17q Gain in Neuroblastoma: A Review of Clinical and Biological Implications.

Neuroblastoma (NB) is the most frequent extracranial solid childhood tumor. Despite advances in the understanding and treatment of this disease, the prognosis in cases of high-risk NB is still poor. 17q gain has been shown to be the most frequent genomic alteration in NB. However, the significance of this remains unclear because of its high frequency and association with other genetic modifications, particularly segmental chromosomal aberrations, 1p and 11q deletions, and MYCN amplification, all of which are also associated with a poor clinical prognosis. This work reviewed the evidence on the clinical and biological significance of 17q gain. It strongly supports the significance of 17q gain in the development of NB and its importance as a clinically relevant marker. However, it is crucial to distinguish between whole and partial chromosome 17q gains. The most important breakpoints appear to be at 17q12 and 17q21. The former distinguishes between whole and partial chromosome 17q gain; the latter is a site of IGF2BP1 and NME1 genes that appear to be the main oncogenes responsible for the functional effects of 17q gain.

Telomere length and verbal learning in bipolar disorders.

INTRODUCTION: Recent studies indicate accelerated ageing processes, shorter telomere length and poorer cognitive functioning in patients with bipolar disorder. The neurobiology underlying cognitive function in bipolar disorder is yet to be established. We anticipated that accelerated ageing as indicated by shortened telomere length, would be associated with reduced cognitive performance in bipolar disorder, particularly for ageing sensitive functions such as memory and learning. METHODS: The study consisted of 647 participants (bipolar disorder [n = 246] and healthy controls [n = 401]). All participants underwent a standardized neuropsychological test battery, including working memory, executive functioning, processing speed, verbal learning, and verbal memory. Leucocyte telomere length was measured via blood and determined by quantitative real-time Polymerase Chain Reaction (qPCR) providing a telomere to single copy ratio (T/S ratio). The T/S ratio was used as an estimate of the mean telomere length of each participant. All analyses were adjusted for medication, Daily Defined Dose (DDD), chronological age, sex, and ethnicity. RESULTS: Patients had shorter telomere lengths than healthy controls (Cohen's d = 0.11, p = 0.01). Within patients', a positive association was observed for verbal learning and telomere length (ß = 0.14, p = 0.025), along with a trend for verbal memory and telomere length (ß = 0.11, p = 0.07). No other associations were observed for telomere length and cognitive functioning in the patient or the control group (p > 0.1). CONCLUSION: Our study may suggest poorer brain health in bipolar disorder as indexed by shorter telomere length and reduced learning correlates. However, the role of telomere length on cognitive functioning in bipolar disorder seems limited.

GSTM1 and GSTT1 double null genotypes determining cell fate and proliferation as potential risk factors of relapse in children with hematological malignancies after hematopoietic stem cell transplantation.

PURPOSE: This study aimed to retrospectively evaluate the genetic association of null variants of glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with hematological malignancies (HMs) undergoing busulfan (BU)- containing allogeneic hematopoietic stem cell transplantation (HSCT) and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST gene-edited cell models. METHODS: GSTM1- and GSTT1-null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1-null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. RESULTS: Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76-15.42; p = 1.9 × 10-5]). BU-induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. CONCLUSION: The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation.

A review of the biological and clinical implications of RAS-MAPK pathway alterations in neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumor in children, representing approximately 8% of all malignant childhood tumors and 15% of pediatric cancer-related deaths. Recent sequencing and transcriptomics studies have demonstrated the RAS-MAPK pathway's contribution to the development and progression of neuroblastoma. This review compiles up-to-date evidence of this pathway's involvement in neuroblastoma. We discuss the RAS-MAPK pathway's general functioning, the clinical implications of its deregulation in neuroblastoma, and current promising therapeutics targeting proteins involved in signaling.

The analysis of GSTA1 promoter genetic and functional diversity of human populations.

GSTA1 encodes a member of a family of enzymes that function to add glutathione to target electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. GSTA1 has several functional SNPs within its promoter region that are responsible for a change in its expression by altering promoter function. This study aims to investigate distributions of GSTA1 promoter haplotypes across different human populations and to assess their impact on the expression of GSTA1. PHASE 2.1.1 was used to infer haplotypes and diplotypes of six GSTA1 promoter SNPs on 2501 individuals from 26 populations classified by the 1000 Genomes Project into five super-populations that included Africa (N = 660), America (N = 347), East Asia (N = 504), Europe (N = 502), and South Asia (N = 488). We used pairwise FST analysis to compare sub-populations and luciferase reporter assay (LRA) to evaluate the impact of each SNP on activation of transcription and interaction with other SNPs. The distributions of GSTA1 promoter haplotypes and diplotypes were significantly different among the different human populations. Three new promoter haplotypes were found in the African super-population. LRA demonstrated that SNPs at -52 and -69 has the most impact on GSTA1 expression, however other SNPs have a significant impact on transcriptional activity. Based on LRA, a new model of cis-elements interaction is presented. Due to the significant differences in GSTA1 diplotype population frequencies, future pharmacogenomics or disease-related studies would benefit from the inclusion of the complete GSTA1 promoter haplotype based on the newly proposed metabolic grouping derived from the LRA results.

The Catalytic Activity of GSTM1 In vitro is Independent of MAPK8.

BACKGROUND: Glutathione S-transferases (GSTs) are phase II metabolic enzymes crucial for the metabolism of electrophilic drugs. Additionally, several GST isoforms are involved in protein- protein interaction with mitogen-activated protein kinases (MAPKs), modulating apoptosis pathways. METHODS: To assess the potential change of enzymatic activity, we performed a GST enzyme assay with human recombinant GSTM1 in the presence and absence of MAPK8. Recently, GSTM1 has been demonstrated to interact with MAPK8 both in silico and in vitro. The binding interface predicted in silico comprised amino acid residues present on the surface of the protein and a few were deep in the active site of the protein. RESULTS: The experiment demonstrated that the GSTM1 activity was conserved even in the presence of MAPK8 in the assay. CONCLUSION: The possible alteration in the activity of MAPK8 in this interaction needs to be evaluated in further experiments.

4th ESPT Conference: pharmacogenomics and personalized medicine - research progress and clinical implementation.

The Fourth European Society of Pharmacogenomics and Personalized Therapy biennial conference was organized in collaboration with the Italian Society of Personalized Medicine (SIMeP) and was held at Benedictine Monastery of San Nicolò l'Arena in Catania, Sicily (Italy) on 4-7 October 2017. The congress addressed the research progress and clinical implementation in pharmacogenomics and personalized medicine. The Fourth European Society of Pharmacogenomics and Personalized Therapy congress brought together leading international scientists and healthcare professionals actively working in the fields of pharmacogenomics and personalized therapy. Altogether, 25 speakers in 15 session comprehensively covered broad spectrum of pharmacogenetics and pharmacogenomics research, clinical applications in different clinical disciplines attended by 270 delegates.

Sex-determining region Y (SRY) attributes to gender differences in RANKL expression and incidence of osteoporosis.

Receptor activator of nuclear factor κB ligand (RANKL) plays a crucial role in bone metabolism. RANKL gene misregulation has been implicated in several bone and cancer diseases. Here, we aimed to identify novel transcription regulators of RANKL expression. We discovered that transcription factors, sex-determining region Y (SRY) and c-Myb, regulate RANKL expression. We demonstrated that c-Myb increases and male-specific SRY decreases RANKL expression through direct binding to its 5'-proximal promoter. These results are corroborated by the gene expression in human bone samples. In osteoporotic men, expression of RANKL is 17-fold higher, which correlates with the drastically reduced expression (200-fold) of Sry, suggesting that in osteoporotic men, the upregulation of RANKL is caused by a decrease of Sry. In healthy men, the expression of RANKL is 20% higher than that in healthy women. Our data suggest that gender differences in RANKL expression and bone quality could be due to the sex-specific transcription factor SRY.

The Biological and Clinical Relevance of G Protein-Coupled Receptors to the Outcomes of Hematopoietic Stem Cell Transplantation: A Systematized Review.

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for several malignant and non-malignant diseases at the cost of serious treatment-related toxicities (TRTs). Recent research on extending the benefits of HSCT to more patients and indications has focused on limiting TRTs and improving immunological effects following proper mobilization and engraftment. Increasing numbers of studies report associations between HSCT outcomes and the expression or the manipulation of G protein-coupled receptors (GPCRs). This large family of cell surface receptors is involved in various human diseases. With ever-better knowledge of their crystal structures and signaling dynamics, GPCRs are already the targets for one third of the current therapeutic arsenal. The present paper assesses the current status of animal and human research on GPCRs in the context of selected HSCT outcomes via a systematized survey and analysis of the literature.

4th ESPT summer school: precision medicine and personalised health.

In September 2018, the European Society of Pharmacogenomics and Personalised Therapy (ESPT), with the support of the Swiss Personalized Health Network (SPHN), organized its 4th biennial summer school, entitled 'Precision Medicine and Personalised Health' (Campus Biotech, Geneva, Switzerland; www.esptsummerschool.eu/ ). The school's comprehensive and innovative educational program aimed to address the fundamentals of pharmacogenomics, the latest knowledge on established and new concepts in the field of precision medicine, as well as its advanced clinical applications in personalized health. The school consisted of 31 lectures, eight interactive workshops, visits to genome center and poster presentations, involving 40 speakers from distinguished international faculties. The meeting was a resounding success by generating informal environments between more than 80 participants from 26 different countries.

Telomerase stability and evaluation of real-time telomeric repeat amplification protocol.

Telomerase is RNA directed polymerase which acts as reverse transcriptase based on its own RNA component. It is considered to be involved in the pathology of many diseases and is recognized as a potential biomarker. The aims were to determine the sample storage conditions and the time frame for samples analysis, then to prove reliability of enzyme activity measurement with real-time telomeric repeat amplification protocol (TRAP) and to evaluate the suitable standard samples for telomerase activity measurements. Samples used for stability and freeze-thaw study were peripheral blood leukocytes, obtained from apparently healthy persons, patients with diagnosed cancer and cell lines. Telomerase activity was measured using TRAP method, while standard evaluation was done using nuclear magnetic resonance (NMR) technique. Storage at -20 °C preserved telomerase activity in samples from cancer patients for at least 14 days (21.46 ± 0.135 versus 21.84 ± 0.357, p = .756), while samples obtained from healthy persons should be stored at -80 °C. We observed significant decrease of telomerase activity at freeze thaw cycle 5 in cancer patients' samples (21.46 ± 0.135 versus 23.09 ± 0.316, p < .05), and in healthy persons' ones already at cycle 3 (22.74 ± 0.107 versus 24.85 ± 0.151, p < .05). Telomerase activity from cell lines samples showed overall greater stability regarding the storage period and freeze-thaw cycles and it was considered for standard sample, which was confirmed by NMR analysis. Telomerase enzyme had adequate stability while efficacy, linearity, and reproducibility of TRAP method were acceptable for bio-analytical methods. All this indicated that telomerase could be a reliable biomarker.

PRIMA-1MET-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death. METHODS: CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential. RESULTS: Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53's role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition. CONCLUSIONS: PRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.

Epigenetic enzymes influenced by oxidative stress and hypoxia mimetic in osteoblasts are differentially expressed in patients with osteoporosis and osteoarthritis.

Epigenetic mechanisms including posttranslational histone modifications and DNA methylation are emerging as important determinants of bone homeostasis. With our case-control study we aimed to identify which chromatin-modifying enzymes could be involved in the pathology of postmenopausal osteoporosis and osteoarthritis while co-regulated by estrogens, oxidative stress and hypoxia. Gene expression of HAT1, KAT5, HDAC6, MBD1 and DNMT3A affected by oxidative stress and hypoxia in an in vitro qPCR screening step performed on an osteoblast cell line was analysed in trabecular bone tissue samples from 96 patients. Their expression was significantly reduced in patients with postmenopausal osteoporosis and osteoarthritis as compared to autopsy controls and significantly correlated with bone mineral density and several bone histomorphometry-derived parameters of bone quality and quantity as well as indicators of oxidative stress, RANK/RANKL/OPG system and angiogenesis. Furthermore, oxidative stress increased DNA methylation levels at the RANKL and OPG promoters while decreasing histone acetylation levels at these two genes. Our study is the first to show that higher expression of HAT1, HDAC6 and MBD1 is associated with superior quantity as well as quality of the bone tissue having a more favourable trabecular structure.

GSTA1 diplotypes affect busulfan clearance and toxicity in children undergoing allogeneic hematopoietic stem cell transplantation: a multicenter study.

Busulfan (BU) dose adjustment following therapeutic drug monitoring contributes to better outcome of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-guided BU dose adjustments. To investigate this aspect, polymorphism within glutathione S transferase genes were assessed. Particularly, promoter haplotypes of the glutathione S transferase A1 (GSTA1) were evaluated in vitro, with reporter gene assays and clinically, in a pediatric multi-center study (N =138) through association with BU pharmacokinetics (PK) and clinical outcomes. Promoter activity significantly differed between the GSTA1 haplotypes (p<0.001) supporting their importance in capturing PK variability. Four GSTA1 diplotype groups that significantly correlated with clearance (p=0.009) were distinguished. Diplotypes underlying fast and slow metabolizing capacity showed higher and lower BU clearance (ml/min/kg), respectively. GSTA1 diplotypes with slow metabolizing capacity were associated with higher incidence of sinusoidal obstruction syndrome, acute graft versus host disease and combined treatment-related toxicity (p<0.0005). Among other GST genes investigated, GSTP1 313GG correlated with acute graft versus host disease grade 1-4 (p=0.01) and GSTM1 non-null genotype was associated with hemorrhagic cystitis (p=0.003). This study further strengthens the hypothesis that GST diplotypes/genotypes could be incorporated into already existing population pharmacokinetic models for improving first BU dose prediction and HSCT outcomes. (No Clinicaltrials.gov identifier: NCT01257854. Registered 8 December 2010, retrospectively registered).

11q deletion in neuroblastoma: a review of biological and clinical implications.

Deletion of the long arm of chromosome 11 (11q deletion) is one of the most frequent events that occur during the development of aggressive neuroblastoma. Clinically, 11q deletion is associated with higher disease stage and decreased survival probability. During the last 25 years, extensive efforts have been invested to identify the precise frequency of 11q aberrations in neuroblastoma, the recurrently involved genes, and to understand the molecular mechanisms of 11q deletion, but definitive answers are still unclear. In this review, it is our intent to compile and review the evidence acquired to date on 11q deletion in neuroblastoma.

The Association of Combined GSTM1 and CYP2C9 Genotype Status with the Occurrence of Hemorrhagic Cystitis in Pediatric Patients Receiving Myeloablative Conditioning Regimen Prior to Allogeneic Hematopoietic Stem Cell Transplantation.

Hemorrhagic cystitis (HC) is one of the complications of busulfan-cyclophosphamide (BU-CY) conditioning regimen during allogeneic hematopoietic stem cell transplantation (HSCT) in children. Identifying children at high risk of developing HC in a HSCT setting could facilitate the evaluation and implementation of effective prophylactic measures. In this retrospective analysis genotyping of selected candidate gene variants was performed in 72 children and plasma Sulfolane (Su, water soluble metabolite of BU) levels were measured in 39 children following treatment with BU-CY regimen. The cytotoxic effects of Su and acrolein (Ac, water soluble metabolite of CY) were tested on human urothelial cells (HUCs). The effect of Su was also tested on cytochrome P 450 (CYP) function in HepaRG hepatic cells. Cumulative incidences of HC before day 30 post HSCT were estimated using Kaplan-Meier curves and log-rank test was used to compare the difference between groups in a univariate analysis. Multivariate Cox regression was used to estimate hazard ratios with 95% confidence intervals (CIs). Multivariate analysis included co-variables that were significantly associated with HC in a univariate analysis. Cumulative incidence of HC was 15.3%. In the univariate analysis, HC incidence was significantly (p < 0.05) higher in children older than 10 years (28.6 vs. 6.8%) or in children with higher Su levels (>40 vs. <11%) or in carriers of both functional GSTM1 and CYP2C9 (33.3 vs. 6.3%) compared to the other group. In a multivariate analysis, combined GSTM1 and CYP2C9 genotype status was associated with HC occurrence with a hazards ratio of 4.8 (95% CI: 1.3-18.4; p = 0.02). Ac was found to be toxic to HUC cells at lower concentrations (33 µM), Su was not toxic to HUC cells at concentrations below 1 mM and did not affect CYP function in HepaRG cells. Our observations suggest that pre-emptive genotyping of CYP2C9 and GSTM1 may aid in selection of more effective prophylaxis to reduce HC development in pediatric patients undergoing allogeneic HSCT. Article summary: (1) Children carrying functional alleles in GSTM1 and CYP2C9 are at high risk for developing hemorrhagic cystitis following treatment with busulfan and cyclophosphamide based conditioning regimen. (2) Identification of children at high risk for developing hemorrhagic cystitis in an allogeneic HSCT setting will enable us to evaluate and implement optimal strategies for its prevention. Trial registration: This study is a part of the trail "clinicaltrials.gov identifier: NCT01257854."

Pharmacogenomics in Pediatric Oncology: Review of Gene-Drug Associations for Clinical Use.

During the 3rd congress of the European Society of Pharmacogenomics and Personalised Therapy (ESPT) in Budapest in 2015, a preliminary meeting was held aimed at establishing a pediatric individualized treatment in oncology and hematology committees. The main purpose was to facilitate the transfer and harmonization of pharmacogenetic testing from research into clinics, to bring together basic and translational research and to educate health professionals throughout Europe. The objective of this review was to provide the attendees of the meeting as well as the larger scientific community an insight into the compiled evidence regarding current pharmacogenomics knowledge in pediatric oncology. This preliminary evaluation will help steer the committee's work and should give the reader an idea at which stage researchers and clinicians are, in terms of personalizing medicine for children with cancer. From the evidence presented here, future recommendations to achieve this goal will also be suggested.