase clinical trials. These often involve the use of drugs authorised for adults but which, however, have been used for many years in paediatrics with no perspective of authorisation in children. In Belgium, medicines are predominantly reimbursed based on their authorised indication. As a consequence, many drugs used in paediatric haemato-oncology are used off-label, despite their status of 'standard of care'. As reimbursement is often not available, alternative ways for funding need to be explored, which causes a significant administrative burden for healthcare providers and emotional distress for the parents. Solutions to organise a systematic reimbursement of standard of care off-label used drugs are described.Conclusion: A number of structural solutions are proposed, and we hope that they might guide health authorities to provide a solution to the problem caused by the lack of reimbursement of some standard of care medicines for children with cancer. What is Known: • Off-label drug use is frequently observed in paediatric haemato-oncology and compromises-in some countries-reimbursement. What is New: • An estimation of the impact of non-reimbursed drugs in Belgium is provided. • Some solutions are presented to overcome this problem in Belgium.

Drug shortages in a pediatric stem cell transplantation ward: Challenges and implications. A 5-year bilan.

This article describes the implications of shortages of pharmaceutical products used in conditioning and supportive care regimens of pediatric patients undergoing a hematopoietic stem cell transplantation in a tertiary care hospital. Between July 2011 and July 2016, a total of 84 individual shortages, affecting 22 different drugs (79.8% supportive care drugs; 20.2% chemotherapeutics) were detected with a mean duration of 85 days (SD 138) per individual drug shortage. Eighteen shortages were critical and very urgent. Sulfamethoxazol/trimethoprim, piperacillin/tazobactam, ranitidine, benzylpenicillin, ondansetron (supportive care) and methotrexate, melphalan (chemotherapeutics) had the longest supply disruptions. A variety of solutions could be identified including the purchase of a generic alternative (36.9%) for both oral and parenteral treatments (in a ratio 3:2). Urgent import from another (European) country was performed in 14 cases (16.7%). High impact solutions such as cohorting of patients and change of ongoing treatments (2.4%) were used for parenteral treatments only. Pharmaceutical modification was sometimes applied for oral treatments (2.4%). Due to persistent occurrence of these shortages, an efficient pharmacy workflow (electronic follow-up by end of 2016) and a multidisciplinary approach were needed.

ß2-adrenergic receptors protect axons during energetic stress but do not influence basal glio-axonal lactate shuttling in mouse white matter.

In vitro studies have demonstrated that ß2-adrenergic receptor activation stimulates glycogen degradation in astrocytes, generating lactate as a potential energy source for neurons. Using in vivo microdialysis in mouse cerebellar white matter we demonstrate continuous axonal lactate uptake and glial-axonal metabolic coupling of glutamate/lactate exchange. However, this physiological lactate production was not influenced by activation (clenbuterol) or blocking (ICI 118551) of ß2-adrenergic receptors. In two-photon imaging experiments on ex vivo mouse corpus callosum subjected to aglycemia, ß2-adrenergic activation rescued axons, whereas inhibition of axonal lactate uptake by α-cyano-4-hydroxycinnamic acid (4-CIN) was associated with severe axonal loss. Our results suggest that axonal protective effects of glial ß2-adrenergic receptor activation are not mediated by enhanced lactate production.

An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours.

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.

MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors.

Increased activity of MYC protein-family members is a common feature in many cancers. Using neuroblastoma as a tumor model, we established a microRNA (miRNA) signature for activated MYCN/c-MYC signaling in two independent primary neuroblastoma tumor cohorts and provide evidence that c-MYC and MYCN have overlapping functions. On the basis of an integrated approach including miRNA and messenger RNA (mRNA) gene expression data we show that miRNA activation contributes to widespread mRNA repression, both in c-MYC- and MYCN-activated tumors. c-MYC/MYCN-induced miRNA activation was shown to be dependent on c-MYC/MYCN promoter binding as evidenced by chromatin immunoprecipitation. Finally, we show that pathways, repressed through c-MYC/MYCN miRNA activation, are highly correlated to tumor aggressiveness and are conserved across different tumor entities suggesting that c-MYC/MYCN activate a core set of miRNAs for cooperative repression of common transcriptional programs related to disease aggressiveness. Our results uncover a widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation and further substantiate the overlapping functions of c-MYC and MYCN in the process of tumorigenesis.

Treatment of localised resectable neuroblastoma. Results of the LNESG1 study by the SIOP Europe Neuroblastoma Group.

Main objective of this study was to confirm that surgery alone is an effective and safe treatment for localised resectable neuroblastoma except stage 2 with amplified MYCN gene (MYCNA). Of 427 eligible stages 1-2 patients, 411 had normal MYCN and 16 had MYCNA. Of the 288 stage 1 patients with normal MYCN, 1 died of complications and 16 relapsed, 2 of whom died; 5-year relapse-free survival (RFS) and overall survival (OS) rates were 94.3% (95% confidence interval (CI): 91.6-97) and 98.9% (95% CI: 97.7-100), respectively. Of the 123 stage 2 patients with normal MYCN, 1 died of sepsis and 22 relapsed, 8 of whom died (RFS 82.8%, 95% CI: 76.2-89.5; OS 93.2%, 95% CI: 88.7-97.8). In stage 2, OS and RFS were worse for patients with elevated LDH and unfavourable histopathology. Of 16 children with MYCNA, 7 were stage 1 (5 relapses and 4 deaths) and 9 were stage 2 (3 relapses and 2 deaths) patients. In conclusion, surgery alone yielded excellent OS for both stage 1 and 2 neuroblastoma without MYCNA, although stage 2 patients with unfavourable histopathology and elevated LDH suffered a high number of relapses. Both stage 1 and 2 patients with MYCNA were at greater risk of relapse.

Surveillance of cytomegalovirus (CMV) DNAemia in pediatric allogeneic stem cell transplantation: incidence and outcome of CMV infection and disease.

Cytomegalovirus (CMV) remains a serious problem after hematopoietic stem cell transplantation (HSCT). To investigate the incidence of CMV infection and outcome we retrospectively analyzed 70 consecutive pediatric allogeneic HSCTs monitored by CMV polymerase chain reaction (PCR), with at least 1-year follow-up or until death. All patients at risk for CMV infection (CMV-seropositive patients and CMV-seronegative recipients transplanted from CMV-seropositive donors) received hyperimmune anti-CMV globulins whereas in the group of HSCT patients with both donor and recipient CMV negativity, polyvalent immunoglobulins were given, both at a dose of 400 mg/kg. All patients received acyclovir at prophylactic doses for at least 6 months. Patients were monitored twice a week by CMV PCR. Patients with 2 positive results for CMV DNAemia received ganciclovir for 14 days and continued until 2 consecutive negative results were obtained. The incidence of CMV DNAemia was 12.8% (9/70) in the whole group, with significant higher risk for patients with CMV-seropositive recipient status, 8 out of 22 (36%), vs. patients with seronegative status, 1 out of 48 (2%) (P=0.0002). Three out of 9 patients with DNAemia developed CMV disease despite adequate preemptive treatment. The transplant-related mortality was higher in the CMV-seropositive recipient group (P=0.05). Age, use of hyperimmune anti-CMV globulins at a high dose, and the low incidence of graft-versus-host disease might be contributing factors to this low incidence.

A case of 'tropical' myelopathy.

We present a case of lower limb sensory disturbances and weakness in a patient originating from Mali. MRI showed a diffuse myelopathy of the cervical and thoracic spinal cord. Serological evaluation of blood and cerebrospinal fluid pointed towards schistosomiasis as the cause. Histological confirmation was made on bladder-biopsy. Treatment with praziquantel and steroids brought marked clinical improvement. This case illustrates the need to keep in mind more exotic causes of myelopathy in those patients coming from endemic regions.

Genome wide measurement of DNA copy number changes in neuroblastoma: dissecting amplicons and mapping losses, gains and breakpoints.

In the past few years high throughput methods for assessment of DNA copy number alterations have witnessed rapid progress. Both 'in house' developed BAC, cDNA, oligonucleotide and commercial arrays are now available and widely applied in the study of the human genome, particularly in the context of disease. Cancer cells are known to exhibit DNA losses, gains and amplifications affecting tumor suppressor genes and proto-oncogenes. Moreover, these patterns of genomic imbalances may be associated with particular tumor types or subtypes and may have prognostic value. Here we summarize recent array CGH findings in neuroblastoma, a pediatric tumor of the sympathetic nervous system. A total of 176 primary tumors and 53 cell lines have been analyzed on different platforms. Through these studies the genomic content and boundaries of deletions, gains and amplifications were characterized with unprecedented accuracy. Furthermore, in conjunction with cytogenetic findings, array CGH allows the mapping of breakpoints of unbalanced translocations at a very high resolution.

Molecular analysis of the putative tumour-suppressor gene EXTL1 in neuroblastoma patients and cell lines.

Although neuroblastoma is the most common extracranial solid tumour of childhood, little is known about its aetiology. Together with MYCN amplification and chromosome 17q gain, chromosome 1p deletion is one of the most frequently occurring genetic abnormalities in neuroblastoma. Based upon mapping of deletion breakpoints, putative tumour suppressor gene loci have been assigned to the distal part of the short arm of chromosome 1. Recently, the EXTL1 gene was suggested as a candidate neuroblastoma-suppressor gene and to evaluate this hypothesis, we performed 1p deletion analysis and mutation screening of the EXTL1-coding region on DNA from 22 primary neuroblastomas and 21 neuroblastoma cell lines. Deletions of the chromosome region 1p36.1, including the EXTL1 gene, were detected in several neuroblastoma cell lines and primary tumours. EXTL1 mutation screening resulted in the detection of one unclassified variant (Ser28Cys) but could not provide additional evidence of EXTL1 being involved in the aetiology of neuroblastoma.

[The neuroblastoma, "enfant terrible" among pediatric tumors]. / Het neuroblastoom, 'enfant terrible' onder de kindertumoren.

Neuroblastoma belongs to the group of small blue round cell tumors and originates in precursor cells of the sympathetic neural tissue. This tumor occurs at the pediatric age and has fascinated and intrigued both clinicians and researchers because of its variable and often unpredictable clinical behaviour. Indeed, the clinical outcome of neuroblastoma patients not only depends on the clinical extension of the disease, but also on other factors including age at diagnosis, presence or absence in the tumor cells of molecular and biological characteristics with prognostic value (e.g. amplification of the oncogene MYCN, frequently associated with chromosome 1p-deletion is predictive for poor survival chance). In 1983 an abdominal stage 3 neuroblastoma was diagnosed in a 9-months old boy. He died of the disease 3 years later. Karyotyping studies in this patient revealed a constitutional chromosome translocation t(1;17) with a breakpoint involving the terminal part of the chromosome 1p arm. We hypothesized that this patient was predisposed to the development of neuroblastoma because he carried in all his somatic cells a chromosomal abnormality involving the region frequently deleted in neuroblastoma tumor cells. We assumed that the chromosomal translocation breakpoints might indicate the regions harbouring genes involved in neuroblastoma development. A somatic cell fusion experiment was performed between the patient's fibroblasts (the only remaining source of patient material) and a fast growing Chinese hamster ovary cell line to assure the possibilities to perform further research. These somatic cell hybrids indeed contained the human translocation chromosomes. Further characterization of the translocation breakpoints by FISH (fluorescent in situ hybridisation) resulted in the identification of NPPA (formerly PND, the gene for pronatriodilatine) and A12M2 (an adenovirus integration site) as flanking markers for the 1p breakpoint. The 17q breakpoint was located between the NF1 (neurofibromatosis 1) gene and the SCYA7 (harboring the gene encoding the monocyte chemotactic protein-3). Starting from these markers chromosome walking experiments furthered the characterization of the chromosomal breakpoint regions and enabled to identify breakpoint overlapping cosmids. Sequence analysis of these markers is ongoing and will reveal if the breakpoint regions indeed harbour a gene involved in neuroblastoma development.

Combined M-FISH and CGH analysis allows comprehensive description of genetic alterations in neuroblastoma cell lines.

Cancer cell lines are essential gene discovery tools and have often served as models in genetic and functional studies of particular tumor types. One of the future challenges is comparison and interpretation of gene expression data with the available knowledge on the genomic abnormalities in these cell lines. In this context, accurate description of these genomic abnormalities is required. Here, we show that a combination of M-FISH with banding analysis, standard FISH, and CGH allowed a detailed description of the genetic alterations in 16 neuroblastoma cell lines. In total, 14 cryptic chromosome rearrangements were detected, including a balanced t(2;4)(p24.3;q34.3) translocation in cell line NBL-S, with the 2p24 breakpoint located at about 40 kb from MYCN. The chromosomal origin of 22 marker chromosomes and 41 cytogenetically undefined translocated segments was determined. Chromosome arm 2 short arm translocations were observed in six cell lines (38%) with and five (31%) without MYCN amplification, leading to partial chromosome arm 2p gain in all but one cell line and loss of material in the various partner chromosomes, including 1p and 11q. These 2p gains were often masked in the GGH profiles due to MYCN amplification. The commonly overrepresented region was chromosome segment 2pter-2p22, which contains the MYCN gene, and five out of eleven 2p breakpoints clustered to the interface of chromosome bands 2p16 and 2p21. In neuroblastoma cell line SJNB-12, with double minutes (dmins) but no MYCN amplification, the dmins were shown to be derived from 16q22-q23 sequences. The ATBF1 gene, an AT-binding transcription factor involved in normal neurogenesis and located at 16q22.2, was shown to be present in the amplicon. This is the first report describing the possible implication of ATBF1 in neuroblastoma cells. We conclude that a combined approach of M-FISH, cytogenetics, and CGH allowed a more complete and accurate description of the genetic alterations occurring in the investigated cell lines.

Multicentre analysis of patterns of DNA gains and losses in 204 neuroblastoma tumors: how many genetic subgroups are there?

PROCEDURE: Analysis of comparative genomic hybridization (CGH) data of 120 tumors from four different studies, and data of 84 previously unpublishied tumors, allowed delineation of at least six different genetic subsets of neuroblastomas. RESULTS AND CONCLUSIONS: A small number of tumors show no detectable imballances. A second group of tumors presents with gains and losses of whole chromosomes and is found predominantly in prognostically favorable stage 1 and 2 tumors. The remaining groups are characterized by the presence of partial chromosome imbalances, and are found mostly in stage 3, 4, and 4S tumors. The third group shows 17q gain without 11q loss, 1p loss, or MYCN amplification (MNA). The fourth group has 1p deletion or MNA, and finally, a fifth group shows 11q loss without 1p deletion or MNA, and is found mainly in stage 4 tumors. The latter group is significantly associated with losses of 3p, 4p, and 14q.

Comparative genomic hybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification.

We have studied the occurrence and association of 11q deletions with other chromosomal imbalances in Stage 4 neuroblastomas. To this purpose we have performed comparative genomic hybridization (CGH) analysis on 50 Stage 4 neuroblastomas and these data were analyzed together with those from 33 previously published cases. We observed a high incidence of 11q deletion in Stage 4 neuroblastoma without MYCN amplification (59%) whereas 11q loss was only observed in 15% of neuroblastomas with MYCN-amplification (p = 0.0002) or 11% of cases with 1p deletion detected by CGH (p = 0.0001). In addition, 11q loss showed significant positive correlation with 3p loss (p = 0.0002). Event-free survival was poor and not significantly different for patients with or without 11q deletion. Our study provides further evidence that Stage 4 neuroblastomas with 11q deletions represent a distinct genetic subgroup that typically shows no MYCN-amplification nor 1p deletion. Moreover, it shows that neuroblastomas with 11q deletion also often present 3p deletion. This genetic subgroup shows a similar poor prognosis as MYCN amplified 4 neuroblastomas.

Chromosome 2 short arm translocations revealed by M-FISH analysis of neuroblastoma cell lines.

PROCEDURE: M-FISH analysis was performed on 18 neuroblastoma cell lines, which were previously studied with cytogenetic, standard FISH and CGH data. RESULTS: One of the most striking findings of this study was the detection of chromosome 2 short arm rearrangements in 61% of the investigated cell lines. These rearrangements resulted from translocations with various partner chromosomes. All translocations, except one were unbalanced, leading to the consistent gain of chromosome segment 2pter-p22. A cryptic balanced translocation t(2;4) was observed with a breakpoint located in the vicinity of MYCN in cell line NBL-S. CONCLUSIONS: Combination of M-FISH results together with cytogenetic, standard FISH and CGH data yielded the most comprehensive description of chromosome 2 short arm rearrangements, leading to a consistent gain of chromosome 2 short arm material.

Long-term results of three randomized trials (58831, 58832, 58881) in childhood acute lymphoblastic leukemia: a CLCG-EORTC report. Children Leukemia Cooperative Group.

We present here the long-term results of three randomized clinical trials conducted on children with newly diagnosed acute lymphoblastic leukemia (ALL) between 1983 and 1998 by the Children Leukemia Cooperative Group (CLCG) from EORTC. In study 58831/32, the overall event-free survival (EFS) rates (+/- s.e.) at 6 and 10 years were 66% +/- 1.8% and 65% +/- 1.8%, respectively, and the risk of isolated central nervous system (CNS) relapse was 6% +/- 1% and 7% +/- 1%, respectively. In patients with a standard risk of relapse the omission of cyclophosphamide had no adverse effect on disease-free survival rates at 10 years (trial 58831). In medium- and high-risk patients the omission of radiotherapy did not increase the risk of CNS or systemic relapse (trial 58832). In study 58881 (1989-1998) the overall EFS rate at 8 years was 68.4% +/- 1.2% and the risk of isolated CNS relapse was 4.2%+/-0.5%. In this trial which adressed three randomized questions, the following results were obtained: the combination of cytarabine at high doses with methotrexate at high doses during interval therapy did not improve prognosis. The addition of 6-mercaptopurine iv during maintenance increased the risk of late relapse. E. coli asparaginase was more toxic and has a higher efficacy than Erwinia asparaginase. Leukocyte counts >100 x 10(9)/l, specific genetic abnormalities, a poor initial response to steroids or a high level of minimal residual disease at early time points were consistently associated with an adverse prognosis in the 58881 trial.

Discrepant flow cytometric expression and function of P-glycoprotein in neuroblastic tumors.

BACKGROUND: Patients suffering from neuroblastic tumors are currently being classified into prognostic subsets based on different clinical and biologic features. In this study, a triple-color flow cytometric assay and a functional test were applied to neuroblastoma cell lines and patients with a neuroblastic tumor, and the value of P-glycoprotein expression and function as potential prognostic characteristics, was determined. METHODS: Twenty-two single-cell suspensions prepared from tumors, and neuroblasts from four bone marrow samples were analyzed by triple-color flow cytometry. Neuroblasts were identified by NB84-positivity and absence of CD45. P-glycoprotein expression was evaluated using 4E3 and MRK16 antibodies. Eighteen samples were tested with a functional assay, based on accumulation and retention of rhodamine-123 with and without the inhibitor verapamil. Six neuroblastoma cell lines were also evaluated. RESULTS: P-glycoprotein expression was seen in 18 of 26 patient samples and in three of six cell lines. The highest expression levels were found in low stage neuroblastoma and well-differentiated tumors; whereas the highest activities were found in stage 4 neuroblastoma and the lowest in ganglioneuroblastoma and ganglioneuroma patients. In 10 of 17 samples, concordant results were found between the flow cytometric immunological test and immunocytochemistry. CONCLUSIONS: The described flow cytometric technique is a new, alternative approach to detect P-glycoprotein expression and function in neural crest tumors. Based on the expression level and the activity value, patients can be segregated into different phenotypic groups. In particular, those patients with high P-glycoprotein activity might benefit from treatment regimens containing reversal agents.

P-glycoprotein is an independent prognostic factor predicting relapse in childhood acute lymphoblastic leukaemia: results of a 6-year prospective study.

P-glycoprotein (P-gp), a cellular drug-efflux pump, is thought to be one of the major causes of multidrug resistance (MDR) in malignancies. Since therapeutic strategies are being developed to circumvent drug resistance by inhibiting P-gp function, large prospective studies evaluating the clinical relevance of P-gp in childhood acute lymphoblastic leukaemia (ALL) are warranted. P-gp expression was evaluated over a period of 6 years in 102 consecutive patients with de novo childhood ALL and in 35 children with relapse of ALL. Bone marrow and blood smears were studied immunocytochemically with two monoclonal antibodies at initial diagnosis and at relapse. P-gp expression was found in 14 (14%) patients at initial diagnosis. After induction treatment, complete remission was achieved in 100/102 patients (98%), of whom 19 relapsed. Cumulative event-free survival was significantly higher in the P-gp-negative group compared with the P-gp-positive population (Logrank P = 0.02). Multivariate analysis showed the results to be independent of age, WBC count and karyotype, and concomitantly underlined the importance of MDR1 phenotype detection in childhood ALL. P-gp expression was more frequently found at relapse (34%) than at primary diagnosis (P = 0.01). In the relapsed patient group, P-gp-positive patients had a 2-fold greater risk for adverse clinical outcome than the P-gp-negative relapsed patients. P-gp expression was not induced by exposure to previous chemotherapy since the majority of P-gp-negative patients remained negative at relapse. P-glycoprotein expression in newly diagnosed childhood ALL is an independent adverse prognostic parameter with a predictive value for relapse.

Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma.

BACKGROUND: Gain of genetic material from chromosome arm 17q (gain of segment 17q21-qter) is the most frequent cytogenetic abnormality of neuroblastoma cells. This gain has been associated with advanced disease, patients who are > or =1 year old, deletion of chromosome arm 1p, and amplification of the N-myc oncogene, all of which predict an adverse outcome. We investigated these associations and evaluated the prognostic importance of the status of chromosome 17. METHODS: We compiled molecular cytogenetic analyses of chromosome 17 in primary neuroblastomas in 313 patients at six European centers. Clinical and survival information were collected, along with data on 1p, N-myc, and ploidy. RESULTS: Unbalanced gain of segment 17q21-qter was found in 53.7 percent of the tumors, whereas the chromosome was normal in 46.3 percent. The gain of 17q was characteristic of advanced tumors and of tumors in children > or =1 year of age and was strongly associated with the deletion of 1p and amplification of N-myc. No tumor showed amplification of N-myc in the absence of either deletion of 1p or gain of 17q. Gain of 17q was a significant predictive factor for adverse outcome in univariate analysis. Among the patients with this abnormality, overall survival at five years was 30.6 percent (95 percent confidence interval, 21 to 40 percent), as compared with 86.0 percent (95 percent confidence interval, 78 to 91 percent) among those with normal 17q status. in multivariate analysis, gain of 17q was the most powerful prognostic factor, followed by the presence of stage 4 disease and deletion of 1p (hazard ratios, 3.4, 2.3, and 1.9, respectively). CONCLUSIONS: Gain of chromosome segment 17q21-qter is an important prognostic factor in children with neuroblastoma.