Bárbara Marques

ArrayCGH-based classification of neuroblastoma into genomic subgroups†

High‐resolution array comparative genomic hybridization (arrayCGH) profiling was performed on 75 primary tumors and 29 cell lines to gain further insight into the genetic heterogeneity of neuroblastoma and to refine genomic subclassification. Using a novel data‐mining strategy, three major and two minor genomic subclasses were delineated. Eighty‐three percent of tumors could be assigned to the three major genomic subclasses, corresponding to the three known clinically and biologically relevant subsets in neuroblastoma. The remaining subclasses represented (1) tumors with no/few copy number alterations or an atypical pattern of aberrations and (2) tumors with 11q13 amplification. Inspection of individual arrayCGH profiles showed that recurrent genomic imbalances were not exclusively associated with a specific subclass. Of particular notice were tumors with numerical imbalances typically observed in subtype 1 neuroblastoma, in association with genomic features of subtype 2A or 2B. A search for prognostically relevant genomic alterations disclosed 1q gain as a predictive marker for therapy failure within the group of subtype 2A and 2B tumors. In cell lines, a high incidence of 6q loss was observed, with a 3.87–5.32 Mb region of common loss within 6q25.1–6q25.2. Our study clearly illustrates the importance of genomic profiling in relation to tumor behavior in neuroblastoma. We propose that genome‐wide assessment of copy number alterations should ideally be included in the genetic workup of neuroblastoma. Further multicentric studies on large tumor series are warranted in order to improve therapeutic stratification in conjunction with other features such as age at diagnosis, tumor stage, and gene expression signatures.

Segmental chromosomal alterations lead to a higher risk of relapse in infants with MYCN-non-amplified localised unresectable/disseminated neuroblastoma (a SIOPEN collaborative study).

Background:In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse.Methods:In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enroled in the prospective European INES trials.Results:Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) (P<0.0001). Progression-free survival was poorer in patients whose tumours harboured SCA, in the whole population and in trials INES99.1 and INES99.2, in the absence of clinical symptoms (log-rank test, P=0.0001, P=0.04 and P=0.0003, respectively). In multivariate analysis, a SCA genomic profile was the strongest predictor of poorer progression-free survival.Conclusion:In infants with stage 4s MYCN-non-amplified NB, a SCA genomic profile identifies patients who will require upfront treatment even in the absence of other clinical indication for therapy, whereas in infants with localised unresectable NB, a genomic profile characterised by the absence of SCA identifies patients in whom treatment reduction might be possible. These findings will be implemented in a future international trial.

Translocation (8;17;15;21)(q22;q23;q15;q22) in acute myeloid leukemia (M2) : A four-way variant of t(8;21)

We report the results of cytogenetic, fluorescence in situ hybridization (FISH) and molecular analyses in a 15-year-old boy diagnosed with acute myeloid leukemia subtype M2 (AML-M2). Cytogenetic and FISH analyses, the latter with whole chromosome painting probes, revealed a complex translocation involving four chromosomes: t(8;17;15;21)(q22;q23;q15;q22). The observation of breakpoints at 8q22 and 21q22 suggested a rearrangement of the ETO and AML1 genes, respectively. Using a dual-color FISH test with ETO and AML1 probes, we demonstrated an AML1/ETO fusion signal on the derivative chromosome 8. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of AML1/ETO fusion transcripts identical to those found in classical t(8;21). The present case highlights the relevant role of the rearranged chromosome 8, which encodes the AML1/ETO fusion product in the pathogenesis of AML-M2.

A Multilocus Technique for Risk Evaluation of Patients with Neuroblastoma

Purpose: Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma. Experimental Design: A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n = 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n = 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level. Results: The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (κ = 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%. Conclusions: The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance. Clin Cancer Res; 17(4); 792–804. ©2011 AACR.

Characterization of two ectrodactyly-associated translocation breakpoints separated by 2.5 Mb on chromosome 2q14.1–q14.2

Split hand-split foot malformation or ectrodactyly is a heterogeneous congenital defect of digit formation. The aim of this study is the mapping of the breakpoints and a detailed molecular characterization of the candidate genes for an isolated and syndromic form of ectrodactyly, both associated with de novo apparently balanced chromosome translocations involving the same chromosome 2 band, [t(2;11)(q14.2;q14.2)] and [t(2;4)(q14.1;q35)], respectively. Breakpoints were mapped by fluorescence in situ hybridization using bacterial artificial chromosome clones. Where possible, these breakpoints were further delimited. Candidate genes were screened for pathogenic mutations and the expression levels of two of them analysed. The isolated bilateral split foot malformation-associated chromosome 2 breakpoint was localized at 120.9 Mb, between the two main candidate genes, encoding GLI-Kruppel family member GLI2 and inhibin-βB. The second breakpoint associated with holoprosencephaly, hypertelorism and ectrodactyly syndrome was mapped 2.5 Mb proximal at 118.4 Mb and the candidate genes identified from this region were the insulin-induced protein 2 and the homeobox protein engrailed-1. No clear pathogenic mutations were identified in any of these genes. The breakpoint between INHBB and GLI2 coincides with a previously identified translocation breakpoint associated with ectrodactyly. We propose a mechanism by which translocations in the 2q14.1–q14.2 region disrupt the specific arrangement of long-range regulatory elements that control the tight quantitative spatiotemporal expression of one or more genes from the breakpoint region.

Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

Background:The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis.Methods:To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol.Results:Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018).Conclusions:The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Three‐way translocation involves MLL, MLLT3, and a novel cell cycle control gene, FLJ10374, in the pathogenesis of acute myeloid leukemia with t(9;11;19)(p22;q23;p13.3)

The MLL gene, at 11q23, undergoes chromosomal translocation with a large number of partner genes in both acute lymphoblastic and acute myeloid leukemia (AML). We report a novel t(9;11;19)(p22;q23;p13.3) disrupting MLL in an infant AML patient. The 5′ end of MLL fused to chromosome 9 sequences on the der(11), whereas the 3′ end was translocated to chromosome 19. We developed long‐distance inverse–polymerase chain reaction assays to investigate the localization of the breakpoints on der(11) and der(19). We found that intron 5 of MLL was fused to intron 5 of MLLT3 at the der(11) genomic breakpoint, resulting in a novel in‐frame MLL exon 5–MLLT3 exon 6 fusion transcript. On the der(19), a novel gene annotated as FLJ10374 was disrupted by the breakpoint. Using reverse transcription–polymerase chain reaction analysis, we showed that FLJ10374 is ubiquitously expressed in human cells. Transfection of the FLJ10374 protein in different cell lines revealed that it localized exclusively to the nucleus. In serum‐starved NIH‐3T3 cells, the expression of FLJ10374 decreased the rate of the G1‐to‐S transition of the cell cycle, whereas the suppression of FLJ10374 through short interfering RNA increased cell proliferation. These results indicate that FLJ10374 negatively regulates cell cycle progression and proliferation. Thus, a single chromosomal rearrangement resulting in formation of the MLL–MLLT3 fusion gene and haplo‐insufficiency of FLJ10374 may have cooperated to promote leukemogenesis in AML with t(9;11;19).

Three-way translocation (X;20;16)(p11;q13;q23) in essential thrombocythemia implicates NFATC2 in dysregulation of CSF2 expression and megakaryocyte proliferation.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm essentially characterized by excessive production of platelets. Molecular pathogenesis of ET is linked in approximately half of the patients to intracellular cytokine signaling dysregulation as a result of thrombopoietin receptor or Janus kinase 2 (JAK2) mutations. However, genetic defects underlying cytokine transcription have not been associated with ET. Using molecular cytogenetics and whole‐genome array analyses, we uncovered a submicroscopic deletion at 20q13.2 in a JAK2V617F‐positive ET patient with an acquired complex chromosome translocation. The deletion encompassed the nuclear factor of activated T‐cells, cytoplasmic, calcineurin‐dependent 2 (NFATC2) gene that encodes a transcription factor involved in the regulation of hematopoietic cytokines. RNA interference‐mediated suppression of NFATC2 mRNA or pharmacological inhibition of NFATC2 protein with 11R‐VIVIT in cultured JAK2V617F‐positive SET‐2 megakaryocytes increased colony stimulating factor 2 (granulocyte‐macrophage) (CSF2) mRNA and promoted cell proliferation. Moreover, impairment of NFATC2–calcineurin interaction with 11R‐VIVIT further reduced the transcription of the NFATC2 gene. Antibody‐mediated neutralization of CSF2 cytokine in inhibitor‐treated cells prevented 11R‐VIVIT‐induced cell proliferation, indicating that impairment of NFATC2–calcineurin interaction promotes megakaryocyte proliferation through up‐regulation of CSF2 transcription. Our results suggest a model in which haplo‐insufficiency of NFATC2 cooperates with activation of the JAK–STAT signaling pathway in the pathogenesis of JAK2V617F‐positive ET with del(20q). These results further indicate that pathogenesis of ET may be linked to genetic defects of other transcription factor genes involved in the regulation of cytokine expression.

Trisomy 15 mosaicism: Challenges in prenatal diagnosis

Keywords: trisomy 15 mosaicism; fetal mosaicism; chorionic villi; prenatal cytogenetics; SNP-array; uniparental disomy

Being a Mother After 35 Years: Will it be Different?

INTRODUCTION Advanced maternal age is defined as maternity after 35 years old and is associated with more complications during pregnancy and neonatal period as well as decreased fertility. This study aims to examine the relationship between advanced maternal age and their maternal and fetal consequences, as well as maternal perception of the risk of pregnancy after 35 years old. MATERIAL AND METHODS Observational, retrospective and comparative study, between two groups: advanced maternal age group (aged ≥ 35 years) and non-advanced maternal age group (age < 35 years), conducted between March and June 2015. Chi-square test and Fishers exact test were used and considered significant if p < 0.05. RESULTS Of the 736 women admitted to the hospital (32.2% with advanced maternal age), 306 were included in the study (153 in each group). In the non-advanced maternal age group there was a greater number of primiparous women (p < 0.01). In the advanced maternal age group, more previous miscarriages were observed (p < 0.001), as well as a higher use of assisted reproductive techniques (p < 0.01), preformed of amniocentesis (p < 0.001) and dystocia, including caesarean sections (p < 0.001). No association was found regarding the presence of maternal complications in pregnancy, birth defects, need for neonatal resuscitation or prematurity. As for the perception of risk in pregnancy, the non-advanced maternal age group considered it to be superior (p < 0.05). DISCUSSION Most women of advanced maternal age have term deliveries without complications. Neonatal outcomes seem not to have been influenced by the advanced maternal age. CONCLUSION The consequences of an advanced maternal age pregnancy in this sample did not have the same clinical expression as described in the literature. In the future, advanced maternal age will possibly be considered after age 40.