Pharmacogenetics in Neuroblastoma: What Can Already Be Clinically Implemented and What Is Coming Next?

Pharmacogenetics is one of the cornerstones of Personalized Precision Medicine that needs to be implemented in the routine of our patients' clinical management in order to tailor their therapies as much as possible, with the aim of maximizing efficacy and minimizing toxicity. This is of great importance, especially in pediatric cancer and even more in complex malignancies such as neuroblastoma, where the rates of therapeutic success are still below those of many other types of tumors. The studies are mainly focused on germline genetic variants and in the present review, state of the art is presented: which are the variants that have a level of evidence high enough to be implemented in the clinic, and how to distinguish them from the ones that still need validation to confirm their utility. Further aspects as relevant characteristics regarding ontogeny and future directions in the research will also be discussed.

Metabolomic profiling in neuroblastoma.

BACKGROUND AND OBJECTIVES: Previous studies on several cancer types show that metabolomics provides a potentially useful noninvasive screening approach for outcome prediction and accurate response to treatment assessment. Neuroblastoma (NB) accounts for at least 15% of cancer-related deaths in children. Although current risk-based treatment approaches in NB have resulted in improved outcome, survival for high-risk patients remains poor. This study aims to evaluate the use of metabolomics for improving patients' risk-group stratification and outcome prediction in NB. DESIGN AND METHODS: Plasma samples from 110 patients with NB were collected at diagnosis prior to starting therapy and at the end of treatment if available. Metabolomic analysis of samples was carried out by ultra-performance liquid chromatography-time of flight mass spectrometry (UPLC-MS). RESULTS: The metabolomic analysis was able to identify different plasma metabolic profiles in high-risk and low-risk NB patients at diagnosis. The metabolic model correctly classified 16 high-risk and 15 low-risk samples in an external validation set providing 84.2% sensitivity (60.4-96.6, 95% CI) and 93.7% specificity (69.8-99.8, 95% CI). Metabolomic profiling could also discriminate high-risk patients with active disease from those in remission. Notably, a plasma metabolomic signature at diagnosis identified a subset of high-risk NB patients who progressed during treatment. CONCLUSIONS: To the best of our knowledge, this is the largest NB study investigating the prognostic power of plasma metabolomics. Our results support the potential of metabolomic profiling for improving NB risk-group stratification and outcome prediction. Additional validating studies with a large cohort are needed.

MTHFR and VDR Polymorphisms Improve the Prognostic Value of MYCN Status on Overall Survival in Neuroblastoma Patients.

Single nucleotide polymorphisms (SNPs) in Pharmacogenetics can play an important role in the outcomes of the chemotherapy treatment in Neuroblastoma, helping doctors maximize efficacy and minimize toxicity. Employing AgenaBioscience MassArray, 96 SNPs were genotyped in 95 patients looking for associations of SNP with response to induction therapy (RIT) and grade 3-4 toxicities, in High Risk patients. Associations of SNPs with overall (OS) and event-free (EFS) survival in the whole cohort were also explored. Cox and logistic regression models with Elastic net penalty were employed. Association with grade 3-4 gastrointestinal and infectious toxicities was found for 8 different SNPs. Better RIT was correlated with rs726501 AG, rs3740066 GG, rs2010963 GG and rs1143684 TT (OR = 2.87, 1.79, 1.23, 1.14, respectively). EFS was affected by rs2032582, rs4880, rs3814058, rs45511401, rs1544410 and rs6539870. OS was influenced by rs 1801133, rs7186128 and rs1544410. Remarkably, rs1801133 in MTHFR (p = 0.02) and rs1544410 in VDR (p = 0.006) also added an important predictive value for OS to the MYCN status, with a more accurate substratification of the patients. Although validation studies in independent cohorts will be required, the data obtained supports the utility of Pharmacogenetics for predicting Neuroblastoma treatment outcomes.

Methodological Approach to Use Fresh and Cryopreserved Vessels as Tools to Analyze Pharmacological Modulation of the Angiogenic Growth.

The sprouting of new vessels is greatly influenced by the procedure chosen. We sought to optimize the experimental conditions of the angiogenic growth of fresh and cryopreserved vessels cultured in Matrigel with the aim to use this system to analyze the pharmacological modulation of the process. Segments of second-order branches of rat mesenteric resistance arteries, thoracic aorta of rat or mouse, and cryopreserved rat aorta and human femoral arteries were cultured in Matrigel for 7-21 days in different mediums, as well as in the absence of endothelial or adventitia layer. Quantification of the angiogenic growth was performed by either direct measurement of the mean length of the neovessels or by calcein AM staining and determination of fluorescence intensity and area. Fresh and cryopreserved arterial rings incubated in Matrigel exhibited a spontaneous angiogenic response that was strongly accelerated by fetal calf serum. Addition of vascular endothelial growth factor, fibroblast growth factor, endothelial growth factor, or recombinant insulin-like growth factor failed to increase aortic sprouting, unless all were added together. Removal of adventitia, but not the endothelial layer, abrogated the angiogenic response of aortic rings. Determination of the mean neovessel length is an easy and accurate method to quantify the angiogenic growth devoid of confounding factors, such as inclusion of other cellular types surrounding the neovessels. Activity of a α1-adrenoceptor agonist (phenylephrine) and its inhibition by a selective antagonist (prazosin) were analyzed to prove the usefulness of the Matrigel system to evaluate the pharmacological modulation of the angiogenic growth.

Emerging drugs for neuroblastoma.

INTRODUCTION: Neuroblastoma accounts for 8 - 10% of pediatric cancers and is responsible for 15% of childhood cancer deaths. Despite multimodality treatment, the overall survival (OS) and event-free survival (EFS) in high-risk patients remain suboptimal. More than half of children diagnosed with high-risk neuroblastoma either do not respond to conventional therapies or relapse after treatment. AREAS COVERED: This review discusses about the unmet medical needs for new therapeutic options against high-risk neuroblastoma. New drugs and therapeutic strategies that are under development in clinical trials, which are currently recruiting patients. EXPERT OPINION: There is a need to improve the response rate of induction chemotherapy, which is not effective in a third of patients and also the other components of the current treatment, little efficacious in avoiding the relapses. Few drugs have been introduced as upfront therapy in the last years. Topotecan, irinotecan and temozolomide are expected to improve the response in high-risk neuroblastoma, but their impact on OS and EFS is unknown. Anti-GD2 antibodies combined with other immunomodulators (IL-2, GM-CSF) are an important advance in the treatment of these children. Nevertheless, the hope is put in the new drugs directed to molecular targets of neuroblastoma. Anti-angiogenic drugs, ALK antagonist and PI3K/Akt/mTOR inhibitors are among the most promising.

Neuroblastoma in Spain: Linking the national clinical database and epidemiological registries - A study by the Joint Action on Rare Cancers.

PURPOSE: Linkage between clinical databases and population-based cancer registries may serve to evaluate European Reference Networks' (ERNs) activity, by monitoring the proportion of patients benefiting from these and their impact on survival at a population level. To test this, a study targeting neuroblastoma (Nb) was conducted in Spain by the European Joint Action on Rare Cancers. MATERIAL AND METHODS: Subjects: Nb cases, incident 1999-2017, aged < 15 years. Linkage included: Spanish Neuroblastoma Clinical Database (NbCDB) (1217 cases); Spanish Registry of Childhood Tumours (RETI) (1514 cases); and 10 regional population-based registries (RPBCRs) which cover 33% of the childhood population (332 cases). Linkage was semiautomatic. We estimated completeness, incidence, contribution, deficit, and 5-year survival in the databases and specific subsets. RESULTS: National completeness estimates for RETI and NbCDB were 91% and 72% respectively, using the Spanish RPBCRs on International Incidence of Childhood Cancer (https://iicc.iarc.fr/) as reference. RPBCRs' specific contribution was 1.6%. Linkage required manual crossover in 54% of the semiautomatic matches. Five-year survival was 74% (0-14 years) and 90% (0-18 months). CONCLUSIONS: All three databases were incomplete as regards Spain as a whole and should therefore be combined to achieve full childhood cancer registration. A unique personal patient identifier could facilitate such linkage. Most children have access to Nb clinical trials. Consolidated interconnections between the national registry and clinical registries (including ERNs and paediatric oncology clinical groups) should be established to evaluate outcomes.

Germline variant in Ctcf links mental retardation to Wilms tumor predisposition.

CTCF germline mutations have been related to MRD21. We report the first bilateral Wilms tumor suffered by a MRD21 patient carrying an unreported CTCF missense variant in a zinc finger domain of CTCF protein. We found that germline heterozygous variant I446K became homozygous in the tumor due to a loss of heterozygosity rearrangement affecting the whole q arm on chromosome 16. Our findings propose CTCF I446K variant as a link between MRD21 and Wilms tumor predisposition.

Suprarenal Masses in Very Young Infants: Is It Safe to Watch and Wait? Report of a SIOPEN Observational Study Results.

Background: To assess whether expectant observation of infants ≤ 90 days old with small suprarenal masses (sSRMs) could avoid unnecessary surgery without impacting outcome. Methods: Infants ≤ 90 days with a ≤ 5 cm mass, without midline extension or lymph node or distant spread were registered (ClinicalTrials.org:NCT01728155). Once staging was completed, they were followed with ultrasound, MRI and urinary catecholamines. Surgical resection was only planned if there was a ≥40% mass volume increase or for a mass persisting after 48 weeks of the planned observation. Results: Over a 5-year period, 128 infants were registered. No infant had detectable MYCN amplification in the peripheral blood. Surgery was performed in 39 (30.5%) patients, in 18 during and in 21 after the planned 48-week observation, and 74% were confirmed to be neuroblastomas. Non-life-threatening surgical complications occurred in two cases. The 3-year overall survival and event-free survival were 100% and 87.1%, respectively. The 16 events observed were volume increase (N = 11) and progression to neuroblastoma stage MS (N = 5). Patients with solid masses or MIBG-positive masses had lower EFS. Conclusions: Expectant observation for infants with sSRMs with clinical follow-up and timely imaging (including MRI scan) is safe and effective, allowing surgery to be avoided in the majority of them.

Retinoblastoma and mosaic 13q deletion: a case report.

BACKGROUND: Patients with 13q-syndrome are at risk of retinoblastoma when the RB1 gene, located in the chromosomal band 13q14.2, is deleted. This syndrome is frequently associated with congenital malformations and developmental delay, although these signs could be mild. Mosaic 13q-deletion patients have been previously reported in the literature; their phenotype is variable, and they may not be recognized. CASE PRESENTATION: Retinoblastoma diagnosed in a child with 13q-mosaicism confirmed in blood, oral mucosa, healthy retina and retinoblastoma. A second RB1 hit is present exclusively in the retinoblastoma sample (RB1 c.958C>T p.Arg320Ter). Other detected molecular events in retinoblastoma are 6p12.3pter gain and 6q25.3qter loss. Clinical examination is unremarkable except for clinodactyly of the right fifth finger. DISCUSSION AND CONCLUSIONS: We describe a case of mosaic 13q deletion syndrome affected by retinoblastoma. Molecular data obtained from the tumor analysis are similar to previous data available about this malignancy. High clinical suspicion is essential for an adequate diagnosis of mosaic cases.

Next-Generation Sequencing Identifies Potential Actionable Targets in Paediatric Sarcomas.

Background: Bone and soft-tissue sarcomas represent 13% of all paediatric malignancies. International contributions to introduce next-generation sequencing (NGS) approaches into clinical application are currently developing. We present the results from the Precision Medicine program for children with sarcomas at a reference centre. Results: Samples of 70 paediatric sarcomas were processed for histopathological analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS) with a consensus gene panel. Pathogenic alterations were reported and, if existing, targeted recommendations were translated to the clinic. Seventy paediatric patients with sarcomas from 10 centres were studied. Median age was 11.5 years (range 1-18). Twenty-two (31%) had at least one pathogenic alteration by NGS. Thirty pathogenic mutations in 18 different genes were detected amongst the 22 patients. The most frequent alterations were found in TP53, followed by FGFR4 and CTNNB1. Combining all biological studies, 18 actionable variants were detected and six patients received targeted treatment observing a disease control rate of 78%. Extrapolating the results to the whole cohort, 23% of the patients would obtain clinical benefit from this approach. Conclusions: Paediatric sarcomas have a different genomic landscape when compared to adult cohorts. Incorporating NGS targets into paediatric sarcomas' therapy is feasible and allows personalized treatments with clinical benefit in the relapse setting.

Germline Predisposition to Pediatric Cancer, from Next Generation Sequencing to Medical Care.

Knowledge about genetic predisposition to pediatric cancer is constantly expanding. The categorization and clinical management of the best-known syndromes has been refined over the years. Meanwhile, new genes for pediatric cancer susceptibility are discovered every year. Our current work shares the results of genetically studying the germline of 170 pediatric patients diagnosed with cancer. Patients were prospectively recruited and studied using a custom panel, OncoNano V2. The well-categorized predisposing syndromes incidence was 9.4%. Likely pathogenic variants for predisposition to the patient's tumor were identified in an additional 5.9% of cases. Additionally, a high number of pathogenic variants associated with recessive diseases was detected, which required family genetic counseling as well. The clinical utility of the Jongmans MC tool was evaluated, showing a high sensitivity for detecting the best-known predisposing syndromes. Our study confirms that the Jongmans MC tool is appropriate for a rapid assessment of patients; however, the updated version of Ripperger T criteria would be more accurate. Meaningfully, based on our findings, up to 9.4% of patients would present genetic alterations predisposing to cancer. Notably, up to 20% of all patients carry germline pathogenic or likely pathogenic variants in genes related to cancer and, thereby, they also require expert genetic counseling. The most important consideration is that the detection rate of genetic causality outside Jongmans MC et al. criteria was very low.

Review: Ewing Sarcoma Predisposition.

Ewing sarcoma is a rare tumor developed in bone and soft tissues of children and teenagers. This entity is biologically led by a chromosomal translocation, typically including EWS and FLI1 genes. Little is known about Ewing sarcoma predisposition, although the role of environmental factors, ethnicity and certain polymorphisms on Ewing sarcoma susceptibility has been studied during the last few years. Its prevalence among cancer predisposition syndromes has also been thoroughly examined. This review summarizes the available evidence on predisposing factors involved in Ewing sarcoma susceptibility. On the basis of these data, an integrated approach of the most influential factors on Ewing sarcoma predisposition is proposed.

Publisher Correction: Clinical Features of Neuroblastoma With 11q Deletion: An Increase in Relapse Probabilities In Localized And 4S Stages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical Features of Neuroblastoma With 11q Deletion: An Increase in Relapse Probabilities In Localized And 4S Stages.

Neuroblastoma (NB) is a heterogeneous tumor with an extremely diverse prognosis according to clinical and genetic factors, such as the presence of an 11q deletion (11q-del). A multicentric study using data from a national neuroblastic tumor database was conducted. This study compared the most important features of NB patients: presence of 11q-del, presence of MYCN amplification (MNA) and remaining cases. A total of 357 patients were followed throughout an 8-year period. 11q-del was found in sixty cases (17%). 11q-del tumors were diagnosed at an older age (median 3.29 years). Overall survival (OS) was lower in 11q-del patients (60% at 5 years), compared to all other cases (76% at 5 years) p = 0.014. Event free survival (EFS) was 35% after 5 years, which is a low number when compared with the remaining cases: 75% after 5 years (p < 0.001). Localized tumors with 11q-del have a higher risk of relapse (HR = 3.312) such as 4 s 11q-del patients (HR 7.581). 11q-del in NB is a dismal prognostic factor. Its presence predicts a bad outcome and increases relapse probability, specially in localized stages and 4 s stages. The presence of 11q aberration should be taken into consideration when stratifying neuroblastoma risk groups.

Activation of α1A -adrenoceptors desensitizes the rat aorta response to phenylephrine through a neuronal NOS pathway, a mechanism lost with ageing.

BACKGROUND AND PURPOSE: A NO-mediated desensitization of vasoconstrictor responses evoked by stimulation of α1 -adrenoceptors has been reported in different vessels. We investigated the involvement of each α1 -adrenoceptor subtype and constitutive NOS isoforms and the influence of ageing and hypertension on this process. EXPERIMENTAL APPROACH: Wistar and spontaneously hypertensive rats (SHR), 16, 32, 52 and 72 weeks-old, were used to evaluate the desensitization process. Expression of α1 -adrenoceptor subtypes, endothelial NOS (eNOS) and neuronal NOS (nNOS) were determined in rat aorta and left ventricle (LV). Expression levels were also evaluated in LV of a group of heart failure patients with a wide age range. KEY RESULTS: Repeated application of phenylephrine decreased subsequent α1 -adrenoceptor-mediated vasoconstriction by increasing nNOS protein expression in aorta, but not in tail or mesenteric resistance arteries, where mRNA levels of nNOS were undetectable. This desensitization process disappeared in the absence of endothelium or in the presence of L-NAME (100 µM), nNOS inhibitors, SMTC (1 µM) and TRIM (100 µM), and 5-methylurapidil (100 nM, α1A -antagonist), but not BMY7378 (10 nM, α1D -antagonist). The α1A /nNOS-mediated desensitization was absent in aged SHR and Wistar animals, where the expression of α1A -adrenoceptors was reduced in aorta and LV. In human LV, a negative correlation was found between age and α1A -adrenoceptor expression. CONCLUSIONS AND IMPLICATIONS: The α1A -adrenoceptor subtype, through endothelial nNOS-derived NO, may act as a physiological 'brake' against the detrimental effects of excessive α1 -adrenoceptor-mediated vasoconstriction. Reduced α1A -adrenoceptor- and nNOS-mediated desensitization in aged patients could be involved in the age-dependent elevation of adrenergic activity.

Two independent epigenetic biomarkers predict survival in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with a highly variable clinical course, ranging from spontaneous regression to life-threatening disease. Survival rates for high-risk NB patients remain disappointingly low despite multimodal treatment. Thus, there is an urgent clinical need for additional biomarkers to improve risk stratification, treatment management, and survival rates in children with aggressive NB. RESULTS: Using gene promoter methylation analysis in 48 neuroblastoma tumors with microarray technology, we found a strong association between survival and gene promoter hypermethylation (P = 0.036). Hypermethylation of 70 genes significantly differentiated high-risk survivor patients from those who died during follow-up time. Sixteen genes with relevant roles in cancer biology were further validated in an additional cohort of 83 neuroblastoma tumors by bisulfite pyrosequencing. High promoter methylation rates of these genes were found in patients with metastatic tumors (either stage metastatic (M) or metastatic special (MS)), 18 months or older at first diagnosis, MYCN amplification, relapsed, and dead. Notably, the degree of methylation of retinoblastoma 1 (RB1) and teratocarcinoma-derived growth factor 1 (TDGF1) predicts event-free and overall survival independently of the established risk factors. In addition, low RB1 mRNA expression levels associate with poor prognosis suggesting that promoter methylation could contribute to the transcriptional silencing of this gene in NB. CONCLUSIONS: We found a new epigenetic signature predictive for NB patients' outcome: the methylation status of RB1 and TDGF1 associate with poorer survival. This information is useful to assess prognosis and improve treatment selection.

Differences in the signaling pathways of α(1A)- and α(1B)-adrenoceptors are related to different endosomal targeting.

AIMS: To compare the constitutive and agonist-dependent endosomal trafficking of α(1A)- and α(1B)-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. METHODS: Using CypHer5 technology and VSV-G epitope tagged α(1A)- and α(1B)-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). RESULTS AND CONCLUSIONS: Constitutive as well as agonist-induced trafficking of α(1A) and α(1B) ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α(1A)-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to ß-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α(1B)-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-ß-cyclodextrin.

α1D-Adrenoceptors are responsible for the high sensitivity and the slow time-course of noradrenaline-mediated contraction in conductance arteries.

The objective of this study was to determine whether the different time-course characteristics of α1-adrenoceptor-mediated contraction in arteries can be related to the subtypes involved. Contractile responses to noradrenaline (NA) were compared with inositol phosphate accumulation and extracellular signal-regulated kinase (ERK)1/2 phosphorylation after α1-agonist stimuli in the same vessels in the presence or absence of α1-antagonists in rat or in α1-subtype knockout (KO) mice. Aorta, where α1D-AR is the main functional subtype, had higher sensitivity to NA (in respect of inositol phosphate [IP], pERK1/2, and contractile response) than tail artery, where the α1A-adrenoceptor subtype is predominant. Furthermore, the contraction in aorta exhibited a slower decay after agonist removal and this was consistent in all strains harboring α1D-adrenoceptors (from rat, α1B-KO, and wild-type [WT] mice) but was not observed in the absence of the α1D-adrenoceptor signal (α1D-adrenoceptor blocked rat aorta or aorta from α1D-KO). IP formation paralleled α1-adrenoceptor-mediated contraction (agonist present or postagonist) in aorta and tail artery. High sensitivity to agonist and persistence of response after agonist removal is a property of α1D-adrenoceptors. Therefore, the preponderance of this subtype in noninnervated conductance arteries such as aorta allows responsiveness to circulating catecholamines and prevents abrupt changes in vessel caliber when the stimulus fluctuates. Conversely, in innervated distributing arteries, high local concentrations of NA are required to activate α1A-adrenoceptors for a response that is rapid but short lived allowing fine adjustment of the contractile tone by perivascular sympathetic nerves.

Treatment of high-risk neuroblastoma with anti-GD2 antibodies.

Anticancer monoclonal antibodies (mAbs) targeting specific antigens on the tumour surface are increasingly being applied in cancer treatment. Potential advantages include long half-life, low toxicity, high affinity and specificity. In order to develop novel immune therapies for high-risk cancers, finding tumour targets that are not widely shared by normal cells is a goal. GD2-disialoganglioside is one of them. It is expressed on the surface of a variety of tumours with no curative therapies for patients with advanced disease. In childhood, neuroblastoma is the most common GD2-expressing tumour. Because of this tumour-selective expression, it is an attractive target for tumour-specific therapies such as antibody therapy. Over the last two decades, several anti-GD2 antibodies have been developed. To reduce both toxicity and development of human anti-mouse antibodies (HAMA), research efforts have primarily focused on exploring anti-GD2 antibodies that substitute mouse components by human ones. This review will examine antibodies currently undergoing clinical testing as well as the most recent advances to improve antibody therapy for patients with high-risk neuroblastoma.