Targeted re-sequencing analysis of 25 genes commonly mutated in myeloid disorders in del(5q) myelodysplastic syndromes

Abstract

Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at great depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 cases of early or advanced del(5q) myelodysplastic syndromes. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases had at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in cases of 5q- syndrome (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. Fifty-two percent of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations had allele frequencies <20%, likely below the detection limit of traditional sequencing methods. Genomic array data showed that cases of advanced del(5q) myelodysplastic syndrome had a complex background of cytogenetic aberrations, often encompassing genes involved in myeloid disorders. Our study is the first to investigate the molecular pathogenesis of early and advanced del(5q) myelodysplastic syndromes using next-generation sequencing technology on a large panel of genes frequently mutated in myeloid malignancies, further illuminating the molecular landscape of del(5q) myelodysplastic syndromes.

Introduction

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies that are characterized by ineffective hematopoiesis resulting in peripheral cytopenias, and typically a hypercellular bone marrow. The MDS are pre-leukemic conditions showing frequent progression (in approximately 40% of patients) to acute myeloid leukemia (AML). In the early stages of the disease, apoptosis of bone marrow precursor cells prevails, but in more advanced disease increased proliferation of immature blasts occurs.1 About 50% of MDS exhibit acquired genomic abnormalities detected by conventional cytogenetic banding techniques. Recent molecular investigations have revealed additional genetic abnormalities in MDS, including micro-deletions and loss of heterozygosity due to acquired uniparental disomy (UPD).2

Interstitial deletion within the long arm of chromosome 5 [del(5q)] is one of the most frequent cytogenetic abnormalities observed in myeloid malignancies, occurring in approximately 10–20% of patients with de novo MDS3 and in a similar proportion of patients with de novo AML.4 In de novo MDS the del(5q) occurs either in isolation or together with other karyotypic abnormalities. Although the 5q- is a good prognostic indicator when found in isolation,5 this is not the case when the 5q- is part of a complex karyotype.6 In a large MDS database, del(5q) was reported as an isolated abnormality in 14% of patients with clonal abnormalities, in 5% with one other abnormality, and in 11% with a complex karyotype.6 The median overall survival in these groups was 80, 47 and 7 months, respectively.6 These findings are consistent with the general notion that the total number of cytogenetic changes found is an independent factor that can allow for the stratification of cohorts of patients into prognostic subgroups.

The 5q- syndrome is the most distinct of all the MDS and is characterized by isolated del(5q), severe macrocytic anemia, frequent thrombocytosis, female predominance, and a lower risk of progression to AML.7 Patients with the 5q- syndrome have one of the best outcomes of any MDS subgroup,8 with a relatively long survival often of several years.7,8 While a small number of gene mutations have been reported in the 5q- syndrome, including mutations of TP53 and JAK2,9,10 the molecular landscape of this disease remains to be fully elucidated. Approximately 10% of patients with the 5q- syndrome show transformation to AML,7 but the genetic aberrations that drive this process have not been fully determined.

The International Prognostic Scoring System (IPSS)11 and its revised version8 are based upon karyotypic abnormalities as well as on morphological data. Recently, a new and more comprehensive cytogenetic scoring system has been developed, which allows for a refined cytogenetic risk prediction.12 Nevertheless, the heterogeneous clinical outcome observed within the karyotypically and morphologically-defined groups in the IPSS indicates that it may be possible to refine the cytogenetic classification by using additional markers. The catalogue of genes that play a role in the molecular pathogenesis of MDS is rapidly expanding, and includes TET2, SF3B1, EZH2 and ASXL1.13–16 Unraveling the genetic complexity of MDS promises to elucidate the pathophysiology of this disease, refine the taxonomy and prognostic scoring systems, and provide novel therapeutic targets.

Technological advances in DNA sequencing provide an important tool to analyze heterogeneous cancer samples. Massively parallel sequencing enables the analysis of independent, clonal, DNA molecules17 and offers the opportunity to adjust the balance between breadth and depth of such assays to identify a wide variety of potentially critical DNA changes in tumors. Broad approaches, such as whole genome and whole exome sequencing, have been used to discover new cancer gene mutations15,18,19 or to study clonal evolution.20 In particular, several such studies in MDS have identified recurrently mutated genes and novel pathways involved in pathogenesis, such as those encoding splicing factors.15,19 However, these genome-wide approaches are still expensive and have relatively low sensitivity. In contrast, a more targeted sequencing approach aimed at detecting selected recurrent mutations in MDS allows for cost-effective and fast sequencing at the profound depth required for accurate characterization of heterogeneous cancer samples.

Here we describe the design, validation and application of a targeted next-generation sequencing approach using a bench-top platform to simultaneously screen 25 genes mutated in a range of myeloid malignancies. We used this method to characterize the mutational profile of a cohort of 43 MDS cases with del(5q).

Methods

Patients’ samples

Nine MDS samples, with known mutations detected by Sanger sequencing, pyrosequencing or amplification refractory mutation system polymerase chain reaction (ARMS-PCR) analysis in at least one of our target genes, were selected to validate our gene panel. The nine samples chosen contained a total of 13 variants across eight genes and included missense (n=4), nonsense (n=1) and frameshift (n=8) mutations (Table 1).

Samples from 43 untreated MDS cases harboring a del(5q) were selected for mutational screening (mean age 66.0 years; range, 24–88). These included 22 patients with 5q- syndrome, nine cases with refractory anemia (RA) with additional karyotypic abnormalities, and 12 cases with advanced MDS [defined as having an increased number of blasts and including 11 RA with excess of blasts (RAEB), and 1 chronic myelomonocytic leukemia (CMML) in transformation]. All karyotypes were determined by conventional G-banding. This study was approved by the ethics committees of the institutes involved and informed consent was obtained.

DNA extraction

Genomic DNA was isolated by phenol-chloroform extraction from peripheral blood neutrophils obtained using Histopaque (Sigma-Aldrich) and pelleted after hypotonic lysis of erythrocytes. The purity of the neutrophil populations was high, >95%, as assessed by standard morphology on Wright-Giemsa-stained cytospin preparations.

Targeted re-sequencing

We designed a TruSeq Custom Amplicon panel (TSCA, Illumina), targeting 25 genes mutated in various myeloid malignancies (Table 2). The panel was developed using the online DesignStudio pipeline (http://designstudio.illumina.com, Illumina), and covers a total of 46,604 bp with 322 amplicons. In genes with well-defined mutational hotspots only these regions were targeted; otherwise the entire coding sequence of the gene was sequenced. Libraries prepared from 250 ng DNA were subjected to 250 bp paired-end sequencing.

Protein sequences resulting from detected DNA-sequence changes were predicted using the insilico.ehu.es on-line tool,21 and Alamut Software (Interactive Biosoftware, San Diego, CA, USA). PolyPhen-2 v2.2.2, also on-line, was used to predict the functional effect of variant calls (Polymorphism Phenotyping v2, http://genet-ics.bwh.harvard.edu/pph2/).22

FLT3 internal tandem duplication fragment analysis

Thirty-three samples in the test cohort with sufficient DNA were screened for internal tandem duplications in the FLT3 gene (FLT3-ITD) using conventional fragment analysis.23 These comprised 20 cases of 5q- syndrome, five cases of del(5q) RA with additional karyotypic abnormalities and eight cases of advanced del(5q) MDS.

Genomic array profiling

Single nucleotide polymorphism array data were available from a previously published study2 for 33 of the 43 samples included in the targeted sequencing analysis. Those data allowed us to identify cryptic copy number changes and regions of UPD.

Results

Quality of the myelodysplastic syndrome del(5q) MiSeq sample run

The number of clusters that passed the quality filter was over 100,000 for the majority of the samples (40/43, 93%) (Online Supplementary Figure S1). Paired-end MiSeq sequencing produced more than 2.2 Gb of sequence data with 91% of reads higher than the quality threshold of Q30, exceeding the expected minimums of 2 Gb and 75%, respectively. The average depth of coverage across all samples was >390×, with 98% of cases (42/43) over 250×, 91% (39/43) over 300× and 49% (21/43) ≥400×. The overall sensitivity of the assay and its background noise were estimated at 1–3% (Online Supplementary Information and Online Supplementary Table S1).

Table 1A.Summary of mutations present in test samples used for the TSCA panel validation. All Q-score values were generated by GATK through the BaseSpace pipeline, with the exception of the TEST009 variant, which was generated by Platypus.

Table 1B.Summary of additional mutations found in the test samples.

Validation of the myeloid gene panel

In order to compare the accuracy and sensitivity of our TSCA assay against standard methods of mutation screening (Sanger sequencing, pyrosequencing, fragment analysis), we re-screened nine test samples (Table 1A), containing 17 variants across 11 genes (ASXL1, DNMT3A, EZH2, FLT3, IDH1, IDH2, KIT, NPM1, NRAS, RUNX1 and TP53).

Using the BaseSpace data analysis pipeline, we were able to successfully identify five missense, three nonsense and seven frameshift mutations in our validation cohort (15 out of 17, 88.2%). In particular, short indel (insertions/deletions) mutations in both ASXL1 and NPM1 (1 bp and 5 bp, respectively) were correctly identified by the BaseSpace analysis software. Analysis of the TEST009 aligned reads in the Integrative Genomics Viewer (IGV, Broad Institute), revealed a dramatically reduced read depth of 30× across TP53, compared to >1000× in other samples, suggesting that there was a failure to align the reads to the reference sequence. The TEST009 sequence data were therefore submitted to a second alignment and variant calling pipeline (Stampy/Platypus24,25), which successfully identified a 19 bp deletion (Online Supplementary Figure S2). Two 109 bp and 64 bp FLT3-ITD in samples TEST003 and TEST004, respectively, were only called after visual inspection of the un-aligned data for reads matching part of the FLT3 target sequence. The presence of FLT3-ITD was subsequently confirmed by fragment analysis.

In addition to the known control mutations, we identified six mutations affecting five genes in five samples (Table 1B). One of these mutations, the C1464X variant in TEST001, was visible in earlier Sanger sequencing traces; however, at the time, the variant had not been called as it was within the range of background noise (Online Supplementary Figure S3). All other additional mutations were confirmed by Sanger sequencing and fragment analysis (Online Supplementary Figure S4 and Online Supplementary Table S2).

Mutations detected in del(5q) cases

Highly purified peripheral blood neutrophil DNA samples from 43 MDS cases harboring a del(5q) were subjected to mutational screening using the 25-gene panel described above. A total of 4036 variant calls were detected by a combination of BaseSpace, Stampy/Platypus and visual inspection of the FLT3 locus. Of these, all non-synonymous variant calls with a COSMIC ID (i.e. recorded in the Catalogue of Somatic Mutations in Cancer26) were considered relevant. We also included in the analysis all non-synonymous variant calls not found in either COSMIC or the dbSNP database (build 135). A total of 29 non-synonymous variants were called in ten different genes: seven affecting TP53, six ASXL1, five TET2, two CBL, two DNMT3A, two SF3B1, two JAK2, one U2AF1, one RUNX1 and one WT1 (Table 3, Figure 1, Online Supplementary Table S3). In addition, 21 synonymous variants with a COSMIC ID were found in five different genes (10 PDGFRA, 5 IDH1, 3 cKIT, 2 FLT3 and 1 TP53) (Online Supplementary Table S4).

Table 2.List of genes targeted for enrichment in the TSCA library.

Distribution of the non-synonymous mutations among disease subgroups

A total of 29 mutations were detected in our cohort of 43 del(5q) MDS cases. Twelve of 29 mutations were found in nine of the 22 5q- syndrome cases (45.0%) (Table 3). Five mutations affected four of the nine cases of del(5q) RA (44.0%) with additional cytogenetic aberrations (Table 3). The more advanced del(5q) MDS cases presented a higher proportion of sequence changes: 12 variant calls were found in eight of the 12 advanced del(5q) cases (66.7%) (Table 3). The genes with the highest mutation frequency in this cohort were TP53 (3/12 patients, 25%; 5 mutations in total as 2 patients had 2 TP53 mutations) and ASXL1 (3/12, 25%). The mutation frequency for these two genes was lower in 5q- syndrome cases (TP53 1/22, 4.5%; ASXL1 3/22, 13.6%).

Other mutations were identified in 5q- syndrome cases (3 TET2, 2 SF3B1, 1 DNMT3A, 1 RUNX1 and 1 WT1), in del(5q) RA with additional cytogenetic abnormalities (1 additional TP53, 1 CBL, 1 DNMT3A, 1 U2AF1 and 1 JAK2) and in advanced del(5q) MDS cases (2 TET2, 1 CBL and 1 JAK2). It is of note that six of the mutations detected in this study present variant frequencies lower than 20%, which are likely to be below the level of detection of Sanger sequencing.27,28 These low frequency mutations were found in the following genes: two in TET2, one in ASXL1, one in DNMT3A, one in JAK2 and one in SF3B1 (Figure 1, Online Supplementary Table S3).

These data show that a number of different gene mutations occur in patients with the 5q- syndrome and that advanced del(5q) MDS cases display a greater mutation frequency than early del(5q) MDS cases, with mutations of TP53 and ASXL1 genes being the most frequent.

Co-occurring mutations: analysis of clonality and timing of mutation acquisition

Clonal evolution has been documented as MDS transforms to AML,29 and when de novo AML relapses after initial chemotherapy.30 The proportion of sequencing reads reporting a given mutation can be used to estimate the fraction of tumor cells carrying that mutation, and to identify whether mutations are clonal (present in all tumor cells) or subclonal (present in a fraction of tumor cells).31 This estimation needs to take into account copy number and loss of heterozygosity data. Five cases in our cohort showed mutations in more than one gene. Whole genome array data were available for all of them.2 The genes with co-occurring mutations were ASXL1, WT1, SF3B1, TET2, DNMT3A, JAK2 and CBL (Figures 1 and 2).

In two cases (1 5q- syndrome, MDS08, and 1 CMML, MDS42) two mutations were present at similar allele frequencies, ASXL1 (44.7%) and WT1 (49.0%) in the 5q- syndrome case, and ASXL1 (45.4%) and CBL (96.0%) - the latter within a region of UPD - in the CMML case. This is suggestive of a dominant clonal population of cells. In this scenario, it is not possible to determine the temporal order of mutations. A third case (MDS29, a del(5q) RA with additional karyotypic abnormalities) had a DNMT3A mutation at a variant allele frequency of ~44%, and a JAK2 mutation at a frequency of ~7%. Since the copy number showed these to have occurred in diploid regions without any loss of heterozygosity, the fraction of cells carrying the mutations would be ~88% and ~14%, respectively. On this basis, we could not infer if the JAK2 mutation was subclonal to the cells carrying the DNMT3A mutation or if, on the contrary, it represented an independent clone. However, assuming that each mutation occurred only once during tumor evolution, it is possible to suggest that the DNMT3A mutation occurred earlier than the JAK2 one in the disease course. Similarly, the fourth case (MDS12, a 5q- syndrome case) had ~80% of cells carrying a SF3B1 mutation, ~20% with ASXL1 mutation and ~10% with TET2 mutation. We can therefore suggest that the SF3B1 mutation occurred before the ASXL1 or TET2 mutation. The variant allele fractions for ASXL1 and TET2 could be consistent with either the TET2 mutation being subclonal to the ASXL1 mutation or on a separate branch of the phylogenetic tree, so we cannot establish the timing of those two mutations relative to each other. Finally, the fifth case [MDS43, a del(5q) RAEB case] had ~86% of cells with an ASXL1 mutation and ~55% with a TET2 mutation. In this case, it was clear that the TET2 mutation was subclonal to the ASXL1 mutation and must have occurred later.

Copy number changes and uniparental disomy analysis

Thirty-three [18 5q- syndrome, 6 del(5q) RA with additional cytogenetic abnormalities, and 9 cases of advanced del(5q) MDS] of the 43 del(5q) MDS samples included in the targeted sequencing analysis had been previously analyzed by single nucleotide polymorphism-arrays to identify cryptic copy number changes and regions of UPD (defined as continuous stretches of homozygous single nucleotide polymorphism calls >2 Mb without copy number loss).2

The results of the analysis are listed in Online Supplementary Table S5. The del(5q) was characterized in all 33 cases. Copy number changes in addition to the del(5q) were observed in six of nine cases of advanced MDS (66.7%) and four of six cases of del(5q) RA with additional cytogenetic abnormalities (66.7%), but in only four of 18 5q- syndrome cases (22.2%). In the 5q- syndrome group, 31 regions of UPD were identified in 17 of 18 patients. All other cases included in this study showed regions of UPD, six regions in all six cases of del(5q) RA with additional cytogenetic aberrations, and 17 in all nine advanced del(5q) cases.2

Table 3.Summary of non-synonymous variant calls with a COSMIC ID or not present in dbSNP.

A proportion of the regions affected by copy number loss encompassed genes that are part of our TSCA gene panel. In advanced del(5q) MDS cases, these were EZH2, NPM1, ETV6, ASXL1 and TP53 (Figure 1, Online Supplementary Table S6). Additional regions of cytogenetic loss encompassed CBL and ETV6 in two different cases of del(5q) RA with additional cytogenetic aberrations (Figure 1, Online Supplementary Table S6). The only DNMT3A loss was seen in a case of 5q- syndrome. In the one case [a del(5q) RAEB] with cytogenetic loss encompassing TP53, the remaining copy had a missense mutation (R273H), predicted to be damaging to the function of the protein (Online Supplementary Table S3).

These results show that advanced del(5q) MDS cases display a more complex landscape of cytogenetic aberrations, both karyotypically evident and cryptic. These regions often contain genes involved in myeloid disease.

Discussion

In this study, we sought to validate an Illumina-based targeted next-generation sequencing platform to simultaneously screen 25 genes relevant to myeloid malignancies for mutations. Once validated, we aimed to use this gene panel to characterize the mutational profile of a cohort of 43 cases of MDS with del(5q), in the context of additional molecular and high-density genomic array data. The prevalence of the mutations detected in complex DNA samples has typically been limited to approximately 20% using Sanger sequencing.27,28 The development of specific mutation enrichment or detection strategies has greatly increased this sensitivity.32,33 In keeping with the improved power of mutation detection of next-generation sequencing over traditional sequencing techniques, we identified previously undetected mutations in the validation cohort (that comprised 9 test samples containing 17 variants across 11 genes) in addition to the previously known mutations in these samples.

Figure 1.Mutations, deletions and loss of heterozygosity in 25 genes analyzed in del(5q) MDS samples. Columns show results for each of the 43 cases analyzed. Gray boxes indicate mutated cases. Black boxes mark samples for which single nucleotide polymorphism-array (SNP) data were available. X: double mutant. Δ: gene encompassed within a region of cytogenetic loss. Θ: gene encompassed within a region of UPD.

Using BaseSpace and Stampy/Platypus24,25 analysis software, we were able to successfully identify all point mutations, short indels and deletions included in the validation cohort. However, FLT3-ITD variants that consist of patient-specific sequence duplications were amplified and sequenced, but were not identified using either bio-informatics pipeline and therefore had to be visually identified. This highlights the need for further refinements to commercially available analysis pipelines before their use in routine clinical practice. The non-alignment of these reads is largely a function of the comparative size of the insertion or deletion compared to the absolute read length. We are hopeful that in the future longer read lengths, in combination with improvements to the alignment algorithms, will greatly increase the ability to detect these important mutations.

Once our panel had been successfully validated, we applied it to study the mutational profile of a series of MDS cases with del(5q). Gene mutation screening in del(5q) MDS has been performed in previous studies, but most of these studies focused on a limited number of genes, and mainly employed traditional sequencing methods.9,34–40 Other studies have investigated a larger number of genes41–43 but did not specifically focus on MDS cases with del(5q). To our knowledge, the present work is the first attempt to screen a large number of genes using a targeted next-generation sequencing approach in both early and advanced del(5q) MDS.

A total of 29 mutations were detected in our cohort of 43 del(5q) MDS cases. Overall, 45% of the cases of 5q-syndrome and 44% of cases of del(5q) RA with additional cytogenetic aberrations had at least one mutation. The more advanced del(5q) cases showed a higher proportion of mutated cases, and 66.7% presented at least one mutation. The genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). The mutation frequency for these two genes was lower in cases of 5q- syndrome (TP53 4.5%, ASXL1 13.6%). We therefore confirmed in our del(5q) cohort the observation made by our group and others that TP53 mutations occur predominantly in MDS with complex karyotype.9,44,45 The increased incidence of TP53 and ASXL1 mutations in advanced del(5q) cases in our present study suggests that these abnormalities may play a role in disease progression in del(5q) MDS. These data are consistent with those of a recent study that showed that TP53 mutations were associated with disease progression in del(5q) MDS.43

The 5q- syndrome is widely considered to be relatively genetically stable compared to other MDS subtypes, on the basis of molecular studies (including genomic array data analysis).2 This is reflected in its relatively good prognosis.8,11 Previous studies have shown mutations in a limited number of genes, including TP53, JAK2 and ASXL19,10,34,46 in this subtype of MDS. The incidence of JAK2 and ASXL1 mutations is ~6%.37 Here, we show that over 40% of patients with the 5q- syndrome in fact harbor a gene mutation, including mutations in TET2, SF3B1, RUNX1, WT1 and ASXL1.

Figure 2.Mutant allele frequencies in individual del(5q) MDS samples. The area of each colored circle indicates the allele frequency of the given mutation. The text under the circles lists the frequency and nature of each mutation in order of decreasing allele frequency.

The SF3B1 mutations detected in this study were identified in cases of 5q- syndrome, and not in the other two groups of patients with del(5q), which are karyotypically or morphologically defined by more advanced disease. SF3B1 mutations have been associated with a relatively benign disease course.15,43,47 It has been suggested that multipotent hematopoietic stem cells initially attain a splicing factor mutation as a founding genetic lesion, and subsequently acquire additional mutations that drive their malignant transformation.43,48 This is consistent with our finding in one 5q- syndrome case with a high SF3B1 mutant allele frequency and two other mutations (ASXL1 and TET2) with lower mutant allele frequencies.

The present study shows that a high proportion of genes involved in the epigenetic regulation of the cell (TET2, ASXL1, DNMT3A and JAK2) are affected by either mutations or cytogenetic losses in del(5q) MDS cases: 15 of 29 genes with non-synonymous mutations (51.7%) and 4 of 10 genes in regions affected by cytogenetic loss (40%) were epigenetic regulators. This observation is consistent with a recent report of mutations in a large cohort of MDS patients (n=117), in which the authors also found 80 mutations in genes predicted to affect the epigenetic regulation of the cell in half of the cohort (52% of cases).43 Genome-wide methylation analysis on a subset of cases with and without mutations in epigenetic factors did not highlight a specific DNA methylation profile associated with these mutations (Online Supplementary Information and Online Supplementary Figure S5).

A total of six of the mutations detected in this study present variant frequencies below the level of detection of Sanger sequencing, which is estimated to be around 15–20%.27,28 For example one patient with the 5q- syndrome showed a DNMT3A mutant allele frequency of 7.8% and another case a SF3B1 mutant allele frequency of 11.4%. Sanger sequencing has been the gold standard for sequencing for many years, and the vast majority of sequencing studies published to date have used this technology. It is likely that previous studies underestimated the prevalence of mutations in MDS. This has recently been illustrated by Jadersten et al.34 who used next-generation sequencing to reveal TP53 mutations (median clone size 11%) in nearly 20% of low-risk MDS patients with del(5q). Our data support the hypothesis that the prevalence of mutations in del(5q) MDS may have also been underestimated for other genes. Here, we have shown that genes involved in the epigenetic regulation of the cell frequently harbor low-frequency mutations in del(5q) MDS, not detectable by means of Sanger sequencing. This has previously been demonstrated for TET2 in MDS and CMML.49

The proportion of variant reads can be used to determine the order of occurrence of multiple mutations and thereby infer the clonal evolution from early stages of the disease. Interestingly, ASXL1 was one of the genes involved in four of the five cases with two or more mutations, with a lower variant frequency than the other co-mutated genes in three of the cases, suggesting that mutation of ASXL1 represented a later event in the disease course in these cases. Our analysis of clonality was based on a small number of single cases with multiple mutations. Studies with a similar sensitivity involving larger MDS cohorts will certainly help to establish the phylogenetic structure of tumor evolution.

In summary, we have successfully developed and validated a panel that allows for the screening of 25 genes frequently mutated in myeloid malignancies. The present study on del(5q) MDS has shown that a number of gene mutations occur in patients with the 5q- syndrome, and that >40% of patients with this low-risk MDS subtype harbor at least one gene mutation. A higher percentage of mutations was found among the more advanced cases of del(5q) MDS, with TP53 and ASXL1 being the more frequently mutated genes. Our study is the first to investigate and compare the molecular pathogenesis of early and advanced del(5q) MDS using targeted next-generation sequencing technology on a large panel of genes frequently mutated in myeloid malignancies.

Acknowledgments

This work was funded by Leukaemia and Lymphoma Research (UK), and by the Oxford Partnership Comprehensive Biomedical Research Centre, with funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health. The authors would like to thank the patients who accepted to participate in this study, all staff in their laboratories for their technical assistance, and all the physicians who referred patients’ material.

Footnotes

• ↵* MFM and AB contributed equally to this manuscript;
• ↵¥ JB and AS were co-senior authors
• The online version of this article has a Supplementary Appendix.
• Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.

• Received February 22, 2013.
• Accepted July 3, 2013.