Henrik Lilljebjörn

Genetic landscape of high hyperdiploid childhood acute lymphoblastic leukemia

High hyperdiploid acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. It is characterized by gain of chromosomes, typically +X, +4, +6, +10, +14, +17, +18, and +21,+21; little is known about additional genetic aberrations. Approximately 20% of the patients relapse; therefore it is clinically important to identify risk-stratifying markers. We used SNP array analysis to investigate a consecutive series of 74 cases of high hyperdiploid ALL. We show that the characteristic chromosomal gains are even more frequent than previously believed, indicating that karyotyping mistakes are common, and that almost 80% of the cases display additional abnormalities detectable by SNP array analysis. Subclonality analysis strongly implied that the numerical aberrations were primary and arose before structural events, suggesting that step-wise evolution of the leukemic clone is common. An association between duplication of 1q and +5 was seen (P = 0.003). Other frequent abnormalities included whole-chromosome uniparental isodisomies (wUPIDs) 9 and 11, gain of 17q not associated with isochromosome formation, extra gain of part of 21q, deletions of ETS variant 6 (ETV6), cyclin-dependent kinase inhibitor 2A (CKDN2A) and paired box 5 (PAX5), and PAN3 poly(A) specific ribonuclease subunit homolog (PAN3) microdeletions. Comparison of whole-chromosome and partial UPID9 suggested different pathogenetic outcomes, with the former not involving CDKN2A. Finally, two cases had partial deletions of AT rich interactive domain 5B (ARID5B), indicating that acquired as well as constitutional variants in this locus may be associated with pediatric ALL. Here we provide a comprehensive characterization of the genetic landscape of high hyperdiploid childhood ALL, including the heterogeneous pattern of secondary genetic events.

The DNA methylome of pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, with high hyperdiploidy [51-67 chromosomes] and the t(12;21)(p13;q22) [ETV6/RUNX1 fusion] representing the most frequent abnormalities. Although these arise in utero, there is long latency before overt ALL, showing that additional changes are needed. Gene dysregulation through hypermethylation may be such an event; however, this has not previously been investigated in a detailed fashion. We performed genome-wide methylation profiling using bacterial artificial chromosome arrays and promoter-specific analyses of high hyperdiploid and ETV6/RUNX1-positive ALLs. In addition, global gene expression analyses were performed to identify associated expression patterns. Unsupervised cluster and principal component analyses of the chromosome-wide methylome profiles could successfully subgroup the two genetic ALL types. Analysis of all currently known promoter-specific CpG islands demonstrated that several B-cell- and neoplasia-associated genes were hypermethylated and underexpressed, indicating that aberrant methylation plays a significant leukemogenic role. Interestingly, methylation hotspots were associated with chromosome bands predicted to harbor imprinted genes and the tri-/tetrasomic chromosomes in the high hyperdiploid ALLs were less methylated than their disomic counterparts. Decreased methylation of gained chromosomes is a previously unknown phenomenon that may have ramifications not only for the pathogenesis of high hyperdiploid ALL but also for other disorders with acquired or constitutional numerical chromosome anomalies.

A novel SERPINE1-FOSB fusion gene results in transcriptional up-regulation of FOSB in pseudomyogenic haemangioendothelioma.

Pseudomyogenic haemangioendothelioma (PHE) is an intermediate malignant vascular soft tissue tumour primarily affecting children and young adults. The molecular basis of this neoplasm is unknown. We here used chromosome banding analysis, fluorescence in situ hybridization (FISH), mRNA sequencing, RT–PCR and quantitative real‐time PCR on a series of morphologically well‐characterized PHEs to show that a balanced translocation, t(7;19)(q22;q13), detected as the sole cytogenetic aberration in two cases, results in fusion of the SERPINE1 and FOSB genes. This translocation has not been observed in any other bone or soft tissue tumour. Interphase FISH on sections from eight additional PHEs identified the same SERPINE1–FOSB fusion in all cases. The role of SERPINE1, which is highly expressed in vascular cells, in this gene fusion is probably to provide a strong promoter for FOSB, which was found to be expressed at higher levels in PHEs than in other soft tissue tumours. FOSB encodes a transcription factor belonging to the FOS family of proteins, which, together with members of the JUN family of transcription factors, are major components of the activating protein 1 (AP‐1) complex. Further studies are needed to understand the cellular impact of the aberrant expression of the FOSB gene, but as the t(7;19) resulting in the SERPINE1–FOSB fusion seems to be pathognomonic for PHE, FISH or RT–PCR could be useful for differential diagnostic purposes. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

Integrative analysis of gene expression and copy number alterations using canonical correlation analysis

BackgroundWith the rapid development of new genetic measurement methods, several types of genetic alterations can be quantified in a high-throughput manner. While the initial focus has been on investigating each data set separately, there is an increasing interest in studying the correlation structure between two or more data sets. Multivariate methods based on Canonical Correlation Analysis (CCA) have been proposed for integrating paired genetic data sets. The high dimensionality of microarray data imposes computational difficulties, which have been addressed for instance by studying the covariance structure of the data, or by reducing the number of variables prior to applying the CCA. In this work, we propose a new method for analyzing high-dimensional paired genetic data sets, which mainly emphasizes the correlation structure and still permits efficient application to very large data sets. The method is implemented by translating a regularized CCA to its dual form, where the computational complexity depends mainly on the number of samples instead of the number of variables. The optimal regularization parameters are chosen by cross-validation. We apply the regularized dual CCA, as well as a classical CCA preceded by a dimension-reducing Principal Components Analysis (PCA), to a paired data set of gene expression changes and copy number alterations in leukemia.ResultsUsing the correlation-maximizing methods, regularized dual CCA and PCA+CCA, we show that without pre-selection of known disease-relevant genes, and without using information about clinical class membership, an exploratory analysis singles out two patient groups, corresponding to well-known leukemia subtypes. Furthermore, the variables showing the highest relevance to the extracted features agree with previous biological knowledge concerning copy number alterations and gene expression changes in these subtypes. Finally, the correlation-maximizing methods are shown to yield results which are more biologically interpretable than those resulting from a covariance-maximizing method, and provide different insight compared to when each variable set is studied separately using PCA.ConclusionsWe conclude that regularized dual CCA as well as PCA+CCA are useful methods for exploratory analysis of paired genetic data sets, and can be efficiently implemented also when the number of variables is very large.

The genomic landscape of high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploid (51–67 chromosomes) acute lymphoblastic leukemia (ALL) is one of the most common childhood malignancies, comprising 30% of all pediatric B cell–precursor ALL. Its characteristic genetic feature is the nonrandom gain of chromosomes X, 4, 6, 10, 14, 17, 18 and 21, with individual trisomies or tetrasomies being seen in over 75% of cases, but the pathogenesis remains poorly understood. We performed whole-genome sequencing (WGS) (n = 16) and/or whole-exome sequencing (WES) (n = 39) of diagnostic and remission samples from 51 cases of high hyperdiploid ALL to further define the genomic landscape of this malignancy. The majority of cases showed involvement of the RTK-RAS pathway and of histone modifiers. No recurrent fusion gene–forming rearrangement was found, and an analysis of mutations on trisomic chromosomes indicated that the chromosomal gains were early events, strengthening the notion that the high hyperdiploid pattern is the main driver event in this common pediatric malignancy.

Identification of ETV6-RUNX1 -like and DUX4 -rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

Fusion gene-mediated truncation of RUNX1 as a potential mechanism underlying disease progression in the 8p11 myeloproliferative syndrome.

The 8p11 myeloproliferative syndrome (EMS) is a chronic myeloproliferative disorder molecularly characterized by fusion of various 5′ partner genes to the 3′ part of the fibroblast growth factor receptor 1 (FGFR1) gene at 8p, resulting in constitutive activation of the tyrosine kinase activity contained within FGFR1. EMS is associated with a high risk of transformation to acute myeloid leukemia (AML), but the mechanisms underlying the disease progression are unknown. In the present study, we have investigated a case of EMS harboring a t(8;22)(p11;q11)/BCR‐FGFR1 rearrangement as well as a t(9;21)(q34;q22) at the time of AML transformation. FISH and RT‐PCR analyses revealed that the t(9;21) leads to a fusion gene consisting of the 5′ part of RUNX1 (exons 1–4) fused to repetitive sequences of a gene with unknown function on chromosome 9, adding 70 amino acids to RUNX1 exon 4. The t(9;21) hence results in a truncation of RUNX1. No point mutations were found in the other RUNX1 allele. The most likely functional outcome of the rearrangement was haploinsufficiency of RUNX1, which thus may be one mechanism by which EMS transforms to AML.

Combined high-resolution array-based comparative genomic hybridization and expression profiling of ETV6/RUNX1 -positive acute lymphoblastic leukemias reveal a high incidence of cryptic Xq duplications and identify several putative target genes within the commonly gained region

Seventeen ETV6/RUNX1-positive pediatric acute lymphoblastic leukemias were investigated by high-resolution array-based comparative genomic hybridization (array CGH), gene expression profiling and fluorescence in situ hybridization. Comparing the array CGH and gene expression patterns revealed that genomic imbalances conferred a great impact on the expression of genes in the affected regions. The array CGH analyses identified a high frequency of cytogenetically cryptic genetic changes, for example, del(9p) and del(12p). Interestingly, a duplication of Xq material, varying between 30 and 60 Mb in size, was found in 6 of 11 males (55%), but not in females. Genes on Xq were found to have a high expression level in cases with dup(Xq); a similar overexpression was confirmed in t(12;21)-positive cases in an external gene expression data set. By studying the expression profile and the proposed function of genes in the minimally gained region, several candidate target genes (SPANXB, HMGB3, FAM50A, HTATSF1 and RAP2C) were identified. Among them, the testis-specific SPANXB gene was the only one showing a high and uniform overexpression, irrespective of gender and presence of Xq duplication, suggesting that this gene plays an important pathogenetic role in t(12;21)-positive leukemia.

Relapsed childhood high hyperdiploid acute lymphoblastic leukemia: presence of preleukemic ancestral clones and the secondary nature of microdeletions and RTK-RAS mutations.

Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% of patients still relapse. It is important to identify these patients already at diagnosis to ensure proper risk stratification. We have investigated 11 paired diagnostic and relapse samples with single nucleotide polymorphism array and mutation analyses of FLT3, KRAS, NRAS and PTPN11 in order to identify changes associated with relapse and to ascertain the genetic evolution patterns. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. On the basis of the genetic relationship between the paired samples, three groups were delineated: (1) identical genetic changes at diagnosis and relapse (2 of 11 cases), (2) clonal evolution with all changes at diagnosis being present at relapse (2 of 11) and (3) clonal evolution with some changes conserved, lost or gained (7 of 11), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern.

Concomitant deletions of tumor suppressor genes MEN1 and AIP are essential for the pathogenesis of the brown fat tumor hibernoma

Hibernomas are benign tumors with morphological features resembling brown fat. They consistently display cytogenetic rearrangements, typically translocations, involving chromosome band 11q13. Here we demonstrate that these aberrations are associated with concomitant deletions of AIP and MEN1, tumor suppressor genes that are located 3 Mb apart and that underlie the hereditary syndromes pituitary adenoma predisposition and multiple endocrine neoplasia type I. MEN1 and AIP displayed a low expression in hibernomas whereas the expression of genes up-regulated in brown fat—PPARA, PPARG, PPARGC1A, and UCP1—was high. Thus, loss of MEN1 and AIP is likely to be pathogenetically essential for hibernoma development. Simultaneous loss of two tumor suppressor genes has not previously been shown to result from a neoplasia-associated translocation. Furthermore, in contrast to the prevailing assumption that benign tumors harbor relatively few genetic aberrations, the present analyses demonstrate that a considerable number of chromosome breaks are involved in the pathogenesis of hibernoma.