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Abstract Pediatric central nervous system tumors remain challenging to diagnose. Imaging approaches do not provide sufficient detail to discriminate between different tumor types, while the histopathological examination of tumor tissue shows high inter-observer variability. Recent studies have demonstrated the accurate classification of central nervous system tumors based on the DNA methylation profile of a tumor biopsy. However, a brain biopsy holds significant risk of bleeding and damaging the surrounding tissues. Liquid biopsy approaches analyzing circulating tumor DNA show high potential as an alternative and less invasive tool to study the DNA methylation pattern of tumors. Here, we explore the potential of classifying pediatric brain tumors based on methylation profiling of the circulating cell-free DNA (cfDNA) in cerebrospinal fluid (CSF). For this proof-of-concept study, we collected cerebrospinal fluid samples from 19 pediatric brain cancer patients via a ventricular drain placed for reasons of increased intracranial pressure. Analyses on the cfDNA showed high variability of cfDNA quantities across patients ranging from levels below the limit of quantification to 40 ng cfDNA per milliliter of CSF. Classification based on methylation profiling of cfDNA from CSF was correct for 7 out of 20 samples in our cohort. Accurate results were mostly observed in samples of high quality, more specifically those with limited high molecular weight DNA contamination. Interestingly, we show that centrifugation of the CSF prior to processing increases the fraction of fragmented cfDNA to high molecular weight DNA. In addition, classification was mostly correct for samples with high tumoral cfDNA fraction as estimated by computational deconvolution (> 40%). In summary, analysis of cfDNA in the CSF shows potential as a tool for diagnosing pediatric nervous system tumors especially in patients with high levels of tumoral cfDNA in the CSF. Further optimization of the collection procedure, experimental workflow and bioinformatic approach is required to also allow classification for patients with low tumoral fractions in the CSF.

Abstract Objectives Cytogenetic abnormalities are predictors of poor prognosis in multiple myeloma (MM). This paper aims to build and validate a multiparametric conventional and functional whole-body MRI-based prediction model for cytogenetic risk classification in newly diagnosed MM. Methods Patients with newly diagnosed MM who underwent multiparametric conventional whole-body MRI, spinal dynamic contrast-enhanced (DCE-)MRI, spinal diffusion-weighted MRI (DWI) and had genetic analysis were retrospectively included (2011–2020/Ghent University Hospital/Belgium). Patients were stratified into standard versus intermediate/high cytogenetic risk groups. After segmentation, 303 MRI features were extracted. Univariate and model-based methods were evaluated for feature and model selection. Testing was performed using receiver operating characteristic (ROC) and precision-recall curves. Models comparing the performance for genetic risk classification of the entire MRI protocol and of all MRI sequences separately were evaluated, including all features. Four final models, including only the top three most predictive features, were evaluated. Results Thirty-one patients were enrolled (mean age 66 ± 7 years, 15 men, 13 intermediate-/high-risk genetics). None of the univariate models and none of the models with all features included achieved good performance. The best performing model with only the three most predictive features and including all MRI sequences reached a ROC-area-under-the-curve of 0.80 and precision-recall-area-under-the-curve of 0.79. The highest statistical performance was reached when all three MRI sequences were combined (conventional whole-body MRI + DCE-MRI + DWI). Conventional MRI always outperformed the other sequences. DCE-MRI always outperformed DWI, except for specificity. Conclusions A multiparametric MRI-based model has a better performance in the noninvasive prediction of high-risk cytogenetics in newly diagnosed MM than conventional MRI alone. Critical relevance statement An elaborate multiparametric MRI-based model performs better than conventional MRI alone for the noninvasive prediction of high-risk cytogenetics in newly diagnosed multiple myeloma; this opens opportunities to assess genetic heterogeneity thus overcoming sampling bias. Key points • Standard genetic techniques in multiple myeloma patients suffer from sampling bias due to tumoral heterogeneity. • Multiparametric MRI noninvasively predicts genetic risk in multiple myeloma. • Combined conventional anatomical MRI, DCE-MRI, and DWI had the highest statistical performance to predict genetic risk. • Conventional MRI alone always outperformed DCE-MRI and DWI separately to predict genetic risk. DCE-MRI alone always outperformed DWI separately, except for the parameter specificity to predict genetic risk. • This multiparametric MRI-based genetic risk prediction model opens opportunities to noninvasively assess genetic heterogeneity thereby overcoming sampling bias in predicting genetic risk in multiple myeloma. Graphical Abstract

Abstract T‐lineage acute lymphoblastic leukemia (T‐ALL) is an aggressive hematological malignancy that accounts for 10%–15% of pediatric and 25% of adult ALL cases. Although the prognosis of T‐ALL has improved over time, the outcome of T‐ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, further progress in the treatment of T‐ALL requires a better understanding of its biology and the development of more effective precision oncologic therapies. The proto‐oncogene MYB is highly expressed in diverse hematologic malignancies, including T‐ALLs with genomic aberrations that further potentiate its expression and activity. Previous studies have associated MYB with a malignant role in the pathogenesis of several cancers. However, its role in the induction and maintenance of T‐ALL remains relatively poorly understood. In this study, we found that an increased copy number of MYB is associated with higher MYB expression levels, and might be associated with inferior event‐free survival of pediatric T‐ALL patients. Using our previously described conditional Myb overexpression mice, we generated two distinct MYB‐driven T‐ALL mouse models. We demonstrated that the overexpression of Myb synergizes with Pten deletion but not with the overexpression of Lmo2 to accelerate the development of T‐cell lymphoblastic leukemias. We also showed that MYB is a dependency factor in T‐ALL since RNA interference of Myb blocked cell cycle progression and induced apoptosis in both human and murine T‐ALL cell lines. Finally, we provide preclinical evidence that targeting the transcriptional activity of MYB can be a useful therapeutic strategy for the treatment of T‐ALL.

The risk stratification of infants with metastatic neuroblastoma (NB) has evolved over time from stage 4/M or IVs/4S/MS/Ms according to various staging systems. Despite these developments for some genetic aberrations, the prognostic value and the impact of soft tissue metastases in infants are not fully understood, nor well described in the different classification systems, hampering the definitions to uniformly treat patients and predict prognosis. A literature review on staging of infants with M/MS disease was performed at the occasion of the diagnosis of NB in an 8-month-old boy who presented with atypical metastatic sites in soft tissue and an aberrant tumor biology. The definitions of stage 4/4S/4s/M/MS/Ms were evaluated and compared to enable tumor risk stratification and inform management. International NB groups use different criteria for defining stage of infants with metastasized NB, resulting in differences in management. Limited literature is available on soft tissue metastases, especially muscular metastases, and is poorly incorporated into management guidelines mainly due to the lack of data. The uncertain prognosis of rare genetic aberrancies may add to the difficulties in treatment decisions. In some rare cases of NB in infants, the international treatment classification is not sufficient for staging and treatment decisions. Based on tumor progression, biology of unknown significance and a lack of evidence to classify a child under 12 months with NB and multiple muscular metastases, the patient was treated as stage 4/M and intermediate-risk protocols with a favorable outcome.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

The development of neuroblastoma (NB) is regulated by multiple core transcription factors. Here, SOX11 is identified as a potential epigenetic master regulator upstream of the core regulatory circuitry in adrenergic high-risk neuroblastoma.

In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.

Abstract This report highlights the importance of integrating clinical, radiological, genetic, and pathological laboratory findings to make a correct diagnosis especially with challenging and rare entities.

Neuroblastoma is a pediatric tumor arising from the sympatho-adrenal lineage and a worldwide leading cause of childhood cancer-related deaths. About half of high-risk patients die from the disease while survivors suffer from multiple therapy-related side-effects. While neuroblastomas present with a low mutational burden, focal and large segmental DNA copy number aberrations are highly recurrent and associated with poor survival. It can be assumed that the affected chromosomal regions contain critical genes implicated in neuroblastoma biology and behavior. More specifically, evidence has emerged that several of these genes are implicated in tumor dependencies thus potentially providing novel therapeutic entry points. In this review, we briefly review the current status of recurrent DNA copy number aberrations in neuroblastoma and provide an overview of the genes affected by these genomic variants for which a direct role in neuroblastoma has been established. Several of these genes are implicated in networks that positively regulate MYCN expression or stability as well as cell cycle control and apoptosis. Finally, we summarize alternative approaches to identify and prioritize candidate copy-number driven dependency genes for neuroblastoma offering novel therapeutic opportunities.

The genus Rosa Linnaeus, 1753 has important economic value in ornamental sector and many breeding activities are going on supported by molecular studies. However, the cytogenetic studies of rose species are scarce and mainly focused on chromosome counting and chromosome morphology-based karyotyping. Due to the small size of the chromosomes and a high frequency of polyploidy in the genus, karyotyping is very challenging for rose species and requires FISH-based cytogenetic markers to be applied. Therefore, in this work the aim is to establish a FISH-based karyotype for Rosa wichurana (Crépin, 1888), a rose species with several benefits for advanced molecular cytogenetic studies of genus Rosa (Kirov et al. 2015a). It is shown that FISH signals from 5S, 45S and an Arabidopsis-type telomeric repeat are distributed on five (1, 2, 4, 5 and 7) of seven chromosome pairs. In addition, it is demonstrated that the interstitial telomeric repeat sequences (ITR) are located in the centromeric regions of four chromosome pairs. Using low hybridization stringency for ITR visualization, we showed that the number of ITR signals increases four times (1–4 signals). This study is the first to propose a FISH-based R. wichurana karyotype for the reliable identification of chromosomes. The possible origin of R. wichurana ITR loci is discussed.

The natural history of primary eosinophilia remains highly variable and is characterized by underlying disease heterogeneity. Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) is a rare and aggressive disease characterized by non-specific cytogenetic abnormalities or elevated blasts, with high risk of transformation to acute leukemia. We describe a case of CEL-NOS with two hierarchically related non-specific cytogenetic rearrangements, associated with an NPM1 mutation and followed by evolution to secondary AML. NPM1 mutations are not previously described in CEL-NOS. Keywords: CEL-NOS, Hypereosinophilia, Secondary AML, NPM1 mutation, Decitabine

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

Posttranslational histone modifications are essential features in epigenetic regulatory networks. One of these modifications has remained largely understudied: regulated histone proteolysis. In analogy to the histone H3 clipping during early mouse embryonic stem cell differentiation, we report for the first time that also in human embryonic stem cells this phenomenon takes place in the two different analyzed cell lines. Employing complementary techniques, different cleavage sites could be identified, namely A21, R26 and residue 31. The enzyme responsible for this cleavage is found to be a serine protease. The formation of cleaved H3 follows a considerably variable pattern, depending on the timeframe, culture conditions and culture media applied. Contrary to earlier findings on H3 clipping, our results disconnect the link between declining Oct4 expression and H3 cleavage.

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23–3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34+ hemogenic endothelial cells rounding up and developing into CD43+ hematopoietic cells without expression of MYB-eGFP. MYB-eGFP+ cells appeared relatively late in embryoid body cultures as CD34+CD43+CD45−/lo cells. These MYB-eGFP+ cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb−/− embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14+ macrophage cells were consistently eGFP− and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB+ hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.

BACKGROUND:With one million new cases of colorectal cancer (CRC) diagnosed annually in the world, CRC is the third most commonly diagnosed cancer in the Western world. Patients with stage I-III CRC can be cured with surgery but are at risk for recurrence. Colorectal cancer is characterized by the presence of chromosomal deletions and gains. Large genomic profiling studies have however not been conducted in this disease. The number of a specific genetic aberration in a tumour sample could correlate with recurrence-free survival or overall survival, possibly leading to its use as biomarker for therapeutic decisions. At this point there are not sufficient markers for prediction of disease recurrence in colorectal cancer, which can be used in the clinic to discriminate between stage II patients who will benefit from adjuvant chemotherapy. For instance, the benefit of adjuvant chemotherapy has been most clearly demonstrated in stage III disease with an approximately 30 percent relative reduction in the risk of disease recurrence. The benefits of adjuvant chemotherapy in stage II disease are less certain, the risk for relapse is much smaller in the overall group and the specific patients at risk are hard to identify. MATERIALS AND METHODS:In this study, array-comparative genomic hybridization analysis (array-CGH) was applied to study high-resolution DNA copy number alterations in 93 colon carcinoma samples. These genomic data were combined with parameters like KRAS mutation status, microsatellite status and clinicopathological characteristics. RESULTS:Both large and small chromosomal losses and gains were identified in our sample cohort. Recurrent gains were found for chromosome 1q, 7, 8q, 13 and 20 and losses were mostly found for 1p, 4, 8p, 14, 15, 17p, 18, 21 and 22. Data analysis demonstrated that loss of chromosome 4 is linked to a worse prognosis in our patients series. Besides these alterations, two interesting small regions of overlap were identified, which could be associated with disease recurrence. Gain of the 16p13.3 locus (including the RNA binding protein, fox-1 homolog gene, RBFOX1) was linked with a worse recurrence-free survival in our patient cohort. On the other hand, loss of RBFOX1 was only found in patients without disease recurrence. Most interestingly, above mentioned characteristics were also found in stage II patients, for whom there is a high medical need for the identification of new prognostic biomarkers. CONCLUSIONS:In conclusion, copy number variation of the 16p13.3 locus seems to be an important parameter for prediction of disease recurrence in colon cancer.

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34+ thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34+ and Notch inactive CD4+CD8+ thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia arises from the leukemic transformation of developing thymocytes and results from cooperative genetic lesions. Inactivation of the PHF6 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting an important tumor suppressive role for PHF6 in the pathobiology of this leukemia. Although the precise function of PHF6 is still unknown, this gene is most likely involved in chromatin regulation, a strongly emerging theme in T-cell acute lymphoblastic leukemia. In this context, our previous description of a cooperative microRNA regulatory network controlling several well-known T-cell acute lymphoblastic leukemia tumor suppressor genes, including PHF6, is of great importance. Given the high frequency of PHF6 lesions in T-cell acute lymphoblastic leukemia and the integration of PHF6 in this microRNA regulatory network, we aimed to identify novel oncogenic microRNAs in T-cell acute lymphoblastic leukemia which suppress PHF6. To this end, we performed an unbiased PHF6 3′UTR-microRNA library screen and combined the results with microRNA profiling data of samples from patients with T-cell acute lymphoblastic leukemia and normal thymocyte subsets. We selected miR-128-3p as a candidate PHF6-targeting, oncogenic microRNA and demonstrated regulation of PHF6 expression upon modulation of this microRNA in T-cell acute lymphoblastic leukemia cell lines. In vivo evidence of an oncogenic role of this microRNA in T-cell acute lymphoblastic leukemia was obtained through accelerated leukemia onset in a NOTCH1-induced T-cell acute lymphoblastic leukemia mouse model upon miR-128-3p over-expression. We conclude that miR-128-3p is a strong novel candidate oncogenic microRNA in T-cell acute lymphoblastic leukemia which targets the PHF6 tumor suppressor gene.

In order to anchor Rosa linkage groups to physical chromosomes, a combination of the Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM) is an efficient approach. Although, Tyramide-FISH is a very promising technique for the visualization of short DNA probes, it is very challenging for plant species with small chromosomes such as Rosa. In this study, we successfully applied the Tyramide-FISH technique for Rosa and compared different detection systems. An indirect detection system exploiting biotinylated tyramides was shown to be the most suitable technique for reliable signal detection. Three gene fragments with a size of 1100 pb-1700 bp (Phenylalanine Ammonia Lyase, Pyrroline-5-Carboxylate Synthase and Orcinol O-Methyl Transferase) have been physically mapped on chromosomes 7, 4 and 1, respectively, of Rosa wichurana. The signal frequency was between 25% and 40%. HRM markers of these 3 gene fragments were used to include the gene fragments on the existing genetic linkage map of Rosa wichurana. As a result, three linkage groups could be anchored to their physical chromosomes. The information was used to check for synteny between the Rosa chromosomes and Fragaria.

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.

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.

One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions, might play a central role in the metastasis of neuroblastoma (NB), a solid tumor of neuroectodermal origin. Using Reverse Transcription Quantitative PCR (RT-qPCR), Western blot, immunocytochemistry and Tissue MicroArrays (TMA) we demonstrate the expression of N-cadherin in neuroblastoma tumors and cell lines. All neuroblastic tumors (n = 356) and cell lines (n = 10) expressed various levels of the adhesion protein. The N-cadherin mRNA expression was significantly lower in tumor samples from patients suffering metastatic disease. Treatment of NB cell lines with the N-cadherin blocking peptide ADH-1 (Exherin, Adherex Technologies Inc.), strongly inhibited tumor cell proliferation in vitro by inducing apoptosis. Our results suggest that N-cadherin signaling may play a role in neuroblastoma disease, marking involvement of metastasis and determining neuroblastoma cell viability.

Fluorescence in situ hybridization was performed to characterize 81 cases of myeloid and lymphoid malignancies with cytogenetic 1p36 alterations not affecting the PRDM16 locus. In total, three subgroups were identified: balanced translocations (N = 27) and telomeric rearrangements (N = 15), both mainly observed in myeloid disorders; and unbalanced non-telomeric rearrangements (N = 39), mainly observed in lymphoid proliferations and frequently associated with a highly complex karyotype. The 1p36 rearrangement was isolated in 12 cases, mainly myeloid disorders. The breakpoints on 1p36 were more widely distributed than previously reported, but with identifiable rare breakpoint cluster regions, such as the TP73 locus. We also found novel partner loci on 1p36 for the known multi-partner genes HMGA2 and RUNX1. We precised the common terminal 1p36 deletion, which has been suggested to have an adverse prognosis, in B-cell lymphomas [follicular lymphomas and diffuse large B-cell lymphomas with t(14;18)(q32;q21) as well as follicular lymphomas without t(14;18)]. Intrachromosomal telomeric repetitive sequences were detected in at least half the cases of telomeric rearrangements. It is unclear how the latter rearrangements occurred and whether they represent oncogenic events or result from chromosomal instability during oncogenesis.

Chromosomal rearrangements involving the EVI1 proto-oncogene are a recurrent finding in myeloid leukemias and are indicative of a poor prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7 or cytogenetic detectable deletions of part of 7q. As EVI1 overexpression alone is not sufficient to induce leukemia, loss of a 7q tumour suppressor gene might be a required cooperating event. To test this hypothesis, we performed high-resolution array comparative genomic hybridization analysis of twelve EVI1 overexpressing patients and three EVI1 deregulated cell lines to search for 7q submicroscopic deletions. This analysis lead to the delineation of two critical regions, one of 0.39 Mb on 7q35 containing the CNTNAP2 gene and one of 1.33 Mb on chromosome bands 7q35-q36 comprising nine genes in EVI1 deregulated cell lines. These findings open the way to further studies aimed at identifying the culprit EVI1 implicated tumour suppressor genes on 7q.

Chromosomal translocations involving the EVI1 locus are a recurrent finding in myeloid leukemia and are associated with poor prognosis. In this study, we performed a detailed molecular characterization of the recurrent translocation t(3;17)(q26;q22) in 13 hematologic malignancies. The EVI1 gene locus was rearranged in all 13 patients and was associated with EVI1 overexpression. In 9 out of 13 patients, the 17q breakpoints clustered in a 250 kb region on band 17q22 encompassing the MSI2 (musashi homologue 2) gene. Expression analyses failed to demonstrate ectopic MSI2 expression or the presence of an MSI2/EVI1 fusion gene. In conclusion, we show for the first time that the t(3;17) is indeed a recurrent chromosomal aberration in myeloid malignancies. In keeping with findings in other recurrent 3q26 rearrangements, overexpression of the EVI1 gene appears to be the major contributor to leukemogenesis in patients with a t(3;17).

The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification of a novel gene that is disrupted by this translocation. This gene, named NBPF1 for Neuroblastoma BreakPoint Family member 1, belongs to a recently described gene family encoding highly similar proteins, the functions of which are unknown. The translocation truncates NBPF1 and gives rise to two chimeric transcripts of NBPF1 sequences fused to sequences derived from chromosome 17. On chromosome 17, the translocation disrupts one of the isoforms of ACCN1, a potential glioma tumor suppressor gene. Expression of the NBPF family in neuroblastoma cell lines is highly variable, but it is decreased in cell lines that have a deletion of chromosome 1p. More importantly, expression profiling of the NBPF1 gene showed that its expression is significantly lower in cell lines with heterozygous NBPF1 loss than in cell lines with a normal 1p chromosome. Meta-analysis of the expression of NBPF and ACCN1 in neuroblastoma tumors indicates a role for the NBPF genes and for ACCN1 in tumor aggressiveness. Additionally, DLD1 cells with inducible NBPF1 expression showed a marked decrease of clonal growth in a soft agar assay. The disruption of both NBPF1 and ACCN1 genes in this neuroblastoma patient indicates that these genes might suppress development of neuroblastoma and possibly other tumor types.

Background and Objectives Classical Hodgkin’s lymphoma (cHL) is a common malignant lymphoma characterized by the presence of large, usually multinucleated malignant Hodgkin and Reed Sternberg (HRS) cells which are thought to be derived from germinal center B-cells. In cHL, the HRS cells constitute less than 1% of the tumor volume; consequently the profile of genetic aberrations in cHL is still poorly understood.Design and Methods In this study, we subjected four commonly used cHL cell lines to array comparative genomic hybridization (aCGH) in order to delineate known chromosomal aberrations in more detail and to search for small hitherto undetected genomic imbalances.Results The aCGH profiles of the four cell lines tested confirmed the complex patterns of rearrangements previously demonstrated with multicolor fluorescence in situ hybridization and chromosomal CGH (cCGH). Importantly, aCGH allowed a much more accurate delineation of imbalances as compared to previous studies performed at a chromosomal level of resolution. Furthermore, we detected 35 hitherto undetected aberrations including a homozygous deletion of chromosomal region 15q26.2 in the cell line HDLM2 encompasing RGMA and CHD2 and an amplification of the STAT6 gene in cell line L1236 leading to STAT6 overexpression. Finally, in cell line KM-H2 we found a 2.35 Mb deletion at 16q12.1 putatively defining a small critical region for the recurrent 16q deletion in cHL. This region contains the CYLD gene, a known suppressor gene of the NF-κB pathway.Interpretation and Conclusions aCGH was performed on four cHL cell lines leading to the improved delineation of known chromosomal imbalances and the detection of 35 hitherto undetected aberrations. More specifically, our results highlight STAT6 as a potential transcriptional target and identified RGMA, CHD2 and CYLD as candidate tumor suppressors in cHL.