Introduction: Recent work has demonstrated that relapses in pediatric acute lymphoblastic leukemia (ALL) can arise from minor subclones present at diagnosis. Several genes have been associated with ...therapy resistance in these subclones, including the Ras pathway genes KRAS, NRAS, and PTPN11, the H3K36 methyltransferase NSD2 (WHSC1), and the 5'-nucleotidase NT5C2. Retrospective backtracking of these relapse-associated alterations has demonstrated that these alterations are frequently present at time of diagnosis in minor subclones, sometimes in less than a few percent of the cells. The prognostic value of subclonal alterations in these genes at time of diagnosis, however, is less well understood. Prospective screening of subclonal mutations, without prior knowledge of the mutation status, requires extra specificity and sensitivity. Accurate quantification of the subclonal burden of these mutations will provide potential for following the subclonal dynamics during early stages of treatment, and could be informative for adapting therapy. The aim of this study was to develop a targeted next generation sequencing assay to perform quantitative detection of subclonal mutations in the selected genes. We used single molecule molecular inversion probes (smMIPs), an approach that applies single molecule tagging to correct for amplification biases (Hiatt et al., Genome Research. 2013, 23: 843-854), an artifact that becomes relevant in case of low mosaic mutations.
Method: We designed a pool of 77 smMIP oligonucleotides targeting the coding sequences of five genes associated with therapy resistance in BCP-ALL, including KRAS, NRAS, PTPN11, NT5C2, and WHSC1. The smMIPs tiled a total of 4124bp of genomic sequence, including hotspot regions of the genes. To demonstrate the potential of this method, we applied this newly designed smMIP panel on 22 BCP-ALL diagnosis samples to retrospective backtrack mutations in KRAS (n=11), NRAS (n=8) and PTPN11 (n=3) that were previously characterized at relapse. We used 100ng of genomic DNA per sample as input, which is the equivalent of 15,000 haploid copies. Sequencing was performed on the Illumina NextSeq platform with pair-end sequencing, data were analyzed by SeqNext v4.2.2.
Result: The average read depth obtained varied per gene from 30,081x (NRAS) to 65,749x (PTPN11). Sequencing reads with the same molecular tag were clustered into one tag-defined read group, in which random errors caused by library construction and sequencing were eliminated. These so-called single molecule consensus reads (smc-reads) were comprised of, on average, 139 individual sequencing reads. Using the smMIP approach, 19 out of the 22 Ras pathway mutations identified at relapse were detectable at diagnosis, of which 10 had a low mutant allele frequency (varying from 0.52-8.31%), which is in line with our previous ultra-deep backtracking result. Taking advantage of the known position of the mutations at relapse, we established the noise level in the diagnosis samples by analyzing variant calls outside the hotspot regions. The noise level was varied between samples from 0.03% to 0.24% (average 0.06%). Based on these background settings, we subsequently searched for novel mutations and identified 1 mutation in NT5C2 (p.P534S, 0.38%), 2 hotspot mutations in WHSC1 (p.E1099K, 0.17% and 0.27%), as well as many additional subclonal mutations in KRAS, NRAS and PTPN11. The latter finding suggests the presence of multiple Ras-mutated subclones in individual cases, of which only a subset survive from chemotherapy and grow out in the relapse clone.
Conclusions: Taken together, single molecule tagging based smMIP technology allows the accurate detection of low mosaic mutations. These findings illustrate the need for the current ongoing prospective mutation screens in unbiased cohorts of diagnosis samples to determine the prognostic value of subclonal mutations in these five genes on the risk of relapse.
No relevant conflicts of interest to declare.
Relapse represents the most common cause of therapy failure in B-cell precursor ALL acute lymphoblastic leukemia (BCP-ALL), and is caused by selective outgrowth of therapy-resistant leukemic cells. ...Two-third of BCP-ALL relapses present after treatment, i.e. after two years. These relapses may originate from leukemic (sub)clones that remained in a quiescent state during treatment or that could not be reached by the chemotherapeutics. Relapses that occur during treatment are different in that they display clonal outgrowth in the presence of chemotherapeutics, and these patients have poorer outcomes. The aim of this study is to explore the genomic abnormalities in leukemia with early relapse, and investigate the clonal dynamics of relapses that arise during treatment.
We included 17 BCP-ALL cases which relapse during treatment (<2yrs) according to DCOG protocols ALL9, ALL10 or ALL11. Median remission time was 1.08 yrs (range 0.48-1.95). Whole exome sequencing was performed on DNA isolated at time of first diagnosis, complete remission and relapse from bone marrow or peripheral blood, with an average read depth on target of 108x. After mapping of the reads, variants were called using HaplotypeCaller. In total, we identified 1771 somatic mutations in 1562 genes. Per case, a median of 21 mutations were detected at diagnosis (range 10-630) and 31 at relapse (range 10-652). A hypermutation profile was observed in one diagnosis-relapse pair, and two additional relapses.
All cases harbored mutations shared between diagnosis and relapse, which were mostly part of the major clone at both time points. However, the fraction of shared mutations varied considerably between cases, ranging from <10% in 3 cases to >80% in 4 cases. Based on the clonal dynamics, 3 distinct groups were recognized. Group I includes two cases, with relapses within 6 months, in which the (sub)clonal mutation spectrum between diagnosis and relapse was identical. Group II (n=10) presented with a relapse closely resembling the major clone at diagnosis. Mostly, these relapses acquired new mutations and they often branched off from the major clone already before the time of diagnosis. Finally, Group III (n=5) consists of cases in which the relapse originates from a minor subclone at diagnosis that hardly resembled the major clone, suggesting a clonal switch during treatment.
Next, we analyzed the genes with mutations that were predicted to be damaging (truncating and non-synonymous conserved missense variants). We performed pathway analysis for these genes and identified RAS pathway genes to be frequently mutated among shared mutations, while mutations in genes involved in epigenetic regulation, chromatin condensation and regulation of transcription were acquired. In total, 7 of the genes with (predicted) pathogenic mutations in relapse were affected in at least two cases, including known genes like KRAS, CREBBP, and WHSC1 (NSD2). CREBBP mutations were never part of the major clone at diagnosis and were present in cases with numerical chromosomal aberrations. Both cases with hotspot E1099K mutation in WHSC1 were t(1;19) translocation-positive.
Recent studies showed that somatic mutational signatures, composed of the six substitution subtypes in a 3-nucleotide context, expose specific biological processes underlying tumor development, including defects in genomic maintenance and repair. Currently 30 mutational signatures have been described http://cancer.sanger.ac.uk/cosmic/signatures. Despite the low number of mutations in most samples, we identified at least 5 of these signatures, including the most common signature 1, a signature associated with aberrant AID/APOBEC activity (signature 2), and three signatures associated with mismatch repair deficiency (6, 15, 26). Most cases carried multiple signatures, but signature 2 was very prominent is one relapse and one diagnosis-relapse pair. Most signatures were preserved from diagnosis to relapse suggesting that the same mutational processes remained active.
Taken together, our results show considerable heterogeneity in the group of children with early relapse of BCP-ALL. Two cases with the shortest remission times relapsed without notifiable somatic changes, whereas most other early relapses appeared to arise from minor or newly appearing subclones. These findings demonstrate the strong clonal selection that occurs during treatment in cases with very early relapse.
No relevant conflicts of interest to declare.
B cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common malignancies in children. In the period 1991-2013, the Dutch Childhood Oncology Group (DCOG) has completed three ...treatment trials for childhood ALL: ALL8, 9 and 10, each protocol with stratifications into risk-groups (details: www.skion.nl). Although the cure rates increased in these subsequent trials, relapses still occurred in a significant number of children. Since consecutive upfront treatment protocols usually change at multiple levels, genomic alterations that are associated with relapse may also be variable, which could provide insight into the biology underlying therapy failure and relapse. In this study, we characterized the genetic architecture of relapsed BCP-ALL patients within the context of these three Dutch upfront protocols.
We identified 3 patient groups based on upfront treatment as follows: Group-1: patients treated upfront with high-amounts of corticosteroids (CS) and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). The number of high-risk patients that relapsed after ALL8 HR and ALL10 HR chemotherapy courses was too low to be included for analysis.
We determined, at relapse, the presence of copy number alterations and sequence mutations in 21 recurrently affected genes involved in B-cell development, cell cycle regulation and RAS signaling, in 123 patients that relapsed after treatment in group-1 (n=56), group-2 (n=20) and group-3 (n=47). The number of CREBBP mutations in patients that relapsed after treatment according to group-1 (ALL9) was significantly lower compared to the other two groups, whereas B-cell development alterations were most common in patients that relapsed after treatment according to group-1, mainly due to a higher number of IKZF1 alterations (Figure 1).
The high number of relapsed patients with leukemic clones carrying IKZF1 alterations in patients treated with high-amounts of CS and relatively mild additional chemotherapy is in line with our recent finding that IKZF1 is a key determinant of GC-induced apoptosis in normal and leukemic B-cells, and that loss of IKZF1 function confers resistance to dexamethasone, the major treatment component in group-1 (Marke et al., submitted). Additionally, in the group-2 patients treated with high-amounts of CS and highly intensive additional chemotherapy, a lower percentage IKZF1-deleted clones was detected at relapse, indicating that more GC-resistant, IKZF1-deleted clones are killed by the intense chemotherapy given in addition to CS in group-2 patients. Similarly, in the group-3 patients relapsing after treatment with lower amounts of CS and moderately-intensive additional chemotherapy, the percentage of surviving IKZF1-deleted clones was lower than in patients treated with high-amounts of CS.
Taken together, our data indicate that the genetic architecture of relapsed BCP-ALL patients depends on the upfront treatment and, in addition, that the poor-prognostic feature of IKZF1-deletions may be more prominent in upfront treatment with high-amounts of CS and relatively mild additional chemotherapy.
Display omitted
No relevant conflicts of interest to declare.
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common cancers in children. The relapse incidence varies between 15% and 25%, dependent on the treatment protocol. It has ...been demonstrated that the presence of mutations in the RAS pathway genes, which regulate signal transduction upon binding of ligand to a variety of membrane receptors, are frequently associated with high-risk ALL and may be relapse drivers (Zhang et al., Blood 2011 118:3080-7; Irving et al., ASH 2013). Relapses in ALL are thought to result from the outgrowth of therapy-resistant residual leukemia cells and recent studies have shown a complex, dynamic architecture of clonal diversity in ALL (Anderson et al., Nature 2011 469:356-61; Notta et al. Nature 2011 496:362-7). In this study, we have investigated the clonal origin of RAS pathway mutations in relapsed BCP-ALL.
We screened 146 relapse samples from children with relapsed BCP-ALL for mutations in five RAS pathway genes using IonTorrent sequencing with a read-depth of approximately 150x. A total of 30% of relapse samples carried RAS pathway mutations, including mutations in KRAS (n=22), NRAS (n=13), PTPN11 (n=8), and FLT3 (n=2). No RAS mutations were found in 103 patients, whereas one patient had both a PTPN11 and a KRAS mutation, and another patient had two KRAS mutations. For 28 mutations we collected matched diagnosis samples and determined mutation presence using Sanger sequencing. We found that 12 of the RAS mutations were also present at diagnosis (43%) and 16 were initially not detected at diagnosis (57%). In order to gain more insight into the clonal evolution of relapse development, we performed amplicon based ultra-deep sequencing on the diagnosis samples, with an average read-depth of 50,000x for each mutation. The ultra-deep sequencing allows for sensitive and accurate detection of relapse-prone clones at diagnosis. A total of 22 mutations were identified at diagnosis samples (79%), of which 10 mutations had a low mutant allele frequency (average 3.8%), and were initially missed by Sanger sequencing (Fig.1). The 12 mutations determined using Sanger sequencing were detected at an average mutant allele frequency of 30.8%. We were unable to detect 6 mutations in the matched diagnosis samples, indicating that these mutations were newly acquired in the relapse clone at a time point after relapse. Our results indicate that cells harboring RAS pathway mutations are recurrently present in subclones at diagnosis. These cells may survive initial therapy and subsequently emerge at relapse. Patients with (minor clones with) RAS pathway mutations identified by ultra-deep sequencing, may benefit from treatment with MEK inhibitors added to the frontline therapy strategy.
Display omitted
No relevant conflicts of interest to declare.
Introduction: Despite risk stratification according to presenting clinical and genetic features, 10-25% of children with acute lymphoblastic leukemia (ALL) relapse, which is associated with a poor ...prognosis. Here, we sought to provide a comprehensive overview of the genetic alterations associated with relapse in ALL.
Methods: We studied 93 children (27 female, 43 male) diagnosed with ALL (62 B-progenitor, 25 T-lineage) between 1987 and 2008 and treated on total therapy studies XI-XVI who experienced relapse and/or a second tumor. Age at diagnosis ranged from 3 months to 18 years. Median time to relapse was 3 years (range 3 months to 10 years). Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B-cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis, relapse and matched normal samples (n=299) were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing.
Results: We found 2692 copy number aberrations (CNAs) with a median of 9 (range 0-109) in the diagnosis samples (n=91) compared to a median of 10 (range 0-112) in the relapse samples (n=89) and 12 (range 0-70) in subsequent samples (n=20). The number of CNAs did not differ significantly between diagnosis, relapse or subsequent samples.
We identified a 7286 non-silent single nucleotide variants (SNVs) and small insertions or deletions (indels) in 5002 genes, 1392 of which were recurrent. The median number of variants was 12 (range 0-70) at diagnosis (n=91), 21 (range 0-858) at relapse (n=91; P=0.0029 v. diagnosis) and 60 (range 10-650) in subsequent samples (n=20; P<0.0001 v. diagnosis). A subset of samples revealed very high (variant range 658-1703; 7 cases, 9 samples: all relapse or subsequent samples), or high mutation burden (variant range 104-290; 12 cases, 16 samples: 2 diagnosis, 10 relapse, and 4 subsequent samples). Genes encoding mediators of DNA repair were affected in all cases with very high mutation burden, compared to 7 of the high burden cases and 18 out of 72 other cases (most commonly genes TP53, MSH2, and MUTYH; P<0.0001).
The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Of the recurrently altered genes, only 87 genes were known to be affected in cancer (Cancer Gene Census, COSMIC database), of which 59 were affected in leukemia and lymphoma tissues, indicating that we have identified 1306 novel recurrently affected genes, most commonly C13orf40 and MKI67. Mutations in epigenetic regulators were particularly frequent, with genes mutated in at least 3 cases altered in over 60% of the cohort (e.g. CREBBP, EP300, MLL2, MLL3, KDM6A/B, CTCF, SETD2, TET2/3, and EZH2).
Clonal evolution analyses showed multiple patterns of evolution, with relapses sharing either few or many variants with the diagnosis sample in a frequency that reflects both predominant clones and minor subclones propagating relapse. Variants in NOTCH1, NRAS, and CREBBP were preserved from a major clone at diagnosis in 4, 6, and 5 cases respectively, but acquired at relapse or grown out from a minor subclone at diagnosis in 3, 5, and 8 cases respectively. In contrast, variants in USH2A (n=4), FOXA1 (n=3), and purine/pyrimidine synthesis pathway genes NT5C2 (n=3), PRPS1 (n=3) and NT5C1B (n=1) were exclusively found in relapse samples. Notably, the NT5C2 mutations, which are thought to confer resistance to thiopurines, were subclonal at relapse in the majority of cases.
We identified 13 cases (10 B-lineage, 3 T-lineage) in which the diagnosis and relapse were fully discordant for all CNAs and sequence mutations, only 4 of which showed a prolonged remission time (>5 years). This suggests that these patients developed a second primary malignancy and may be predisposed to leukemia development. Indeed, one case revealed focal amplifications on chromosome 1q21.1 encompassing the neuroblastoma breakpoint family genes, which are implicated in cancer development. Comprehensive germline analyses are underway.
Conclusion: This study has provided detailed insight into the genetic basis of relapse, implicating multiple new genes and pathways involved in treatment resistance, demonstrating multiple patterns of clonal evolution, and revealing an unexpectedly high frequency of genetically discordant second malignancy in relapse in ALL.
Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.
Despite high survival rates for children with acute lymphoblastic leukemia (ALL), only 40% of adult patients will achieve long-term disease-free survival, and relapses in both pediatric and adult ALL ...are often fatal. Most current therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells, yet recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. The functional properties and clinical relevance of these neoplastic subclones remain undefined. Genome wide copy number analysis of matched diagnostic and relapse ALL samples identified that in 50% of patients, the clones present at relapse are not the dominant clones at diagnosis, but have evolved from an ancestral pre-leukemic clone (Mullighan et al., 2008). In order to investigate the functional consequences of clonal evolution in disease progression and therapy resistance, we performed limiting dilution analysis of 3 diagnostic and 14 paired diagnostic/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics, by transplantation into immune-deficient mice (xenografts). In one patient, the leukemia-initiating cell (LIC) frequency was 7.65 fold higher in the relapse sample than at diagnosis, while another patient showed the reverse with a 5.81 fold higher LIC frequency in the diagnostic sample. Two patients showed no significant differences in LIC frequency from diagnosis to relapse. LIC frequency varied from 1 in 14.2 to 1 in 4802 CD19+ blast cells. Interestingly, in 50% of the paired patient samples, transplantation of cells from the relapse sample gave rise to greater leukemic dissemination to the spleen and/or central nervous system of recipient mice in comparison to the diagnostic sample, despite similar levels of engraftment in the bone marrow. This data suggests that although the LIC frequency in B-ALL remains high and relatively static between diagnosis and relapse, relapse cells acquire increased invasive properties. To investigate the clonal composition of 3 diagnostic B-ALL samples, we undertook copy number variation (CNV) analysis of xenografts generated at both limiting and high transplanted cell doses. In all 3 samples, we detected subclones in the xenografts that were distinct from the predominant clone in the primary patient sample. We performed network analysis on these subclones and identified differentially enriched pathways, including differential expression of anti-apoptotic and apoptosis regulation pathways, providing evidence of putative functional differences. These results support the existence of functionally diverse subclones within diagnostic samples as well as functional diversity between the subclones present at diagnosis and relapse. Ongoing in depth genomic analysis of the diagnosis/relapse paired samples will add to our understanding of the functional role of the subclones identified at diagnosis in the establishment of disease relapse. In summary, these experiments will provide further insight into the functional heterogeneity present in B-ALL and the drivers of lymphoid leukemogenesis that lead to therapy failure and disease relapse.
Danska:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.
Introduction. Prior studies have described a subset of B-progenitor ALL cases with a distinct gene expression profile and/or deletions involving ERG (encoding the ETS family member v-ets avian ...erythroblastosis virus E26 oncogene), however the relationship of these alterations and their role in leukemogenesis are poorly understood. We performed integrated genomic and epigenetic analyses, biochemical studies and leukemogenesis assays to define the genetic basis of this form of ALL.
Methods. We studied 1674 childhood, adolescent and young adult B-progenitor ALL cases with microarray gene expression profiling and/or RNA-sequencing data to enable the identification of ERG ALL by unsupervised clustering and predictive analysis of microarrays. Detailed genomic analysis was performed for 144 ERG ALL cases, including whole genome (N=38), exome (n=46) and/or RNA-sequencing (n=57) cases, and single nucleotide polymorphism array analysis. Epigenetic profiling, including whole genome bisulfite sequencing, chromatin immunoprecipitation and sequencing for ERG and histone modifications and ATAC-sequencing were performed for a subset of 8 xenografted ERG tumors and reference cell lines. ERG transcript expression was measured by analysis of RNA-seq analysis and quantitative RT-PCR assays, and by interrogation of TCGA and PCGP RNA-seq data. The function of ERG isoforms was evaluated by EMSA and transcriptional reporter assays, immunofluoresence, colony forming assays and retroviral bone marrow transplant assays.
Results. One hundred and forty four cases (8.6%) of B-ALL cases exhibited a distinct gene expression profile and lacked known chromosomal rearrangements (ERG ALL). Such cases had favorable outcome. Eighty cases (55.6%) had focal deletions of ERG with no evidence of oncogenic or chimeric ERG fusions. The deletions were most commonly heterozygous and involving exons 3-7 (n=27) or 3-9 (n=22) of 10 coding exons, and less commonly involving exon 1, or a larger region of the gene. No ERG deletions were identified in non-ERG ALL. Two cases harbored missense mutations in the ETS domain. Analysis of whole genome and exome sequencing data of 71 cases identified a high frequency of alterations of lymphoid transcription factors (46.5%; IKZF1 36.7%, PAX5 11.3%); mutation of transcription factors otherwise uncommon in ALL (21%; MYC, MYCBP2, MGA, ZEB2, GATA3); activation of signaling pathways, most commonly NRAS or KRAS (35.2%); cell cycle regulation (22.5%); and epigenetic modifiers (56.3%), most commonly KMT2D, SETD2, ARID2 and NCOR1. Notably, the five year event-free survival of ERG ALL cases with IKZF1 alterations exceeded 85% in both St Jude and Children's Oncology Group cohorts.
We observed striking transcriptional deregulation at the ERG locus. Most (51/56) ERG- deleted cases expressed an ERG isoform encoded by a novel exon in intron 6 that splices in frame to distal exons, resulting in expression of a truncated C-terminal ERG protein that lacks the pointed and central regulatory domains, but retains the ETS and transactivation domain (ERGalt). ERGalt was also present in most (36/44) cases lacking an ERG deletion, and was strongly associated with presence of ERGalt protein in leukemic cells. We also identified expression of an Antisense Long non-coding RNA associated with the ERG locus (ALE) in ERG ALL. ERGalt and ALE were absent, or uncommonly expressed at very low levels in non-ERG ALL. ERGalt was absent, and ALE rarely expressed in non-ALL PCGP and TCGA samples.
ERGalt and point mutant ERG were retained in the nucleus, bound DNA targets and acted as competitive inhibitors of wild type (WT) ERG in transcriptional reporter assays. Lineage-negative Arf -null bone marrow cells transduced with ERG WT induced an aggressive erythro-megakaryoblastic leukemia; in contrast ERGalt induced an immature lymphoid progenitor leukemia.
Conclusions. Genomic alterations drive aberrant transcription of ERG, resulting on expression of a truncated, C-terminal oncogenic ERG protein. This represents a novel mechanism of transcription factor deregulation in leukemia. As a subset of ERG ALL cases lack ERG deletion, and as IKZF1 alterations are not associated with inferior outcome in this form of ALL, diagnostic approaches must incorporate gene expression profiling in addition to identification of ERG and IKZF1 alterations to accurately identify this form of leukemia.
Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Stock:Gilead: Membership on an entity's Board of Directors or advisory committees. Voorhees:Oncopeptides: Consultancy; Onyx Pharmaceuticals: Research Funding; GSK: Consultancy; Oncopeptides: Research Funding; Janssen: Research Funding; A Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy; Millennium Pharmaceuticals: Consultancy, Research Funding; Acetylon Pharmaceuticals, Inc.: Research Funding; Novartis: Consultancy; Array BioPharma: Consultancy; GSK: Research Funding; Celgene: Research Funding. Hunger:Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership. Mullighan:Incyte: Consultancy; Amgen: Honoraria.
PDF
