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Genomic characterization of pediatric acute lymphoblastic leukemia by deep sequencing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine. (Syvänen)ORCID iD: 0000-0003-3887-9519
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children, with close to 200 cases per year in the Nordic countries. Despite recent advances in modern chemotherapies, 20% of the ALL patients experience a relapse. ALL has traditionally been stratified into subtypes with different risk classification and therapy using large genomic aberrations such as translocations and aneuploidies. In recent years technological advances have enabled the detection of smaller genetic variants, such as point mutations and small insertions/deletions. This thesis focuses on the detection of these smaller variants and their potential impact for ALL.

The present work includes four studies. In the first study we investigated the effects of whole genome amplification and non-indexed pooling strategies to maximize the output of targeted sequencing. We found that whole genome amplified DNA is equivalent to genomic DNA when screening for point mutations in targeted sequencing data. We were able to accurately detect variants in non-indexed pools with up to ten samples. The second study describes further work on non-indexed pools where we pooled samples in an overlapping scheme and identified carriers of rare variants. The third study describes the whole genome and RNA sequencing of four patients with ALL and validated the results in a cohort of 168 additional ALL patients. In the whole genome sequenced patients we found somatic mutations in both known cancer driver-genes (KRAS and NOTCH1) and in putative driver-genes (KMT2D and KIF1B) after analysis of the additional ALL patients. We validated point mutations genome-wide and observed a large number of C>A mutations in one patient, in contrast to C>T mutations that are more common in cancer in general. The fourth study analyzed the same cohort as the third study and expanded the target to 872 genes linked to cancer, ALL or epigenetic regulation recorded in the literature. We found distinctive differences between BCP-ALL and T-ALL both in number and types of mutations. In addition we observed an association between mutations in the Notch signaling pathway and relapse.

These results will have an impact on future studies of cancer, and add another piece to the genetic puzzle of ALL.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 35 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1170
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-269760ISBN: 978-91-554-9444-5 (print)OAI: oai:DiVA.org:uu-269760DiVA: diva2:885128
Public defence
2016-02-19, E10:1307-1309, Navet, BMC, Husargatan 3, Uppsala, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2016-01-26 Created: 2015-12-18 Last updated: 2016-02-12Bibliographically approved
List of papers
1. Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment
Open this publication in new window or tab >>Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment
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2013 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 14, no 1, 856- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Target enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing.

RESULTS:

We captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792--1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools.

CONCLUSION:

Our work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-212775 (URN)10.1186/1471-2164-14-856 (DOI)000328647700003 ()24314227 (PubMedID)
Funder
Swedish Cancer Society, CAN2010/592EU, European Research Council, 262055Swedish Foundation for Strategic Research , RBc08-008Swedish Research Council, 90559401
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2013-12-13 Created: 2013-12-13 Last updated: 2017-12-06Bibliographically approved
2. Identification of somatic variants by targeted sequencing of pooled cancer samples
Open this publication in new window or tab >>Identification of somatic variants by targeted sequencing of pooled cancer samples
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-269752 (URN)
Available from: 2015-12-18 Created: 2015-12-18 Last updated: 2016-02-02
3. The Mutational Landscape in Pediatric Acute Lymphoblastic Leukemia Deciphered by Whole Genome Sequencing
Open this publication in new window or tab >>The Mutational Landscape in Pediatric Acute Lymphoblastic Leukemia Deciphered by Whole Genome Sequencing
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2015 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 36, no 1, 118-128 p.Article in journal (Refereed) Published
Abstract [en]

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well-defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B-cell precursor patients, of which one carried the t(12;21)ETV6-RUNX1 translocation and two lacked a known primary genetic aberration, and one T-ALL patient. We found that each patient had a unique genome, with a combination of well-known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non-coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

National Category
Medical Genetics Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-238183 (URN)10.1002/humu.22719 (DOI)000347076700016 ()25355294 (PubMedID)
Available from: 2014-12-10 Created: 2014-12-10 Last updated: 2017-12-05Bibliographically approved
4. Distinct mutational spectrum in genetic subtypes of pediatric acute lymphoblastic leukemia uncovered by deep targeted sequencing
Open this publication in new window or tab >>Distinct mutational spectrum in genetic subtypes of pediatric acute lymphoblastic leukemia uncovered by deep targeted sequencing
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-269757 (URN)
Available from: 2015-12-18 Created: 2015-12-18 Last updated: 2016-02-02

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Lindqvist, Carl Mårten

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