Recurrent somatic mutations and low germline predisposition mutations in Korean ALL patients

In addition to somatic mutations, germline genetic predisposition to hematologic malignancies is currently emerging as an area attracting high research interest. In this study, we investigated genetic alterations in Korean acute lymphoblastic leukemia/lymphoma (ALL) patients using targeted gene panel sequencing. To this end, a gene panel consisting of 81 genes that are known to be associated with 23 predisposition syndromes was investigated. In addition to sequence variants, gene-level copy number variations (CNVs) were investigated as well. We identified 197 somatic sequence variants and 223 somatic CNVs. The IKZF1 alteration was found to have an adverse effect on overall survival (OS) and relapse-free survival (RFS) in childhood ALL. We found recurrent somatic alterations in Korean ALL patients similar to previous studies on both prevalence and prognostic impact. Six patients were found to be carriers of variants in six genes associated with primary immunodeficiency disorder (PID). Of the 81 genes associated with 23 predisposition syndromes, this study found only one predisposition germline mutation (TP53) (1.1%). Altogether, our study demonstrated a low probability of germline mutation predisposition to ALL in Korean ALL patients.

B-cell precursor and T-cell precursor acute lymphoblastic leukemia/lymphoma (B-ALL, T-ALL) are two of the most common malignancies in children. ALL can be classified by genetic alterations, which are highly various and heterogeneous. Chromosome aneuploidy, structural alterations and rearrangements, copy number variations (CNVs), and sequence mutations all contribute to leukemogenesis. In 2016, the fourth edition of the World Health Organization (WHO) classification of lymphoid and myeloid neoplasms and acute leukemia included new provision entities of ALL: BCR-ABL1-like (or Ph-like) ALL, iAMP21 (intrachromosomal amplification of chromosome 21), and early T-cell precursor ALL (ETP-ALL) 1 .
BCR-ABL1-like ALL is a high-risk form of ALL with its peak incidence in young adults. IKZF1 deletions, mutations of JAK-STAT and RAS signaling genes (NRAS, KRAS, PTPN11, NF1, etc.), and structural rearrangements (CRLF2, ABL-class tyrosine kinase genes, JAK2, EPOR, etc.) have been identified in this group 2 . iAMP21 accounts for about 2% of ALL in older children, and it is associated with a number of adverse outcomes 3 . In iAMP21, three or more additional copies of RUNX1 (AML1) are observed on chromosome 21 in metaphase fluorescence in situ hybridization (FISH). ETP-ALL is defined as CD1a − , CD8 − , CD5 − (dim), and positive for one or more stem-cell or myeloid antigens. Genetic alterations in ETP are somewhat different than those in non-ETP, and FLT3, DNMT3A, and WT1 mutations are more often found in ETP than in non-ETP 4,5 .
In addition to somatic mutations, germline genetic predisposition to hematologic malignancies has emerged as an area of research interest. Genes found to be associated with predisposition to myeloid malignancy have been included in the WHO classification, "Myeloid neoplasms with germ line predisposition" 1 ; this category includes CEBPA, DDX41, RUNX1, ANKRD26, ETV6, GATA2, and others. A number of syndromes, such as bone marrow failure syndrome and telomere biology disorders, are also included in that category. One early study estimated that childhood leukemia with hereditary genetic causes accounted for 2.6% of all cancers 6 . Down syndrome (DS) is the most common underlying genetic predisposition for ALL 6,7 . There are a number of other syndromes that also increase susceptibility to ALL, such as Li Fraumeni (TP53), Bloom syndrome (BLM), Wiskott Aldrich www.nature.com/scientificreports/ PID-associated germline sequence variants. Five PID-associated gene variants were identified in five patients (Supplement Table S1). All these variants were heterozygous autosomal recessive (AR) PID associated variants. Three variants (IL12RB1, CTC1, and LPIN2) have yet to be published, while the other variants (TYK2 and LIG4) were known variants.

CDKN2A/B by NGS and FISH. FISH for CDKN2A/B was performed upon the initial diagnosis of ALL.
We compared the CDKN2A/B results between the FISH and NGS CNVs analyses. The overall agreement rate for CDKN2A/B was 83.7% (Table 4). Nine cases were positive for CDKN2A/B deletion according to NGS, but negative by FISH. Meanwhile, six cases were normal by NGS analysis, but deletion/duplication was confirmed by FISH.

Clinical effects of genetic alteration. Overall survival and relapse-free survival. Overall survival (OS)
and relapse-free survival (RFS) are shown by cytogenetic groups in Fig. 3. There were statistically significant differences in OS and RFS in childhood ALL, but not in adult ALL.
Clinical impact of IKZF1. IKZF1 alteration had adverse effects on OS and RFS only in childhood ALL ( Fig. 4). No other genes had a consistent clinical effect in childhood or adult ALL (data not shown).

Discussion
NGS technology has been applied to a number of hematologic diseases. Many gene panels and several methods have been used to detect not only sequence variants, but also large gene deletions and duplications or gene fusions [15][16][17][18][19] . In this study, we found a significant agreement rate between NGS and FISH for CDKN2A/B CNV detection. We also found significant IKZF1 deletions using an NGS CNV analysis. A presumed diagnosis of BCR-ABL1-like ALL can be enabled using an NGS CNV analysis to test for genetic alterations in IKZF1 and JAK1/ JAK2 because the former (68%) and latter (55% among patients with CRFL2 rearrangement) are more prevalent in BCR-ABL1-like ALL than in other B-ALL sub-types 2 . Although we did not perform gene expression profiling and FISH, or RT-PCR for alterations commonly found in BCR-ABL1-like ALL, we found seven cases with IKZF1 alterations in non-ALL with BCR-ABL, and these showed adverse clinical effects. We assume that the seven non-ALL patients with BCR-ABL1 and an IKZF1 alteration are likely BCR-ABL1-like ALL.
Skin fibroblasts are the only recommended control sample for germline mutations, because peripheral blood (PB) and bone marrow can be contaminated with leukemic cells, while other samples, such as saliva or buccal swab, can be also contaminated with PB. Further, clonal hematopoiesis can be observed in ~ 10% of the healthy population, and this rate increases with age 25 . However, a skin biopsy is an invasive procedure, and as a result, such samples are not readily available. We therefore used CR-state bone marrow slides (acquired to test for residual leukemic cells) as the control for germline mutations. No apparent leukemic samples were obtained in our review of bone marrow morphology, or in the FISH, chromosome, flow-cytometry, and RT-PCR results. The variant allele fraction (VAF) and public population databases were used as filtering tools 26 . Germline variants can have a VAF of > 33%, even in tumor samples 26 ; variants within that range have a high possibility of germline origin. Some presumably somatic variants registered in public databases, such as the Single Nucleotide Polymorphism database (dbSNP), the 1000 Genomes Project, the Exome Aggregation Consortium (ExAC) database, and the Human Gene Mutation Database (HGMD), may be of germline origin. We double checked the germline variants in both CR and leukemic samples. True germline variants (identified in CR samples) were also identified in paired leukemic samples with similar VAF. By contrast, true somatic variants (identified in leukemic samples) were either not found, or were found with very low VAF (< 1%) in paired CR.
Various syndromes increase the risk of ALL, with variable penetrance and preference. DS is the most common genetic cause of childhood leukemia. In an analysis of the National Registry of childhood tumors in the   26 .
In this study, we did identify only one pathogenic variant (TP53) among 81 genes associated with 23 syndromes that are well known for their connection to hematologic malignancy. For example, Li-Fraumeni syndrome (TP53) is a well-known rare cancer syndrome. The most common cancers in patients with Li-Fraumeni syndrome are solid cancers (such as breast cancer, lung cancer, and bladder cancer) 12 . However, a somatic TP53 alteration is strongly associated with low hypodiploidy ALL (~ 90%), disease relapse, and germline origin (~ 40%) 29 . Although hypodiploid ALL accounts for only 5% of childhood ALL cases, hypodiploid ALL patients should be tested for Li-Fraumeni syndrome because of its poor prognosis and the possibility of a germline TP53 mutation 30 .
We found germline copy number variation in CASP10. CASP10 is a causative gene for autoimmune lymphoproliferative syndrome (ALPS) type IIa, which is a very rare PID (primary immunodeficiency disorder). However, the association between exonic deletion of CASP10 and leukemia in our patient is unclear in this study.   www.nature.com/scientificreports/ In a previous study, similar exonic deletion of CASP10 had only been found in a patient with systemic juvenile idiopathic arthritis with incomplete penetrance (healthy relative with the same CNV) 13 . In this case, we could not find any medical history associated with ALPS in our patient. More than 300 distinct disorders and genes of PID have been classified by the International Union of Immunological Societies PID expert committee 31 . An increase in leukemia/lymphoma with PID (including ALPS) is well known and expected 32 . Although the mechanism of leukemogenesis in PID remains unclear, intrinsic (cancer predisposition parallel to the immunological defect) and extrinsic (chronic infections, inflammation, or loss of immunosurveillance) mechanisms have been proposed by Hauck et al. 33 . Notably, in this study, we identified five PID-associated sequence variants and one CNV. All these sequence variants were heterozygous autosomal recessive PID associated variants. Therefore, the association between these variants and leukemia in our patients is unclear. However, some studies have reported an increased risk of cancer in heterozygous carriers of autosomal recessive PID associated variants, heterozygous BLM (Bloom syndrome, AR) mutations, and  www.nature.com/scientificreports/ heterozygous ATM (Ataxia-telangiectasia, AR) mutations 34,35 . Moreover, Qin, N. et al. have reported a risk of subsequent cancer among long-term survivors of childhood cancer with germline pathogenic/likely pathogenic mutations (DNA repairs genes which are mostly included in PID-associated genes, such as BLM, FANCA, BRCA2 LIG4, NBN, etc.) 36 . Therefore, further studies should continue elucidating these uncertain significant variants of PID-associated variants. Our study had several limitations. First, we enrolled a relatively small number of cases. Second, we did not use skin fibroblasts in our search for germline mutations. Third, we did not perform a familial study or any clinical or physical investigations of the identified germline variants.

Conclusion
We found recurrent somatic alterations in Korean ALL patients. Further, we identified the low probability of germline mutation predisposition in unselected sporadic Korean ALL patients. We also demonstrated the usefulness of NGS technology, which provides comprehensive genetic information.

Methods
Study population and samples. We selected paired initial-diagnosis and complete remission (CR) bone marrow samples from patients diagnosed with ALL at Samsung Medical Center from 2008 to 2012.
The Institutional Review Board at Samsung Medical Center approved this study (IRB No. 2015-11-053), and informed consent was obtained from all participants. All experiments were performed in accordance with the relevant guidelines and regulations.
To detect germline mutations, we used bone marrow slides that were obtained when the patients were in CR. In total, 31 (33.3%) patients received allogenic stem cell transplantation. CR status bone marrow slides before allogenic stem cell transplantation were used to accurately detect patient germline mutations. The morphology, chromosome, FISH, and immunophenotyping results were reviewed, and bone marrow slides with no apparent residual leukemic cells were selected as control samples.
Conventional study. A chromosome study was conducted using a standard method, and the karyotypes are described according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed to detect recurrent translocation (HemaVision kit, DNA Technology, Aarhus, Denmark). FLT-ITD mutation analyses (by fragment length polymorphism) and FISH for CDKN2A/B were performed as well.
Targeted gene sequencing. Gene panel. From a literature review, we selected 500 genes found to be significantly mutated in ALL (Supplement Table S5). Our gene panel 17  Data analysis. The data analysis was conducted using previously described methods (Supplement Fig. S9) 17 .
After sequencing, we aligned the reads to human genomic reference sequences (GRCh37) using the Burrows-Wheeler alignment tool. The Genome Analysis Tool Kit (Broad Institute) was used for variant calling. Pindel was used for crosscheck insertion and mutation deletion. All mutations were annotated using ANNOVAR and VEP software. Variants were further examined by visual inspection using the Integrative Genomic Viewer. Annotated variants were classified using automated algorithm software, DxSeq Analyzer (Dxome, Seoul, Korea), by applying the standards and guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology 37 . ExomeDepth (1.1.10), an R package, was used to detect exon-and genelevel CNVs in target regions, followed by visualization using a base-level read depth normalization algorithm www.nature.com/scientificreports/