Detection of Dual IDH1 and IDH2 Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes
2015; Elsevier BV; Volume: 17; Issue: 6 Linguagem: Inglês
10.1016/j.jmoldx.2015.06.004
ISSN1943-7811
AutoresMia Y. Platt, Amir T. Fathi, Darrell R. Borger, Andrew M. Brunner, Robert P. Hasserjian, Leonora Balaj, Amy Lum, Stephen Yip, Dora Dias‐Santagata, Zongli Zheng, Long P. Le, Timothy A. Graubert, A. John Iafrate, Valentina Nardi,
Tópico(s)Neutropenia and Cancer Infections
ResumoStudies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications. Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications. In recent years, whole-genome sequencing of acute myeloid leukemias (AMLs) led to the identification of frequent heterozygous mutations in isocitrate dehydrogenase 1 gene (IDH1).1Mardis E.R. Ding L. Dooling D.J. Larson D.E. 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Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation.Science. 2013; 340: 622-626Crossref PubMed Scopus (629) Google Scholar Clinical trials with IDH1 and IDH2 inhibitors for patients with IDH1/2 mutations are ongoing.16Hansen E. Quivoron C. Straley K. Lemieux R.M. Popovici-Muller J. Sadrzadeh H. Fathi A.T. Gliser C. David M. Saada V. Micol J.-B. Bernard O. Dorsch M. Yang H. Su M. Agresta S. de Botton S. Penard-Lacronique V. Yen K. AG-120, an oral, selective, first-in-class, potent inhibitor of mutant IDH1, reduces intracellular 2HG and induces cellular differentiation in TF-1 R132H cells and primary human IDH1 mutant AML patient samples treated ex vivo.Blood. 2014; 124: 3734Google Scholar, 17Shih A.H. Shank K.R. Meydan C. Intlekofer A.M. Ward P. Thompson C.B. Melnick A.M. Travins J. Straley K. Gliser C. Yen K. Levine R.L. AG-221, a small molecule mutant IDH2 inhibitor, remodels the epigenetic state of IDH2-mutant cells and induces alterations in self-renewal/differentiation in IDH2-mutant AML model in vivo.Blood. 2014; 124: 437Crossref PubMed Scopus (28) Google Scholar, 18Quivoron C. David M. Straley K. Travins J. Kim H. Chen Y. Zhu D. Saada V. Bawa O. Opolon P. Polrot M. Micol J.-B. Willekens C. Bernard O. Yang H. Agresta S. de Botton S. Yen K. Penard-Lacronique V. AG-221, an oral, selective, first-in-class, potent IDH2-R140Q mutant inhibitor, induces differentiation in a xenotransplant model.Blood. 2014; 124: 3735Google Scholar, 19Stein E.M. Altman J.K. Collins R. DeAngelo D.J. Fathi A.T. Flinn I. Frankel A. Levine R.L. Medeiros B.C. Patel M. Pollyea D.A. Roboz G.J. Stone R.M. Swords R.T. Tallman M.S. Agresta S. Fan B. Yang H. Yen K. de Botton S. AG-221, an oral, selective, first-in-class, potent inhibitor of the IDH2 mutant metabolic enzyme, induces durable remissions in a phase I study in patients with IDH2 mutation positive advanced hematologic malignancies.Blood. 2014; 124: 115Google Scholar, 20Fan B. Chen Y. Wang F. Yen K. Utley L. Almon C. Biller S. Agresta S. Yang H. Evaluation of pharmacokinetic-pharmacodynamic (PKPD) relationship of an oral, selective, first-in-class, potent IDH2 inhibitor, AG-221, from a phase 1 trial in patients with advanced IDH2 mutant positive hematologic malignancies.Blood. 2014; 124: 3737Google Scholar Because our laboratory launched a next-generation sequencing (NGS)-based tumor genotyping assay in early 2014, we found that IDH1 and IDH2 mutations co-occur in the same tumors more frequently than was reported. This study is a detailed description of five cases of dual mutations we have thus far identified and of the potential implications of these results. The Partners HealthCare Institutional Review Board granted approval for this study before its initiation. The electronic files of the Massachusetts General Hospital Pathology Department were queried for all cases run with the use of the NGS assay (SNAPSHOT NGS) since its launch in April 2014 until October 3, 2014, with a primary diagnosis of AML, MDS, or chronic myelomonocytic leukemia. A subsequent query was run to identify the subset of cases with either IDH1 or IDH2 single nucleotide variants (SNVs). Unique patients were enumerated such that multiple specimens sent on a single patient were not individually counted. The SNAPSHOT NGS assay uses a multiplex PCR technology called Anchored Multiplex PCR for SNVs and insertion/deletion detection in genomic DNA with the use of NGS.21Zheng Z. Liebers M. Zhelyazkova B. Cao Y. Panditi D. Lynch K.D. Chen J. Robinson H.E. Shim H.S. Chmielecki J. Pao W. Engelman J.A. Iafrate A.J. Le L.P. Anchored multiplex PCR for targeted next-generation sequencing.Nat Med. 2014; 20: 1479-1484Crossref PubMed Scopus (590) Google Scholar Briefly, genomic DNA was isolated from blood or bone marrow aspirates (QIAcube; Qiagen, Valencia, CA). The genomic DNA was sheared with the Covaris (Woburn, MA) M220 instrument followed by end-repair, adenylation, and ligation with an adapter. A sequencing library that targeted hotspots and exons in 39 genes (Supplemental Table S1) was generated with two hemi-nested PCR reactions with the use of one primer specific to a sequence in the gene of interest and one specific to a universal sequence in the adapter, for each PCR reaction.21Zheng Z. Liebers M. Zhelyazkova B. Cao Y. Panditi D. Lynch K.D. Chen J. Robinson H.E. Shim H.S. Chmielecki J. Pao W. Engelman J.A. Iafrate A.J. Le L.P. Anchored multiplex PCR for targeted next-generation sequencing.Nat Med. 2014; 20: 1479-1484Crossref PubMed Scopus (590) Google Scholar Illumina (San Diego, CA) MiSeq 2 × 151 bp paired-end sequencing results were aligned to the hg19 human genome reference with the use of BWA-MEM.22Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar PCR/optical duplicates were removed on the basis of unique start sites of sequenced molecules. MuTect23Cibulskis K. Lawrence M.S. Carter S.L. Sivachenko A. Jaffe D. Sougnez C. Gabriel S. Meyerson M. Lander E.S. Getz G. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples.Nat Biotechnol. 2013; 31: 213-219Crossref PubMed Scopus (2894) Google Scholar was used for SNV detection and Oncotator was used for mutation annotation (http://www.broadinstitute.org/oncotator, last accessed May 6, 2015). A minimum threshold of five unique reads that supported the canonical mutation and visualization in JBrowse24Westesson O. Skinner M. Holmes I. Visualizing next-generation sequencing data with JBrowse.Brief Bioinform. 2013; 14: 172-177Crossref PubMed Scopus (53) Google Scholar were required to make a variant call. Barcoded and tagged unidirectional PCR primers targeting codons 132 of IDH1 and 140 of IDH2 were designed to generate amplicons approximately 100 bp in length (Table 1). Genomic DNA samples were quantitated with the Qubit high-sensitivity DNA assay kit (Thermo Fisher Scientific, Waltham, MA), individually amplified with Platinum Taq HiFi, purified with Ampure XL beads (Agencourt, Brea, CA), and quantitated with High-Sensitivity DNA chip on the Agilent Technologies BioAnalyzer (Santa Clara, CA). The amplicons were normalized and pooled to generate emulsion PCR libraries on the Ion Torrent OneTouch2 platform with the use of the Ion PGM Template OT2 400 kit (Thermo Fisher Scientific). The libraries were enriched with the Ion Torrent ES platform, and all sequencing was performed with the Ion PGM Hi-Q Sequencing kit on the Ion Torrent PGM platform, analyzed, and visualized with the IGV version 2.3. The average base pair coverage of the amplicon was approximately 100,000 times. Relative frequencies of the mutant alleles were derived from dividing the number of mutant calls by total calls of the relevant position (Supplemental Table S2).Table 1PCR Primers for Focused Ultra-Deep Amplicon Sequencing Using the Ion Torrent PGM PlatformPrimersSequenceIDH1 primers IDH1_R132_1F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGGCATGTCACATTATTGCCAACATGACT-3′ IDH1_R132_2F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGCGATCTCACATTATTGCCAACATGACT-3′ IDH1_R132_3F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCTATCACATTATTGCCAACATGACT-3′ IDH1_R132_4F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGACACATCACATTATTGCCAACATGACT-3′ IDH1_R132_5F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCTTCACATTATTGCCAACATGACT-3′ IDH1_R132_6F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTCAGTTCACATTATTGCCAACATGACT-3′ IDH1_R132_7F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTATATTCACATTATTGCCAACATGACT-3′ IDH1_R132_8F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGTCATCACATTATTGCCAACATGACT-3′ IDH1_R132_9F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTAGCTTCACATTATTGCCAACATGACT-3′ IDH1_R132_10F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTGTGTTCACATTATTGCCAACATGACT-3′ IDH1_R132_R5′-CCTCTCTATGGGCAGTCGGTGATGCATGCGGTCTTCAGAGAAGCCATT-3′IDH2 primers IDH2_R140_1F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGGCATGCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_2F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGCGATCCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_3F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCTACTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_4F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGACACACTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_5F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCTCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_6F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTCAGTCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_7F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTATATCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_8F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGTCACTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_9F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTAGCTCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_10F5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTGTGTCTAGGCGTGGGATGTTTTTG-3′ IDH2_R140_R5′-CCTCTCTATGGGCAGTCGGTGATGCATGTCTGTCCTCACAGAGTTCAAGC-3′Bold text indicates sequence of the adapter and barcode. Open table in a new tab Bold text indicates sequence of the adapter and barcode. Genomic DNA was used as input for digital PCR at 10 ng per reaction. Primers spanned an intron/exon junction and were as follows: IDH1 forward primer, 5′-CTGCAAAAATATCCCCCGGC-3′; IDH1 reverse primer, 5′-CAAGTTGGAAATTTCTGGGCCAT-3′; mutant probe, 5′-ATCATAGGTCGTCATGCTTAT-3′; and wild-type probe, 5′-ATCATAGGTCATCATGCTTAT-3′. Reactions were performed in a 20-μL reaction with the use of the droplet digital PCR Supermix for Probes (no dUTP; Bio-Rad, Hercules, CA). Final concentration of primers and probes were 900 nmol/L and 250 nmol/L, respectively. The relation between the presence or absence of mutant or wild-type molecules in a droplet is defined by the Poisson distribution and allows robust, digital quantification of the two molecules relative to one another (Supplemental Table S3). Droplet generation was performed according to the manufacturer's instructions. Cycling conditions were 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 64°C for 2 minutes, and then a final 10-minute incubation at 98°C. The temperature ramp rate was 2.5°C/second. Droplet reading was performed on a QX200 droplet digital PCR droplet reader (Bio-Rad), and the analysis was performed with manual mode on a QuantaSoft Analysis software version 1.4 (Bio-Rad) by adjusting the gates to the no template control samples. Specimens were sent to LabCorp (Research Triangle Park, NC) for FLT3, NPM1, and CEBPA mutation analysis. Briefly, PCR amplification for the detection of FLT3 internal tandem duplication mutations (FLT3-ITDs) was performed, and the products were run on the ABI 3500xl genetic analyzer (Thermo Fisher Scientific) for size determination. The ITD wild-type produces a fragment that is approximately 327 bp, whereas the presence of an insertion produces a fragment that is ≥330 bp. PCR and size determination was also performed for NPM1 analysis to detect a 4-bp insertion at nucleotide position 959 (exon 12). For CEBPA analysis, the CEBPA coding region was PCR-amplified and was Sanger sequenced to identify sequence variations. Two micron-thick formalin-fixed, paraffin-embedded bone marrow (BM) core biopsy sections were stained with hematoxylin and eosin. Immunohistochemistry for IDH1 was performed on 5-μm–thick formalin-fixed, paraffin-embedded BM biopsy sections with an antibody specific for the mutant IDH1 p.Arg132His protein (dilution 1:150; H09; Dianova, Hamburg, Germany). A labeled streptavidin biotin kit was used as a detection system (Leica Biosystem, Nussloch, Germany). Combined cytoplasmic and nuclear staining was interpreted as immunopositive. Currently, antibodies are only available against IDH1 p.Arg132His mutant protein; no antibodies specific for any of the IDH2 mutated proteins are available for use in immunohistochemistry. Clinical, cytogenetic, and molecular characteristics of the patients with dual mutations in IDH1 and IDH2 are listed in Table 2. Three of the five patients, all men, exhibited diagnoses of AML. Two additional patients (one female and one male) were diagnosed with MDS (both refractory anemia with excess blasts-2; RAEB-2). The age at diagnosis ranged from 61 to 73 years. The AML patients had BM aspirate or (when not available) peripheral blood blast counts that ranged from 22% to 33% at diagnosis. Blast counts from the RAEB-2 patients were not available for the sequenced specimens. All five patients had normal karyotypes.Table 2Patient Characteristics and IDH1 and IDH2 Single Nucleotide VariantsCase No.DxAge, years/sexSpeci-men typeBlast, %IDH1 mutation (allelic frequency, %)No. of Reads (ALT/REF)IDH2 mutation (allelic frequency, %)No. of Reads (ALT/REF)Other (allelic frequency, %)Karyo-typeFLT3-ITD, NPM1, CEBPA status1AML63/MBM22p.Arg132His (c.395G>A) (12)588/4228p.Arg140Gln (c.419G>A) (2)232/12310SMAD4 p.Ala458Val (c.1373C>T) (45),CDH1 p.Ala592Thr (c.1774G>A) (46)46,XYFLT3 wild-typeNPM1 wild-typeCEBPA wild-type2AML73/MBM30∗BM was unavailable for use to determine blast percentage by structural characteristics because of extensive necrosis. Instead, the blast percentage from PB is shown.(PB)p.Arg132His (c.395G>A) (44)191/256p.Arg140Trp (c.418C>T) (5)50/104446,XYFLT3 wild-typeNPM1 mutated (4 bp insertion at c.959)CEBPA wild-type3AML61/MBM33p.Arg132Cys (c.394C>T) (3)7/237p.Arg140Gln (c.419G>A) (3)19/589NRAS p.Gly12Asp (c.35G>A) (34),NRAS p.Gly12Ser (c.34G>A) (2)46,XYFLT3-ITD mutated [PCR product of 429 bp (wild-type ∼327 bp)]NPM1 wild-typeCEBPA double mutant [p.Asn74X (c.219insT), p.Ala295Thr (c.883G>A)]4RAEB-268/FPB13†The blast count was unavailable for the specimen submitted for sequencing. The blast percentage shown is from a different specimen on a different day (from PB 16 days before the sequencing sample for patient 4, and from BM 13 days before the sequencing sample for patient 5.).p.Arg132His (c.395G>A) (4)8/199p.Arg140Gln (c.419G>A) (12)60/48246,XXNA5RAEB-264/MBM13†The blast count was unavailable for the specimen submitted for sequencing. The blast percentage shown is from a different specimen on a different day (from PB 16 days before the sequencing sample for patient 4, and from BM 13 days before the sequencing sample for patient 5.).p.Arg140Gln (c.419G>A) (27);p.Arg140Trp (c.418C>T) (13)337/943,169/113846,XYFLT3 wild-typeNPM1 wild-typeCEBPA wild-typeF, female; M, male; ALT, alternative allele; AML, acute myeloid leukemia; BM, bone marrow; Dx, diagnosis; ITD, internal duplication mutation; NA, not applicable; PB, peripheral blood; RAEB-2, refractory anemia with excess blasts-2; REF, reference allele.∗ BM was unavailable for use to determine blast percentage by structural characteristics because of extensive necrosis. Instead, the blast percentage from PB is shown.† The blast count was unavailable for the specimen submitted for sequencing. The blast percentage shown is from a different specimen on a different day (from PB 16 days before the sequencing sample for patient 4, and from BM 13 days before the sequencing sample for patient 5.). Open table in a new tab F, female; M, male; ALT, alternative allele; AML, acute myeloid leukemia; BM, bone marrow; Dx, diagnosis; ITD, internal duplication mutation; NA, not applicable; PB, peripheral blood; RAEB-2, refractory anemia with excess blasts-2; REF, reference allele. Of note, patient 2 had a history of bladder cancer, treated with surgery and Bacille Calmette-Guérin. None of the patients had any reported environmental exposures or a family history of blood disorders or hematologic neoplasms. This study included all AML (n = 53), MDS (n = 34), and chronic myelomonocytic leukemia (n = 5) patients with specimens submitted for the SNAPSHOT NGS assay during the 6-month period from the assay's launch in April 2014 through October 2014 (total n = 92). Of these 92 patients, 21 (23%) harbored IDH mutations (14 AML, 6 MDS, 1 chronic myelomonocytic leukemia). Of these 21 patients, 4 (19%) had coexisting IDH1 and IDH2 mutations. A fifth patient (with MDS) who was included in our study was previously identified as harboring two different IDH2 mutations on a previously used hotspot profiling platform, which were confirmed by the current SNAPSHOT NGS assay. The dual IDH1 and IDH2 variants identified were missense mutations at the canonically mutated codons, Arg132 and Arg140, respectively (Table 2). Although several IDH mutations were observed at low AF, our confidence in these calls was based on a high depth of coverage (average target region coverage for each of the five samples, 3453; range, 690 to 8909), with a high number of unique molecules supporting the calls (well above background), the mutations' location at canonical sites, and the identification of the mutations on multiple samples from the same patient on repeat testing in some instances (data not shown). In addition, the mutations were verified by an orthogonal sequencing method (see Focused Ultra-Deep Amplicon Sequencing Using the Ion Torrent PGM Platform) (Supplemental Table S2). The IDH1/2 mutational burden for each case (as represented by the variant AF) is described in the sections below along with non-IDH mutations. Patient 1 had AML with normal karyotype and without FLT3, NPM1, or CEBPA mutations and showed an IDH1 p.Arg132His (c.395G>A) mutation at 12% AF and an IDH2 p.Arg140Gln (c.419G>A) mutation at 2%, in the setting of a marrow blast percentage of 22% (Table 2 and Figure 1). Although the blast count does not necessarily reflect the size of the clonal population, if we assumed a heterozygous IDH1 mutation were present in every cell of the blast population, the marrow involvement by 22% blasts would predict a variant AF of approximately 11%. The calculated AF of 12% by SNAPSHOT NGS, combined with the observation of mutant IDH protein by immunohistochemistry in nearly all of the blasts, is consistent with this assumption (Supplemental Figure S1). SNVs in SMAD family member 4 and CDH1 genes were also detected by the SNAPSHOT NGS assay. The variant AFs of the SNVs in SMAD family member 4 and cadherin 1, type 1 genes, however, were close to 50% (45% and 46%, respectively), suggesting these were not somatic tumor mutations. Patient 2 had AML with normal karyotype and mutated NPM1, but wild-type FLT3 and CEBPA, and showed a predominant IDH1 p.Arg132His (c.395G>A) mutation on the BM aspirate with a 44% AF, whereas an IDH2 p.Arg140Trp (c.418C>T) mutation was present at 5% AF. The marrow blast percentage could not be determined because of extensive necrosis, but a concurrent peripheral blood sample showed a blast percentage of 30% (Table 2 and Figure 1). No other SNVs were detected by SNAPSHOT NGS. Patient 3 had AML with normal karyotype, a FLT3 ITD mutation, two CEBPA mutations, and no NPM1 mutation. Both an IDH1 p.Arg132Cys (c.394C>T) and an IDH2 p.Arg140Gln (c.419G>A) mutation were present, each at an equally low AF of 3%, in the setting
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