Long-term follow-up for childhood cancer survivors: the Geneva experience.

AIMS OF THE STUDY: Although the 5-year survival for pediatric cancer in Switzerland today is over 85%, two thirds of the survivors will develop chronic health conditions due to the disease or to the toxicity of treatments. In this context, a long-term personalized follow-up program (LTFU program), was set up at the University Hospitals of Geneva (HUG) since 2015. We aimed to describe this program, more particularly the specialized follow-ups set up, the cumulative burden of the chronic health conditions, and finally assess the satisfaction of patients and/or their parents with it. METHODS: A monocentric retrospective study was performed where data on follow-ups and chronic health conditions were collected from medical charts of people who had childhood cancer and who participated in the LTFU program. Chronic health conditions were classified and graded in severity with the Common Terminology Criteria of Adverse Events (CTCAE) classification, version 5.0. This study was completed by a satisfaction survey among patients and/or their parents. RESULTS: Out of 83 eligible patients, 51 (61.4%) accepted to participate, with an average age of 17.4 years (range, 10 to 35) at the time of study. Mean delay since end of treatment was 9.8 years (range: 4.5-31). The prevalence of any chronic health condition is 82.3%, 43.1% for having 1 or 2 chronic health conditions and 39.2% for having more than 3 chronic health conditions. The total number of Grade CTCAE 1-4 chronic health conditions was 118 for the 51 participants, with a mean of 2.3 (range, 0 to 7) disorders per patient. The most frequently affected systems were neurological (14.4%), musculoskeletal (13.6%), endocrine (9.3%) and renal (9.3%) systems. Sarcoma, central nervous system tumors and neuroblastoma were the diagnoses associated with the highest average number of chronic health conditions. Among the 118 questionnaires sent to patients and/or parents, we received 82 (69.5%) responses. The level of satisfaction was good to excellent for more than 90% of the participants, for all the items evaluated. CONCLUSIONS: Childhood cancer survivors present a significant number of chronic health conditions, confirming the need for appropriate long-term, multidisciplinary and patient-specific medical follow-up based on the primary diagnosis and therapies received. Moreover, the LTFU program at the HUG was highly appreciated by patients and/or their parents and this motivates its permanent conduct.

Cohort-based association study of germline genetic variants with acute and chronic health complications of childhood cancer and its treatment: Genetic Risks for Childhood Cancer Complications Switzerland (GECCOS) study protocol.

INTRODUCTION: Childhood cancer and its treatment may lead to various health complications. Related impairment in quality of life, excess in deaths and accumulated healthcare costs are relevant. Genetic variations are suggested to contribute to the wide inter-individual variability of complications but have been used only rarely to risk-stratify treatment and follow-up care. This study aims to identify germline genetic variants associated with acute and late complications of childhood cancer. METHODS AND ANALYSIS: The Genetic Risks for Childhood Cancer Complications Switzerland (GECCOS) study is a nationwide cohort study. Eligible are patients and survivors who were diagnosed with childhood cancers or Langerhans cell histiocytosis before age 21 years, were registered in the Swiss Childhood Cancer Registry (SCCR) since 1976 and have consented to the Paediatric Biobank for Research in Haematology and Oncology, Geneva, host of the national Germline DNA Biobank Switzerland for Childhood Cancer and Blood Disorders (BISKIDS).GECCOS uses demographic and clinical data from the SCCR and the associated Swiss Childhood Cancer Survivor Study. Clinical outcome data consists of organ function testing, health conditions diagnosed by physicians, second primary neoplasms and self-reported information from participants. Germline genetic samples and sequencing data are collected in BISKIDS. We will perform association analyses using primarily whole-exome or whole-genome sequencing to identify genetic variants associated with specified health conditions. We will use clustering and machine-learning techniques and assess multiple health conditions in different models. DISCUSSION: GECCOS will improve knowledge of germline genetic variants associated with childhood cancer-associated health conditions and help to further individualise cancer treatment and follow-up care, potentially resulting in improved efficacy and reduced side effects. ETHICS AND DISSEMINATION: The Geneva Cantonal Commission for Research Ethics has approved the GECCOS study.Research findings will be disseminated through national and international conferences, publications in peer-reviewed journals and in lay language online. TRIAL REGISTRATION NUMBER: NCT04702321.

Predictors for participation in DNA self-sampling of childhood cancer survivors in Switzerland.

BACKGROUND: Research on germline genetic variants relies on enough eligible participants which is difficult to achieve for rare diseases such as childhood cancer. With self-collection kits, participants can contribute genetic samples conveniently from their home. Demographic and clinical factors were identified previously that influenced participation in mailed self-collection. People with pre-existing heritable diagnoses might participate differently in germline DNA collection which might render sampling biased in this group. In this nationwide cross-sectional study, we analysed predictive factors of participation in DNA self-collection including heritable diagnoses. METHODS: We identified childhood cancer survivors from the Swiss Childhood Cancer Registry for invitation to germline DNA self-sampling in September 2019. Participants received saliva sampling kits by postal mail at their home, were asked to fill them, sign an informed consent, and send them back by mail. Two reminders were sent to non-participants by mail. We compared demographic, clinical, and treatment information of participants with non-participants using univariable and multivariable logistic regression models. RESULTS: We invited 928 childhood cancer survivors in Switzerland with a median age of 26.5 years (interquartile range 19-37), of which 463 (50%) participated. After the initial send out of the sampling kit, 291 (63%) had participated, while reminder letters led to 172 additional participants (37%). Foreign nationality (odds ratio [OR] 0.5; 95%-confidence interval [CI] 0.4-0.7), survivors aged 30-39 years at study versus other age groups (OR 0.5; CI 0.4-0.8), and survivors with a known cancer predisposition syndrome (OR 0.5; CI 0.3-1.0) were less likely to participate in germline DNA collection. Survivors with a second primary neoplasm (OR 1.9; CI 1.0-3.8) or those living in a French or Italian speaking region (OR 1.3; CI 1.0-1.8) tended to participate more. CONCLUSIONS: We showed that half of childhood cancer survivors participated in germline DNA self-sampling relying completely on mailing of sample kits. Written reminders increased the response by about one third. More targeted recruitment strategies may be advocated for people of foreign nationality, aged 30-39 years, and those with cancer predisposition syndromes. Perceptions of genetic research and potential barriers to participation of survivors need to be better understood. TRIAL REGISTRATION: Biobank: https://directory.bbmri-eric.eu/#/collection/bbmri-eric:ID:CH_HopitauxUniversitairesGeneve:collection:CH_BaHOP Research project : Clinicaltrials.gov: NCT04702321 .

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.

Therapeutic Drug Monitoring of Busulfan for the Management of Pediatric Patients: Cross-Validation of Methods and Long-Term Performance.

BACKGROUND: Busulfan (Bu) is an alkylating agent used as part of the conditioning regimen in pediatric patients before hematopoietic stem cell transplantation. Despite intravenous (IV) administration and dosing recommendations based on age and weight, reports have revealed interindividual variability in Bu pharmacokinetics and the outcomes of hematopoietic stem cell transplantation. In this context, adjusting doses to Bu's narrow therapeutic window is advised. We aimed to assess the utility of therapeutic drug monitoring (TDM) of Bu in children, the reliability of Bu quantification methods, and its stability in plasma when stored for up to 5 years. METHODS: Eighteen patients from our TDM center (252 samples) were included. All of them received a 2-hour Bu IV infusion 4 times daily for a total of 16 doses. The first dose of Bu was age/weight-based, and the subsequent doses were adjusted from third or fifth dose onward based on the estimated first dose pharmacokinetic parameters to target steady-state concentrations (Css) of 600-900 ng/mL. The performance of our unit's high-performance liquid chromatography with tandem mass spectrometry method was assessed using a quality control (QC, 35 series) chart. International, multicenter, cross-validation test (n = 21) was conducted to validate different analytical methods. To assess Bu stability, regression analyses and Bland-Altman plots were performed on measurements at repeated time points on samples stored at -80°C for up to 5 years. RESULTS: We observed a 4.2-fold interindividual variability in Bu Css after the first dose, with only 28% of children having a Css within the target range. During the 4 days of conditioning, 83% of children had their doses modified according to TDM recommendations. This achieved a Css within the target range in 75% of the children. Routine QC measurements were generally within the ±15% range around theoretical values, showing the optimal robustness of our center's analytical method. Two of the 21 Bu TDM centers returned inadequate results during cross-validation testing; both used a UV detection method. Storage at -80°C led to a fall in Bu content of 14.9% ± 13.4% at 2-4 years and of 20% ± 5% by 5 years (roverall = 0.92). CONCLUSIONS: We conclude that TDM is an effective method of achieving targeted Bu levels in children. QC programs are crucial to monitoring and maintaining the quality of an analytical method.

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."

Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism.

Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma.

In vivo brain macromolecule signals in healthy and glioblastoma mouse models: 1H magnetic resonance spectroscopy, post-processing and metabolite quantification at 14.1 T.

In (1)H magnetic resonance spectroscopy, macromolecule signals underlay metabolite signals, and knowing their contribution is necessary for reliable metabolite quantification. When macromolecule signals are measured using an inversion-recovery pulse sequence, special care needs to be taken to correctly remove residual metabolite signals to obtain a pure macromolecule spectrum. Furthermore, since a single spectrum is commonly used for quantification in multiple experiments, the impact of potential macromolecule signal variability, because of regional differences or pathologies, on metabolite quantification has to be assessed. In this study, we introduced a novel method to post-process measured macromolecule signals that offers a flexible and robust way of removing residual metabolite signals. This method was applied to investigate regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account. However, since no significant differences in metabolite quantification were detected, it was concluded that a single macromolecule spectrum can be generally used for the quantification of healthy mouse brain spectra. Alternatively, the study of a mouse model of human glioma showed several alterations of the macromolecule spectrum, including, but not limited to, increased mobile lipid signals, which had to be taken into account to avoid significant metabolite quantification errors.

Quantification, self-renewal, and genetic tracing of FL1⁺ tumor-initiating cells in a large cohort of human gliomas.

Evidence has emerged that the initiation and growth of gliomas is sustained by a subpopulation of cancer-initiating cells (CICs). Because of the difficulty of using markers to tag CICs in gliomas, we have previously exploited more robust phenotypic characteristics, including a specific morphology and intrincic autofluorescence, to identify and isolate a subpopulation of glioma CICs, called FL1(+). The objective of this study was to further validate our method in a large cohort of human glioma and a mouse model of glioma. Seventy-four human gliomas of all grades and the GFAP-V(12)HA-ras B8 mouse model were analyzed for in vitro self-renewal capacity and their content of FL1(+). Nonneoplastic brain tissue and embryonic mouse brain were used as control. Genetic traceability along passages was assessed with microsatellite analysis. We found that FL1(+) cells from low-grade gliomas and from control nonneoplasic brain tissue show a lower level of autofluorescence and undergo a restricted number of cell divisions before dying in culture. In contrast, we found that FL1(+) cells derived from many but not all high-grade gliomas acquire high levels of autofluorescence and can be propagated in long-term cultures. Moreover, FL1(+) cells show a remarkable traceability over time in vitro and in vivo. Our results show that FL1(+) cells can be found in all specimens of a large cohort of human gliomas of different grades and in a model of genetically induced mouse glioma as well as nonneoplastic brain. However, their self-renewal capacity is variable and seems to be dependent on the tumor grade.

In vivo metabolic profiling of glioma-initiating cells using proton magnetic resonance spectroscopy at 14.1 Tesla.

In the last decade, evidence has emerged indicating that the growth of a vast majority of tumors including gliomas is sustained by a subpopulation of cancer cells with stem cell properties called cancer initiating cells. These cells are able to initiate and propagate tumors and constitute only a fraction of all tumor cells. In the present study, we showed that intracerebral injection of cultured glioma-initiating cells into nude mice produced fast growing tumors showing necrosis and gadolinium enhancement in MR images, whereas gliomas produced by injecting freshly purified glioma-initiating cells grew slowly and showed no necrosis and very little gadolinium enhancement. Using proton localized spectroscopy at 14.1 Tesla, decreasing trends of N-acetylaspartate, glutamate and glucose concentrations and an increasing trend of glycine concentration were observed near the injection site after injecting cultured glioma-initiating cells. In contrast to the spectra of tumors grown from fresh cells, those from cultured cells showed intense peaks of lipids, increased absolute concentrations of glycine and choline-containing compounds, and decreased concentrations of glutamine, taurine and total creatine, when compared with a contralateral non-tumor-bearing brain tissue. A decrease in concentrations of N-acetylaspartate and γ-aminobutyrate was found in both tumor phenotypes after solid tumor formation. Further investigation is needed to determine the cause of the dissimilarities between the tumors grown from cultured glioma-initiating cells and those from freshly purified glioma-initiating cells, both derived from human glioblastomas.

Marker-independent identification of glioma-initiating cells.

Tumor-initiating cells with stem cell properties are believed to sustain the growth of gliomas, but proposed markers such as CD133 cannot be used to identify these cells with sufficient specificity. We report an alternative isolation method purely based on phenotypic qualities of glioma-initiating cells (GICs), avoiding the use of molecular markers. We exploited intrinsic autofluorescence properties and a distinctive morphology to isolate a subpopulation of cells (FL1(+)) from human glioma or glioma cultures. FL1(+) cells are capable of self-renewal in vitro, tumorigenesis in vivo and preferentially express stem cell genes. The FL1(+) phenotype did not correlate with the expression of proposed GIC markers. Our data propose an alternative approach to investigate tumor-initiating potential in gliomas and to advance the development of new therapies and diagnostics.

EZH2 is essential for glioblastoma cancer stem cell maintenance.

Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) occurs in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma multiforme (GBM). Based on its ability to modulate transcription of key genes implicated in cell cycle control, DNA repair, and cell differentiation, EZH2 is believed to play a crucial role in tissue-specific stem cell maintenance and tumor development. Here, we show that targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep), or its specific downregulation by short hairpin RNA (shRNA), strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. Using genome-wide expression analysis of DZNep-treated GBM CSCs, we found the expression of c-myc, recently reported to be essential for GBM CSCs, to be strongly repressed upon EZH2 depletion. Specific shRNA-mediated downregulation of EZH2 in combination with chromatin immunoprecipitation experiments revealed that c-myc is a direct target of EZH2 in GBM CSCs. Taken together, our observations provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a novel mechanism underlying GBM CSC maintenance and suggest that EZH2 may be a valuable new therapeutic target for GBM management.

Limits of CD133 as a marker of glioma self-renewing cells.

In human gliomas, self-renewing and tumor-initiating cells are characterized by the expression marker CD133. Although, widely used, the validity of CD133 is debated as recent data show that CD133(+) and CD133(-) cells share similar stemness and tumorigenic properties. To clarify this "CD133 controversy", we reexamined the methods of purification and the stem behavior of both CD133 compartments in fresh gliomas and gliomasphere cultures. Using human anti-CD133-coupled microbeads and magnetic activated cell sorting, we observed a nonspecific sorting of glioma cells irrespective of their CD133 expression. In contrast, when purified by fluorescence activating cell sorting, a specific expression and enrichment of CD133 was successfully observed in fresh human gliomas and gliomasphere cultures. However, neither the expression of stemness genes nor the long-term self-renewal capacities of CD133(+) and CD133(-) cells were significantly different, even after fresh isolation. Altogether, our data show that purification of CD133(+) glioma cells using hCD133-microbeads presents a lack of specificity and demonstrate that the use of CD133 as a unique glioma stem cell marker is likely not sufficient to tag the whole self-renewing tumor cell reservoir.

Urinary alpha1-antichymotrypsin: a biomarker of prion infection.

The occurrence of blood-borne prion transmission incidents calls for identification of potential prion carriers. However, current methods for intravital diagnosis of prion disease rely on invasive tissue biopsies and are unsuitable for large-scale screening. Sensitive biomarkers may help meeting this need. Here we scanned the genome for transcripts elevated upon prion infection and encoding secreted proteins. We found that alpha(1)-antichymotrypsin (alpha(1)-ACT) was highly upregulated in brains of scrapie-infected mice. Furthermore, alpha(1)-ACT levels were dramatically increased in urine of patients suffering from sporadic Creutzfeldt-Jakob disease, and increased progressively throughout the disease. Increased alpha(1)-ACT excretion was also found in cases of natural prion disease of animals. Therefore measurement of urinary alpha(1)-ACT levels may be useful for monitoring the efficacy of therapeutic regimens for prion disease, and possibly also for deferring blood and organ donors that may be at risk of transmitting prion infections.

Experimental autoimmune encephalomyelitis repressed by microglial paralysis.

Although microglial activation occurs in inflammatory, degenerative and neoplastic central nervous system (CNS) disorders, its role in pathogenesis is unclear. We studied this question by generating CD11b-HSVTK transgenic mice, which express herpes simplex thymidine kinase in macrophages and microglia. Ganciclovir treatment of organotypic brain slice cultures derived from CD11b-HSVTK mice abolished microglial release of nitrite, proinflammatory cytokines and chemokines. Systemic ganciclovir administration to CD11b-HSVTK mice elicited hematopoietic toxicity, which was prevented by transfer of wild-type bone marrow. In bone marrow chimeras, ganciclovir blocked microglial activation in the facial nucleus upon axotomy and repressed the development of experimental autoimmune encephalomyelitis. We conclude that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions. The microglial compartment thus provides a potential therapeutic target in inflammatory CNS disorders. These results validate CD11b-HSVTK mice as a tool to study the impact of microglial activation on CNS diseases in vivo.