Identification and evaluation of novel prognostic genetic markers for childhood acute lymphoblastic leukemia

Childhood acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer today. Due to advances in risk stratification and treatment, survival rates have increased drastically the last decades. Currently, children with acute leukemia in the Nordic countries are diagnosed and treated according to the NOPHO-2008 treatment protocol. In this protocol, a number of cytogenetic markers are used for risk stratification and guidance of treatment intensity. However, genetic markers associated with high risk are infrequent and relapses occur across all genetic subtypes, including those associated with a favorable outcome. Importantly, over 25% of childhood ALL cases harbor none of the currently used genetic risk markers in their bone marrow cells at diagnosis. The aim of this thesis was to generate a greater understanding of the genetic landscape in ALL, as well as to identify novel genetic markers of prognostic relevance, with special focus on the group of patients lacking risk-stratifying markers. In paper I, we investigated the frequency and prognostic impact of IKZF1 deletions in patients diagnosed with B-cell precursor (BCP) ALL in the Stockholm region; IKZF1 deletions were present in 15% of cases and significantly associated with inferior outcome. These results led to paper II, where the cohort was extended to include BCP ALL cases with available IKZF1 data from other centers in Sweden. This study verified that IKZF1 deletion was an independent risk factor for decreased survival, and could confirm that the frequency and prognostic effect was most pronounced in patients without risk-stratifying markers. A high frequency of IKZF1 deletions could also be detected in paper III, where we investigated the genetic copy number landscape of BCP ALL across the different cytogenetic subtypes. This study showed that a majority of cases without risk-stratifying markers harbor deletions with potential prognostic significance, suggesting that a large proportion of this group could be assigned to distinct genetic subtypes. Intrachromosomal amplification of chromosome 21 (iAMP21) is an intermediate/high-risk subtype for which the biological cause of the high relapse risk is unknown. In paper IV, we used an integrated molecular approach to investigate the iAMP21 subtype, and identified significant overexpression of tree potential candidate genes, i.e. DYRK1A, SON and CHAF1B, with leukemia-relevant functions that could represent future targets for therapy. Together, these studies have identified a number of potential novel prognostic genetic markers that may contribute to the clinical risk-evaluation of children diagnosed with BCP ALL, and to our understanding of the biology behind relapse and poor outcome in this disease

Validation of the United Kingdom copy-number alteration classifier in 3239 children with B-cell precursor ALL

Genetic abnormalities provide vital diagnostic and prognostic information in pediatric acute lymphoblastic leukemia (ALL) and are increasingly used to assign patients to risk groups. We recently proposed a novel classifier based on the copy-number alteration (CNA) profile of the 8 most commonly deleted genes in B-cell precursor ALL. This classifier defined 3 CNA subgroups in consecutive UK trials and was able to discriminate patients with intermediate-risk cytogenetics. In this study, we sought to validate the United Kingdom ALL (UKALL)-CNA classifier and reevaluate the interaction with cytogenetic risk groups using individual patient data from 3239 cases collected from 12 groups within the International BFM Study Group. The classifier was validated and defined 3 risk groups with distinct event-free survival (EFS) rates: good (88%), intermediate (76%), and poor (68%) (P < .001). There was no evidence of heterogeneity, even within trials that used minimal residual disease to guide therapy. By integrating CNA and cytogenetic data, we replicated our original key observation that patients with intermediate-risk cytogenetics can be stratified into 2 prognostic subgroups. Group A had an EFS rate of 86% (similar to patients with good-risk cytogenetics), while group B patients had a significantly inferior rate (73%, P < .001). Finally, we revised the overall genetic classification by defining 4 risk groups with distinct EFS rates: very good (91%), good (81%), intermediate (73%), and poor (54%), P < .001. In conclusion, the UKALL-CNA classifier is a robust prognostic tool that can be deployed in different trial settings and used to refine established cytogenetic risk groups

Detailed gene dose analysis reveals recurrent focal gene deletions in pediatric B-cell precursor acute lymphoblastic leukemia

To identify copy number alterations (CNAs) in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL), array comparative genomic hybridization was performed on 50 cases; detected CNAs were validated in a cohort of 191 cases analyzed by single nucleotide polymorphism arrays. Apart from CNAs involving leukemia-associated genes, recurrent deletions targeting genes not previously implicated in BCP ALL, e.g. INIP, IRF1 and PDE4B, were identified. Deletions of the DNA repair gene INIP were exclusively found in cases with t(12;21), and deletions of SH2B3 were associated with intrachromosomal amplification of chromosome 21 (

Validated MP-WGS findings.

<p>Validated MP-WGS findings.</p

High-resolution detection of chromosomal rearrangements in leukemias through mate pair whole genome sequencing

<div><p>The detection of recurrent somatic chromosomal rearrangements is standard of care for most leukemia types. Even though karyotype analysis—a low-resolution genome-wide chromosome analysis—is still the gold standard, it often needs to be complemented with other methods to increase resolution. To evaluate the feasibility and applicability of mate pair whole genome sequencing (MP-WGS) to detect structural chromosomal rearrangements in the diagnostic setting, we sequenced ten bone marrow samples from leukemia patients with recurrent rearrangements. Samples were selected based on cytogenetic and FISH results at leukemia diagnosis to include common rearrangements of prognostic relevance. Using MP-WGS and in-house bioinformatic analysis all sought rearrangements were successfully detected. In addition, unexpected complexity or additional, previously undetected rearrangements was unraveled in three samples. Finally, the MP-WGS analysis pinpointed the location of chromosome junctions at high resolution and we were able to identify the exact exons involved in the resulting fusion genes in all samples and the specific junction at the nucleotide level in half of the samples. The results show that our approach combines the screening character from karyotype analysis with the specificity and resolution of cytogenetic and molecular methods. As a result of the straightforward analysis and high-resolution detection of clinically relevant rearrangements, we conclude that MP-WGS is a feasible method for routine leukemia diagnostics of structural chromosomal rearrangements.</p></div

High-resolution detection of chromosomal rearrangements in leukemias through mate pair whole genome sequencing - Fig 2

<p><b>(a) Graphic representation of MP-WGS events in ALL1.</b> Circos plot representing unique events detected by MP-WGS in ALL1 and graphic representation of <i>TCF3</i> (green) and <i>PBX1</i> (red) gene structure and the mate pair reads supporting the link between both genes. The red line in circos plot represents the event between <i>PBX1</i> (1q23) and <i>TCF3</i> (19p13.3). (<b>b)</b> Junction sequence of ALL1 Sanger sequence showing the 11 base-pair insertion at the junction between <i>TCF3</i> (green) and <i>PBX1</i> (red). <b>(c)</b> Circos plots of the remaining samples. Red lines: Unique events previously detected by FISH and/or karyotyping and novel unique events validated with Sanger; Gray lines: Unique events not further investigated.</p

Karyotype, FISH and sequencing results.

<p>Karyotype and FISH results together with sequence composition at the junction region of the canonical fusion genes for samples ALL1, ALL3 and ALL5. Genome coordinates are based on hg19. Upper and lower sequences correspond to the reference sequences of the genes involved in the fusion. Patient sequences are shown in between in purple and orange. Insertions, deletions and microhomologies are shown in green.</p

Schematic representation summarizing the workflow.

<p>Schematic representation summarizing the workflow.</p

Intragenic amplification of PAX5: a novel subgroup in B-cell precursor acute lymphoblastic leukemia?

Intragenic PAX5 amplification defines a novel, relapse-prone subtype of B-cell precursor acute lymphoblastic leukemia with a poor outcome