Publications by authors named "Candy Kumps"

28 Publications

  • Page 1 of 1

IQSEC2 disorder: A new disease entity or a Rett spectrum continuum?

Clin Genet 2021 Mar 9;99(3):462-474. Epub 2021 Jan 9.

Medical Genetics, University of Siena, Siena, Italy.

IQSEC2 mutations are associated with IQSEC2-related intellectual disability (ID). Phenotypic spectrum has been better defined in the last few years by the increasing number of reported cases although the genotype-phenotype relationship for IQSEC2 remains overall complex. As for IQSEC2-related ID a wide phenotypic diversity has been described in Rett syndrome (RTT). Several patients harboring IQSEC2 mutations present with clinical symptoms similar to RTT and some cases meet most of the criteria for classic RTT. With the aim of establishing a genotype-phenotype correlation, we collected data of 16 patients harboring IQSEC2 point mutations (15 of them previously unreported) and of five novel patients carrying CNVs encompassing IQSEC2. Most of our patients surprisingly shared a moderate-to-mild phenotype. The similarities in the clinical course between our mild cases and patients with milder forms of atypical RTT reinforce the hypothesis that also IQSEC2 mutated patients may lay under the wide clinical spectrum of RTT and thus IQSEC2 should be considered in the differential diagnosis. Our data confirm that position, type of variant and gender are crucial for IQSEC2-associated phenotype delineation.
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http://dx.doi.org/10.1111/cge.13908DOI Listing
March 2021

Phenotypic spectrum of the RBM10-mediated intellectual disability and congenital malformation syndrome beyond classic TARP syndrome features.

Clin Genet 2021 Mar 5;99(3):449-456. Epub 2021 Jan 5.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Pathogenic variants in the RBM10 gene cause a rare X-linked disorder described as TARP (Talipes equinovarus, Atrial septal defect, Robin sequence, and Persistent left vena cava superior) syndrome. We report two novel patients with truncating RBM10 variants in view of the literature, presenting a total of 26 patients from 15 unrelated families. Our results illustrate the highly pleiotropic nature of RBM10 pathogenic variants, beyond the classic TARP syndrome features. Major clinical characteristics include severe developmental delay, failure to thrive, brain malformations, neurological symptoms, respiratory issues, and facial dysmorphism. Minor features are growth retardation, cardiac, gastrointestinal, limb, and skeletal abnormalities. Additional recurrent features include genital and renal abnormalities as well as hearing and visual impairment. Thus, RBM10 loss of function variants typically cause an intellectual disability and congenital malformation syndrome that requires assessment of multiple organ systems at diagnosis and for which provided clinical features might simplify diagnostic assessment. Furthermore, evidence for an RBM10-related genotype-phenotype correlation is emerging, which can be important for prognosis.
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http://dx.doi.org/10.1111/cge.13901DOI Listing
March 2021

Phenotyping nocturnal polyuria: circadian and age-related variations in diuresis rate, free water clearance and sodium clearance.

Age Ageing 2020 04;49(3):439-445

Department of Urology, Ghent University Hospital, Ghent, Belgium.

Background: this study compares diuresis rate, sodium clearance and free water clearance (FWC) by age and time of day (nighttime vs. daytime) in subjects with and without nocturnal polyuria (NP) to determine whether these variables affect the phenotype of NP.

Methods: post hoc analysis of two prospective observational studies. Eight urine samples collected at 3-h intervals and a single blood sample were used to calculate daytime (10a/1p/4p/7p/10p) and nighttime (1a/4a/7a) diuresis rates, sodium clearance and FWC. Three mixed linear models were constructed for diuresis rate, sodium clearance and FWC using four predictor variables: NP status (present [nocturnal urine production >90 ml/h] vs. absent [≤90 ml/h]), time of day, age and study identification.

Results: subjects with NP experienced higher nighttime versus daytime diuresis rates, sodium clearance and FWC. Regardless of NP status, increased age was accompanied by an increase in the ratio of nighttime/daytime diuresis rate, nighttime sodium clearance and daytime sodium clearance. FWC showed a complex age effect, which was independent of time of day or NP status.

Conclusions: age-related increases in nighttime/daytime diuresis rate, 24-h sodium clearance and 24-h FWC are not specific to subjects with NP. The age-related surge in either nocturnal sodium clearance or nocturnal FWC may represent the relevant substrate for behavioural or pharmacologic interventions targeting sodium diuresis or free water diuresis, respectively. Increases in FWC in older age groups may reflect impaired circadian rhythmicity of endogenous AVP or changes in responsiveness of the aged nephron to water clearance.
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http://dx.doi.org/10.1093/ageing/afz200DOI Listing
April 2020

The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness.

Sci Rep 2020 01 14;10(1):218. Epub 2020 Jan 14.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Neuroblastoma is an aggressive childhood cancer arising from sympatho-adrenergic neuronal progenitors. The low survival rates for high-risk disease point to an urgent need for novel targeted therapeutic approaches. Detailed molecular characterization of the neuroblastoma genomic landscape indicates that ALK-activating mutations are present in 10% of primary tumours. Together with other mutations causing RAS/MAPK pathway activation, ALK mutations are also enriched in relapsed cases and ALK activation was shown to accelerate MYCN-driven tumour formation through hitherto unknown ALK-driven target genes. To gain further insight into how ALK contributes to neuroblastoma aggressiveness, we searched for known oncogenes in our previously reported ALK-driven gene signature. We identified ETV5, a bona fide oncogene in prostate cancer, as robustly upregulated in neuroblastoma cells harbouring ALK mutations, and show high ETV5 levels downstream of the RAS/MAPK axis. Increased ETV5 expression significantly impacted migration, invasion and colony formation in vitro, and ETV5 knockdown reduced proliferation in a murine xenograft model. We also established a gene signature associated with ETV5 knockdown that correlates with poor patient survival. Taken together, our data highlight ETV5 as an intrinsic component of oncogenic ALK-driven signalling through the MAPK axis and propose that ETV5 upregulation in neuroblastoma may contribute to tumour aggressiveness.
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http://dx.doi.org/10.1038/s41598-019-57076-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959226PMC
January 2020

Nocturnal voiding frequency does not describe nocturia-related bother.

Neurourol Urodyn 2019 08 4;38(6):1648-1656. Epub 2019 Jun 4.

Department of Medicine & Aged Care, Royal Melbourne Hospital, Parkville, Victoria, Australia.

Aim: Nocturia frequency has been used as a measure of treatment efficacy for nocturia even though fluctuation of the symptom over time has been well described in the literature. Additionally, given the multifactorial causal pathway and clinically relevant comorbidities, frequency alone may be an insufficient marker of treatment direction. The aim of this study was to investigate factors associated with nocturia-related bother to identify additional variables that may capture the impact of nocturia, direct clinical care and have potential to quantify treatment outcome.

Methods: Prospective data from tertiary hospital Urology and Continence cohorts were matched for identical variables to generate a sample of 204 datasets. Descriptive statistics were obtained to describe the two cohorts. Characteristics of patients were evaluated across levels of nocturia frequency and nocturia-related bother using nonparametric methods; statistically significant differences between groups in each cohort were established.

Results: Nocturia frequency alone does not comprehensively reflect attributable bother. Five sleep variables (poor quality sleep, short time to first awakening to void, less than 7 hours of total sleep, primary sleep latency, and daytime sleepiness) and daily urinary urgency were significantly associated with high nocturia-related bother. Attributable bother, despite high-frequency nocturia, was minimized by male gender, lack of daily urinary urgency and good sleep quality. Poor health status, urinary urgency and sleep latency were associated with nocturia frequency.

Conclusions: Items of importance to individuals with nocturia have been identified from patient data. These variables have the potential to sit alongside change in nocturia frequency as potential markers of treatment response.
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http://dx.doi.org/10.1002/nau.24029DOI Listing
August 2019

ALK positively regulates MYCN activity through repression of HBP1 expression.

Oncogene 2019 04 11;38(15):2690-2705. Epub 2018 Dec 11.

Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PIK-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PIK antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41388-018-0595-3DOI Listing
April 2019

Smart diapers for nursing home residents with dementia: a pilot study.

Acta Clin Belg 2019 Aug 27;74(4):258-262. Epub 2018 Aug 27.

d Department of Urology , Ghent University Hospital , Ghent , Belgium.

: The objective of the study is to evaluate the use of an experimental smart diaper as an indicator of saturation for diaper change in persons with dementia living in nursing homes. : A multicenter prospective study was conducted in 3 nursing homes amongst 18 residents with dementia. For each resident, a frequency-volume urine chart (FVUC) was kept for 24 h including voided volume and diaper weights, wearing smart diapers. A comparative study was set up between results obtained by smart diapers and data registered in FVUCs. : Analysis based on quantification of the agreement between saturation calculated by smart diaper and determined by FVUC indicates that measurements reported by sensor do not correspond with measurements based on FVUC. For the regular diaper, the saturation measured by sensor may be 26% below or 39% above saturation based on FVUC and for the super diaper, respectively, 34% below or 30% above. : This study indicates that the sensor detects and notifies wetness but is not sensitive enough for using it as an indicator for diaper change in people with severe dementia.
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http://dx.doi.org/10.1080/17843286.2018.1511279DOI Listing
August 2019

Prognostic and Therapeutic Implications of Circulating Androgen Receptor Gene Copy Number in Prostate Cancer Patients Using Droplet Digital Polymerase Chain Reaction.

Clin Genitourin Cancer 2018 06 29;16(3):197-205.e5. Epub 2017 Dec 29.

Department of Urology, Ghent University Hospital, Ghent, Belgium.

Background: Resistance mechanisms in the androgen receptor (AR) signaling pathway remain key drivers in the progression to castration-resistant prostate cancer (CRPC) and relapse under antihormonal therapy.

Materials And Methods: We evaluated the circulating AR gene copy number (CN) gain using droplet digital polymerase chain reaction in 21 control and 91 prostate cancer serum samples and its prognostic and therapeutic implications in prostate cancer.

Results: In CRPC, AR CN gain was associated with faster progression to CRPC (P = .026), a greater number of previous treatments (P = .045), and previous chemotherapy (P = .016). Comparing patients with and without CN gain, the median progression-free survival (PFS) in the abiraterone subgroup was 1.7 months versus not reached (P = .004), and the median overall survival (OS) was 7 months versus 20.9 months (P = .020). In the enzalutamide subgroup, PFS was 1.7 versus 10.8 months (P = .006), and OS was 6.1 versus 16.5 months (P = .042). In the taxane subgroup, PFS was 3.2 versus 6.5 months (P = .093), and OS was 3.9 months versus not reached (P = .026). The presence of more AR copies correlated with shorter androgen deprivation (P = .002), abiraterone (P = .022), enzalutamide (P = .008), and taxane (P = .039) therapy.

Conclusion: Circulating AR CN gain predicts for a poor prognosis in CRPC. It is a promising biomarker predetermining rapid CRPC progression and predicting worse abiraterone and enzalutamide outcomes. Furthermore, it is associated with multiple previous treatments and previous chemotherapy.
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http://dx.doi.org/10.1016/j.clgc.2017.12.008DOI Listing
June 2018

Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

Oncotarget 2017 Dec 6;8(63):106820-106832. Epub 2017 Nov 6.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Background: Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours after TAE684 administration, resulting in the 77-gene ALK signature, which was shown to gradually decrease from 120 minutes after TAE684 treatment, to gain deeper insight into the molecular effects of oncogenic ALK signaling.

Aim: Here, we further dissected the transcriptional dynamic profiles of neuroblastoma cells upon TAE684 treatment in a detailed timeframe of ten minutes up to six hours after inhibition, in order to identify additional early targets for combination treatment.

Results: We observed an unexpected initial upregulation of positively regulated MYCN target genes following subsequent downregulation of overall MYCN activity. In addition, we identified adrenomedullin (ADM), previously shown to be implicated in sunitinib resistance, as the earliest response gene upon ALK inhibition.

Conclusions: We describe the early and late effects of ALK inhibitor TAE684 treatment on the neuroblastoma transcriptome. The observed unexpected upregulation of ADM warrants further investigation in relation to putative ALK resistance in neuroblastoma patients currently undergoing ALK inhibitor treatment.
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http://dx.doi.org/10.18632/oncotarget.22423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739776PMC
December 2017

Clean Intermittent Self-Catheterization as a Treatment Modality for Urinary Retention: Perceptions of Urologists.

Int Neurourol J 2017 Sep 12;21(3):189-196. Epub 2017 Sep 12.

Urology Department, Ghent University Hospital, Ghent, Belgium.

Purpose: Clean intermittent self-catheterization (CISC) is now considered the gold standard for the management of urinary retention. In the literature, several articles on patients' perspectives on CISC and adherence to this technique have been published. No studies have yet explored the points of view of professional caregivers, such as nurses and doctors. The aim of this study was to explore the opinions of urologists about CISC and to evaluate the need for dedicated nurses specialized in CISC through a self-administered questionnaire.

Methods: A questionnaire was developed to explore the opinions of professional caregivers about self-catheterization and to evaluate the need to provide nurses with specialized education in CISC. Questionnaires were sent to 244 urologists through email. We received 101 completed questionnaires. The response rate was 41.4%.

Results: Hand function, the presence or absence of tremor, and visual acuity were rated as the most important determinants for proposing CISC to a patient. Twenty-five percent of the urologists reported that financial remuneration would give them a greater incentive to propose CISC. The lack of dedicated nurses was reported by half of the urologists as a factor preventing them from proposing CISC. A meaningful number of urologists thought that patients perceive CISC as invasive and unpleasant. Although most urologists would choose CISC as a treatment option for themselves, almost 1 urologist out of 5 would prefer a permanent catheter.

Conclusions: This questionnaire gave valuable insights into urologists' perceptions of CISC, and could serve as the basis for a subsequent broader international study. Further research should also focus on the opinions of nurses and other caregivers involved in incontinence management. Apart from financial remuneration, it is also clear that ensuring sufficient expertise and time for high-quality CISC care is important. This could be a potential role for dedicated nurses.
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http://dx.doi.org/10.5213/inj.1734824.412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636956PMC
September 2017

Pathophysiology of nocturnal lower urinary tract symptoms in older patients with urinary incontinence.

Int J Urol 2017 11 16;24(11):808-815. Epub 2017 Aug 16.

Department of Urology, Ghent University Hospital, Ghent, Belgium.

Objectives: To explore the mismatch between functional bladder capacity and nocturnal urine production, and to study the pathophysiology of an increased nocturnal urine production in older patients with urinary incontinence.

Methods: The present prospective observational study included adults aged ≥65 years with urinary incontinence. Participants completed questionnaires, frequency volume charts and renal function profiles. The nocturnal lower urinary tract symptom index was defined as nocturnal urine output/maximum voided volume; the nocturnal polyuria index as nocturnal/24 h urine output.

Results: The median age (n = 95) was 74 years (69-79), 87% were women and 73% had nocturnal lower urinary tract symptoms (nocturnal urinary incontinence or nocturia ≥2). Participants with nocturnal lower urinary tract symptoms had a significantly higher nocturnal urine output (809 mL vs 650 mL; P = 0.001) and no significant difference in maximum voided volume (350 mL vs 437 mL; P = 0.079) compared with participants without nocturnal lower urinary tract symptoms. Participants (nocturnal polyuria index >33% [n = 56], nocturnal polyuria index >40% [n = 42], nocturnal lower urinary tract symptom index >1.87 [n = 51]) showed higher night-time diuresis rates, free water and sodium clearance compared with during the daytime. Controls (nocturnal polyuria index ≤33% [n = 26], nocturnal polyuria index ≤40% [n = 40], nocturnal lower urinary tract symptom index ≤1.87 [n = 44]) had no circadian rhythm in their diuresis rate or sodium clearance, but more nocturnal free water clearance compared with during the daytime.

Conclusions: The majority of older adults with urinary incontinence present nocturnal lower urinary tract symptoms. An increased nocturnal sodium diuresis seems to be the only mechanism differentiating patients with nocturnal lower urinary tract symptoms from controls.
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http://dx.doi.org/10.1111/iju.13431DOI Listing
November 2017

The independent oncological role for cytoreductive nephrectomy in metastatic renal cell carcinoma: Prognostic features in the era of targeted therapies.

Urol Oncol 2017 04 30;35(4):152.e13-152.e22. Epub 2017 Jan 30.

Department of Urology, Ghent University Hospital, Gent, Belgium.

Objectives: To describe the effects of cytoreductive nephrectomy (CN) on the natural course of metastatic renal cell carcinoma (mRCC). CN appears to stabilize metastatic lesions in mRCC in a subgroup of patients and we hypothesize that systemic treatment might be deferred in these patients with stable disease after CN.

Subjects And Methods: Overall, 45 patients with mRCC who underwent CN and subsequent oncologic follow-up were included in this retrospective, single-center analysis. After CN, patients were followed at least every 3 months with clinical evaluation, contrast-enhanced computerized tomography scan of chest and abdomen, with additional imaging if needed. At 3 months, patients were radiographically evaluated and categorized into nonresponders (death or progression) or responders (stable disease or remission). Kaplan-Meier and Cox proportional hazards regression statistics were used to describe prognostic factors for overall survival (OS) and systemic therapy-free survival (STFS).

Results: Median OS was 31(3-121) months. Further, 24 (53.3%) and 21 (46.7%) patients were classified as responders and nonresponders at 3 months, respectively. Responders had a significant better 2-year OS compared with nonresponders (81.7% vs. 26.5%, P = 0.005). Responders also had a better 2-year STFS (40.3% vs. 6.3%, P = 0.005). On Cox regression analysis, worse OS was found to be associated with low preoperative hemoglobin levels, the absence of postoperative radiographical response, and the presence of non-clear cell pathology. The presence of postoperative radiographical response, normal preoperative lactate dehydrogenase levels, the presence of a single metastasis, and performing metastasis-directed therapy was found to be associated with a longer systemic therapy-free period.

Conclusion: A beneficial oncologic response is observed in approximately half of the patients undergoing CN. Absence of radiographic progression at 3 months is an important marker for OS and STFS. Therefore, systemic treatment might be postponed in selected patients.
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http://dx.doi.org/10.1016/j.urolonc.2016.10.013DOI Listing
April 2017

Circadian Variation in Post Void Residual in Nursing Home Residents With Moderate Impairment in Activities of Daily Living.

J Am Med Dir Assoc 2017 May 17;18(5):433-437. Epub 2017 Jan 17.

Department of Urology, Ghent University Hospital, Ghent, Belgium.

Background: Despite the conflicting evidence about postvoid residual (PVR) and its variation in time and corresponding voided volume (VV), studies with urinary diaries and systematic measurements of PVR after each void have never been conducted in nursing home (NH) residents.

Objective: To describe the circadian rhythm of PVR and residual fraction (RF, the net quantity of PVR) and to identify the time window with the highest PVR and RF.

Design, Setting, And Participants: A multicentre prospective study conducted between 2014 and 2015 in 5 Belgian NHs. A convenience sample of cognitively intact residents completed a 24-hour frequency volume chart with PVR.

Results: Participants (n = 73) had a median age of 84 years (interquartile range 82-89) and moderate impairment of activities of daily living; 69% were women. In residents with nocturia, mean PVR was higher during the night [45 mL (26-80)] than during the day [36 mL (18-61)]. In residents without nocturia no difference was detected. In spite of the variation between diurnal and nocturnal VV and PVR in residents with nocturia, all residents emptied their bladder as effectively during daytime as during nighttime [mean RF = 20% (12-32)]. Maximum PVR and RF in residents with nocturia (n = 57) showed a circadian variation. The highest PVR and RF were found during the day. The PVR and RF of the first morning void were an indicator of the maximum nocturnal PVR and RF.

Conclusions: PVR and VV should be measured in NH residents during the waking hours (first morning void excepted) to detect the clinically relevant maximum PVR and RF.
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http://dx.doi.org/10.1016/j.jamda.2016.11.022DOI Listing
May 2017

Androgen Receptor Gene Copy Number and Protein Expression in Treatment-Naïve Prostate Cancer.

Urol Int 2017 5;99(2):222-228. Epub 2017 Jan 5.

Department of Urology, Ghent University Hospital, Ghent, Belgium.

Objectives: To evaluate the androgen receptor (AR) gene copy number in androgen deprivation therapy (ADT) treatment-naïve prostate cancer (PCa) patients and to evaluate the corresponding AR protein expression and assess the association between these features and prognostic factors.

Materials And Methods: Chromosome X and AR gene copy number, using fluorescence-in-situ-hybridization, and epithelial-stromal AR expression, using AR immunohistochemistry, were analyzed in 62 ADT treatment-naïve PCa patients and 8 castration-refractory patients.

Results: In ADT treatment-naïve PCa patients, the AR expression was higher in tumor epithelial cells versus surrounding stromal cells (p < 0.001) and versus normal epithelium in the same patient (p = 0.043). The difference between tumoral AR expression and expression in normal epithelium was higher in patients with ≥15% of tumor cells with increased AR copy number (p = 0.019). Peritumoral stroma had lower AR expression in patients with lymph-node or distant metastases compared to those without metastases (p = 0.038).

Conclusions: This research evaluates the link between AR gene status, expression profile, and possible prognostic factors. Furthermore, it highlights the importance of the peritumoral environment in PCa. Additional research is needed to further clarify the role of stromal AR in PCa dissemination and identify possible therapeutic strategies to target this mechanism.
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http://dx.doi.org/10.1159/000455158DOI Listing
June 2018

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment.

Clin Cancer Res 2015 Jul 24;21(14):3327-39. Epub 2015 Mar 24.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Purpose: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies.

Experimental Design: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALK(F1174L) or ALK(R1275Q) hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALK(F1174L) double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas.

Results: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALK(F1174L) and ALK(R1275Q) regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage.

Conclusions: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-2024DOI Listing
July 2015

Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma.

Oncotarget 2014 May;5(9):2688-702

Inserm U830, 26 rue d'Ulm, 75005 Paris, France.

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058037PMC
http://dx.doi.org/10.18632/oncotarget.1883DOI Listing
May 2014

The need for transparency and good practices in the qPCR literature.

Nat Methods 2013 Nov;10(11):1063-7

Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford, UK.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
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http://dx.doi.org/10.1038/nmeth.2697DOI Listing
November 2013

Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.

PLoS One 2013 14;8(8):e71776. Epub 2013 Aug 14.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.

Results: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5).

Conclusions: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743747PMC
April 2014

Focal DNA copy number changes in neuroblastoma target MYCN regulated genes.

PLoS One 2013 4;8(1):e52321. Epub 2013 Jan 4.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052321PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537730PMC
July 2013

Targeted expression of mutated ALK induces neuroblastoma in transgenic mice.

Sci Transl Med 2012 Jul;4(141):141ra91

Institute of Pathology, University Hospital Cologne, Kerpenerstrasse 62, 50924 Cologne, Germany.

Activating anaplastic lymphoma kinase (ALK) mutations were recently detected in most familial and 10% of sporadic neuroblastomas. However, the role of mutated ALK in tumorigenesis remains elusive. We demonstrate that targeted expression of the most frequent and aggressive variant, ALK(F1174L), is tumorigenic in mice. Tumors resembled human neuroblastomas in morphology, metastasis pattern, gene expression, and the presence of neurosecretory vesicles as well as synaptic structures. This ALK-driven neuroblastoma mouse model precisely recapitulated the genetic spectrum of the disease. Chromosomal aberrations were syntenic to those in human neuroblastoma, including 17q gain and MYCN oncogene amplification. Targeted ALK(F1174L) and MYCN coexpression revealed a strong synergism in inducing neuroblastoma with minimal chromosomal aberrations, suggesting that fewer secondary hits are required for tumor induction if both oncoproteins are targeted. Treatment of ALK(F1174L) transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALK(F1174L) activity. We conclude that an activating mutation within the ALK kinase domain is sufficient to induce neuroblastoma development, and ALK inhibitors show promise for treating human neuroblastomas harboring ALK mutations.
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http://dx.doi.org/10.1126/scitranslmed.3003967DOI Listing
July 2012

Identification of tumoral glial precursor cells in neuroblastoma.

Cancer Lett 2011 Dec 17;312(1):73-81. Epub 2011 Aug 17.

Developmental Tumor Biology Laboratory, Hospital Sant Joan de Déu, Fundació Sant Joan de Déu, Barcelona, Spain.

Neuroblastic tumors (NBT) are composed by neuroblasts and Schwannian-like stroma. The origin of these two cell subtypes remains unclear. In this study, we describe, a neuroblastic-like subpopulation in neuroblastoma (NB) coexpressing GD2 and S100A6, neuroblastic and glial lineage markers respectively. The GD2(+)/S100A6(+) neuroblastic subpopulation was found to be enriched in low risk NB, distributed around the perivascular niche. Some stromal bundles showed GD2(+)/S100A6 costaining. Metastatic bone marrow specimens also showed GD2(+)/S100A6(+) cells. During in vitro retinoic acid induced differentiation of NB cell lines, rare GD2(+)/S100A6 neuroblatic cells appeared. We conclude that GD2(+)/S100A6(+) neuroblasts may represent a tumoral glial precursor subpopulation in NBT.
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http://dx.doi.org/10.1016/j.canlet.2011.08.004DOI Listing
December 2011

Identification of a novel recurrent 1q42.2-1qter deletion in high risk MYCN single copy 11q deleted neuroblastomas.

Int J Cancer 2012 Jun 29;130(11):2599-606. Epub 2011 Aug 29.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Neuroblastoma is an aggressive embryonal tumor that accounts for ∼15% of childhood cancer deaths. Hitherto, despite the availability of comprehensive genomic data on DNA copy number changes in neuroblastoma, relatively little is known about the genes driving neuroblastoma tumorigenesis. In this study, high resolution array comparative genome hybridization (CGH) was performed on 188 primary neuroblastoma tumors and 33 neuroblastoma cell lines to search for previously undetected recurrent DNA copy number gains and losses. A new recurrent distal chromosome 1q deletion (del(1)(q42.2qter)) was detected in seven cases. Further analysis of available array CGH datasets revealed 13 additional similar distal 1q deletions. The majority of all detected 1q deletions was found in high risk 11q deleted tumors without MYCN amplification (Fisher exact test p = 5.61 × 10(-5) ). Using ultra-high resolution (∼115 bp resolution) custom arrays covering the breakpoints on 1q for 11 samples, clustering of nine breakpoints was observed within a 12.5-kb region, of which eight were found in a 7-kb copy number variable region, whereas the remaining two breakpoints were colocated 1.4-Mb proximal. The commonly deleted region contains one miRNA (hsa-mir-1537), four transcribed ultra conserved region elements (uc.43-uc.46) and 130 protein coding genes including at least two bona fide tumor suppressor genes, EGLN1 (or PHD2) and FH. This finding further contributes to the delineation of the genomic profile of aggressive neuroblastoma, offers perspectives for the identification of genes contributing to the disease phenotype and may be relevant in the light of assessment of response to new molecular treatments.
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http://dx.doi.org/10.1002/ijc.26317DOI Listing
June 2012

Isolation of disseminated neuroblastoma cells from bone marrow aspirates for pretreatment risk assessment by array comparative genomic hybridization.

Int J Cancer 2012 Mar 9;130(5):1098-108. Epub 2011 Jun 9.

Faculty of Pharmaceutical Sciences, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.

In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior.
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http://dx.doi.org/10.1002/ijc.26133DOI Listing
March 2012

The quassinoid derivative NBT-272 targets both the AKT and ERK signaling pathways in embryonal tumors.

Mol Cancer Ther 2010 Dec 1;9(12):3145-57. Epub 2010 Oct 1.

Department of Oncology, University Children's Hospital of Zurich, Switzerland.

The quassinoid analogue NBT-272 has been reported to inhibit MYC, thus warranting a further effort 7to better understand its preclinical properties in models of embryonal tumors (ET), a family of childhood malignancies sharing relevant biological and genetic features such as deregulated expression of MYC oncogenes. In our study, NBT-272 displayed a strong antiproliferative activity in vitro that resulted from the combination of diverse biological effects, ranging from G(1)/S arrest of the cell cycle to apoptosis and autophagy. The compound prevented the full activation of both eukaryotic translation initiation factor 4E (eIF4E) and its binding protein 4EBP-1, regulating cap-dependent protein translation. Interestingly, all responses induced by NBT-272 in ET could be attributed to interference with 2 main proproliferative signaling pathways, that is, the AKT and the MEK/extracellular signal-regulated kinase pathways. These findings also suggested that the depleting effect of NBT-272 on MYC protein expression occurred via indirect mechanisms, rather than selective inhibition. Finally, the ability of NBT-272 to arrest tumor growth in a xenograft model of neuroblastoma plays a role in the strong antitumor activity of this compound, both in vitro and in vivo, with its potential to target cell-survival pathways that are relevant for the development and progression of ET.
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http://dx.doi.org/10.1158/1535-7163.MCT-10-0539DOI Listing
December 2010

Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

Clin Cancer Res 2010 Sep 18;16(17):4353-62. Epub 2010 Aug 18.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression.

Experimental Design: The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis.

Results: ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival.

Conclusions: ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-2660DOI Listing
September 2010

Multiplex Amplicon Quantification (MAQ), a fast and efficient method for the simultaneous detection of copy number alterations in neuroblastoma.

BMC Genomics 2010 May 12;11:298. Epub 2010 May 12.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Background: Cancer genomes display characteristic patterns of chromosomal imbalances, often with diagnostic and prognostic relevance. Therefore assays for genome-wide copy number screening and simultaneous detection of copy number alterations in specific chromosomal regions are of increasing importance in the diagnostic work-up of tumors.

Results: We tested the performance of Multiplex Amplicon Quantification, a newly developed low-cost, closed-tube and high-throughput PCR-based technique for detection of copy number alterations in regions with prognostic relevance for neuroblastoma. Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method on 52 neuroblastoma tumors showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations.

Conclusion: Multiplex Amplicon Quantification is a low-cost and high-throughput PCR-based technique that can reliably detect copy number alterations in regions with prognostic relevance for neuroblastoma.
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http://dx.doi.org/10.1186/1471-2164-11-298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879279PMC
May 2010

Smoothing waves in array CGH tumor profiles.

Bioinformatics 2009 May 10;25(9):1099-104. Epub 2009 Mar 10.

Department of Epidemiology & Biostatistics, VU University Medical Center, PO Box 7057, 1007MB Amsterdam, The Netherlands.

Motivation: Many high-resolution array comparative genomic hybridization tumor profiles contain a wave bias, which makes accurate detection of breakpoints in such profiles more difficult.

Results: An efficient and highly effective algorithm that largely removes the wave bias from tumor profiles by regressing the tumor profile data on data of profiles from the clinical genetics practice. Results are illustrated on two independent datasets. The algorithm is shown to be robust against the presence of true copy number aberrations. Moreover, the smoothed profiles are able to recapitulate the aberration location and signal for simulated tumor profiles.

Availability: Easy-to-use R scripts, user instructions and data are available from http://www.few.vu.nl/~mavdwiel/nowaves.html.

Supplementary Information: Supplementary information are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btp132DOI Listing
May 2009

Low-cost dedicated mini-arrays for high-throughput analysis of DNA copy-number alterations in neuroblastoma.

Cancer Lett 2008 Sep 13;269(1):111-6. Epub 2008 Jun 13.

Center for Medical Genetics Ghent, Ghent University Hospital, 2nd Floor, Room 120.038, De Pintelaan 185, MRB2, 9000 Ghent, Belgium.

ArrayCGH is commonly used for high-resolution detection of copy-number alterations in tumours, allowing identification of chromosomal aberrations with prognostic or diagnostic relevance. Currently available arrayCGH platforms are still very expensive for analysis of large sets of samples. For this purpose, we have constructed a dedicated mini-array that is enriched for BAC/PAC clones in the prognostic important regions for neuroblastoma and that only covers a small area on the slide, allowing down-scaling of the labelling and hybridisation reagents and hence reducing the price. The mini-arrays were validated on neuroblastoma samples and comparison with high-resolution whole-genome arrayCGH data yielded complete concordant results.
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http://dx.doi.org/10.1016/j.canlet.2008.04.031DOI Listing
September 2008