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A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing
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The Journal of Molecular Diagnostics, Vol. -, No. -, - 2014
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A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing: Report of a Large Series of Myeloid Neoplasms Q29
Amir Behdad, Helmut C. Weigelin, Kojo S.J. Elenitoba-Johnson, and Bryan L. Betz From the Department of Pathology, University of Michigan, Ann Arbor, Michigan Accepted for publication September 29, 2014. Address correspondence to Bryan L. Betz, Ph.D., Department of Pathology, University of Michigan, Traverwood II, 2910 Huron Pkwy., Ste. C, Ann Arbor, MI 48105. E-mail: [email protected].
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Diagnostic testing for CEBPA mutations is the standard of care for cytogenetically normal acute myeloid leukemia. Widespread implementation of this testing is hampered by technical challenges associated with the GC content of the gene, the variability of the mutations, and the complexity of the sequence analysis and variant interpretation. We developed a robust Sanger-sequencing test to detect CEBPA mutations in diagnostic acute myeloid leukemia specimens. Our experience with testing 2393 cases of suspected myeloid neoplasms is presented. NPM1, FLT3einternal tandem duplication (ITD), and FLT3D835 mutation status were determined in parallel; 160 (6.7%) cases harbored CEBPA mutations, including 86 with a single mutation and 74 with double mutations. Nineteen single-mutant cases and 3 double-mutant cases showed only nucleotide substitutions that could not be detected by fragmentanalysisebased tests. A subset of cases harbored double mutations with uneven mutant allele frequency and required careful interpretation because of possible leukemic heterogeneity. NPM1 and FLT3-ITD mutations were more frequent in single- compared with double-mutation cases (31% versus 5% for NPM1, and 28% versus 16% for FLT3-ITD). This sequencing-based assay provides an efficient and reliable CEBPA mutation testing platform, permitting detection of all mutations with immediate distinction of single- and double-mutation cases. Given the technical challenges, robust Sangersequencing assays continue to maintain an important role in clinical CEBPA testing despite the development of multigene next-generation sequencing assays. (J Mol Diagn 2014, -: 1e9; http:// dx.doi.org/10.1016/j.jmoldx.2014.09.007)
Acute myeloid leukemia (AML) is a heterogeneous group of diseases with variable survival rates, disease course, and response to therapy. Inclusion of cytogenetic information in the classification of AML has helped tremendously to improve risk stratification of this disease,1e5 which can guide therapeutic decisions. However, 40% to 50% of AMLs are cytogenetically normal (CN-AML),1,6 and molecular markers are becoming increasingly important for further classification of this subgroup.7 Among the molecular alterations in AML, mutations of the CEBPA, NPM1, and FLT3 genes are among the most common and bear important prognostic significance. CEBPA, a single-exon gene, encodes a leucine zipper transcription factor with an important role in myeloid differentiation.8,9 CEBPA mutations are encountered in
approximately 8% to 15% of AML and are one of the most common mutations in CN-AML.7,10,11 Patients with CEBPAmutated AML (CEBPA-AML) may carry one (single-mutated CEPBA) or two mutated alleles (double-mutated CEPBA). The majority of double-mutated CEPBA cases harbor a truncating mutation in the N-terminal region of the protein combined with an inframe mutation in the C-terminal region. The N-terminal mutations abolish translation of full-length p42 protein, leading to production of only a smaller p30 isoform coded from an internal ATG start codon at amino acid 120. The p30 protein is thought to have a dominant-negative effect Supported by the Department of Pathology, University of Michigan, Ann Q2 Arbor, MI. Disclosure: None declared. Q3
Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.09.007
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Behdad et al and play a crucial role in leukemogenesis.10 In contrast, C-terminal inframe mutations lead to proteins with impaired DNA binding and dimerization. Recent studies have shown that the favorable outcomes associated with CEBPA mutations are limited to double-mutated CEPBA cases.12e14 Given its prognostic importance, AML with mutated CEBPA represents a provisional entity in the 2008 World Health Organization classification of tumors of the hematopoietic and lymphoid tissues. Consequently, a reliable and accurate testing method is necessary to assess CEBPA mutations in the work-up of all CN-AML patients. CEBPA mutations are highly variable and can occur across the entire coding region of the gene. The development of robust assays for CEBPA mutation testing is hampered by the high GC content of the gene (75% in the coding region), the presence of a trinucleotide repeat region, the complexity of the mutations, and the frequent occurrence of mutations in mononucleotide repeats. Similarly, inclusion of CEBPA as part of next-generation sequencing panels has been impeded by poor amplicon coverage and technical challenges associated with identification and calling of variants. Techniques that have been used successfully to screen for CEBPA mutations include Sanger sequencing or PCR-based fragment-length assays.11,15e18 Although efficient and sensitive, the fragmentlength assays only detect mutations that result in a net insertion or deletion, and not substitution mutations.19 Fragment-length analysis-based tests also cannot distinguish a common 6-bp duplication polymorphism (p.H195_P196dup) from an actual insertion or duplication mutation.20 Sequencing-based assays, despite their ability to detect all mutations, are more labor-intensive and expensive. Expertise in sequence annotation and interpretation also is required, which can be difficult in cases with unusual variants or complex length-affecting mutations. Consequently, many laboratories struggle with implementing a reliable and sensitive sequencing-based CEBPA assay for routine diagnostic purposes. Here, we describe a clinical-grade, Sanger-sequencinge based test to detect CEBPA mutations in diagnostic AML specimens. We evaluated the performance of this test over a duration of 4 years and describe our experience with interpreting one of the largest series of reported CEBPA mutations. In addition, we correlated the co-occurrence of these mutations with FLT3 internal tandem duplication (FLT3-ITD), FLT3D835, and NPM1 mutations.
other diagnostic data were available for only a limited number of patients and thus were not included for correlation.
DNA Extraction Genomic DNA was extracted from peripheral blood or bone marrow using the Qiagen Biorobot EZ1 (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions.
CEBPA Mutation Assay
The entire coding sequence of the CEBPA gene was amplified by PCR in two overlapping fragments, using the primers with sequences listed in Table 1. Each 30 mL PCR reaction ½T1 contained 50 ng DNA, 300 nmol/L of each primer, 1X Phusion GC mastermix (Finnzymes), and 8% dimethyl Q6 sulfoxide. Cycling conditions were as follows: 98 C for 30 seconds, 35 cycles of 99 C for 5 seconds, 68 C for 20 seconds, 72 C for 30 seconds, and final elongation at 72 C for 2 minutes. An aliquot of each PCR product was confirmed by gel electrophoresis. The rest was purified by the QIAquick PCR purification kit (Qiagen) and subjected to bidirectional sequencing using nested sequencing primers (Table 1) with ABI BigDye v1.1 terminators on an ABI 3130xl genetic analyzer (Applied Biosystems). Sequences spanning the Q7 entire CEBPA coding region were analyzed for mutations with software-assisted review (Mutation Surveyor, SoftGe- Q8 netics) in comparison with a GenBank reference sequence (https://www.ncbi.nlm.nih.gov/nuccore; GenBank accession Q9 number NM_004364). For interpretative purposes the coding region of CEBPA was divided into three regions: N-terminal (amino acids 1 to 120), midregion (amino acids 121 to 277), and C-terminal (amino acids 278 to 358), as previously described.14 Lengthaffecting frameshift mutations and nonsense point mutations in any region of the gene were considered pathogenic. Inframe length-affecting mutations and missense point mutations in the C-terminal region also were considered pathogenic. Inframe and missense mutations in the N-terminal and midregions were reported as variants of uncertain significance. Pathogenic mutations in which the mutant sequence trace was present at a higher level than the wildtype trace were considered homozygous and interpreted as
After approval by the University of Michigan Institutional Review Board, we conducted a retrospective study of the results of our clinical diagnostic testing for CEBPA mutations. Bone marrow or blood specimens in EDTA from 2420 patients with suspected myeloid neoplasm were submitted to our laboratory for routine diagnostic purposes. Clinical and
Table 1 CEBPA Assay Primers PCR primers CEBPA P1 Forward: 50 -GAGCAGGGTCTCCGGGTGG-30 Reverse: 50 -CCAGCTGCTTGGCTTCATCC-30 CEBPA P2 Forward: 50 -GCAGGCTGGAGCCCCTGTAC-30 Reverse: 50 -GACCCCAAACCACTCCCTGG-30 Sequencing primers CEBPA P1-SEQ Forward: 50 -AGGCTGGAGGCCGCCGAG-30 Reverse: 50 -GCTGCTTGGCTTCATCCTCC-30 CEBPA P2-SEQ Forward: 50 -GGCTGGAGCCCCTGTACGAG-30 Reverse: 50 -AACCACTCCCTGGGTCCCCGC-30
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Materials and Methods Patients
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double mutations.12,21,22 Synonymous variants and polymorphisms including c.402G>A (p.A134A), c.573C>T (p.H191H), c.690G>T (p.T230T), and c.584_589dup (p.H195_P196dup)20 were scored as wild type.
CEBPA-P2 721 bp CEBPA-P1 609 bp TAD1
TAD2
1
DBD/ZIP 1077 bp
(GCC) 7
FLT3 and NPM1 Mutation Testing Q10
When available, results of FLT3-ITD, FLT3-D835, and NPM1 mutations were retrieved for cases that tested positive for a CEBPA mutation. FLT3 and NPM1 testing was performed as previously described.23,24
3% DMSO 8% DMSO GC Buffer
CEBPA-P1
CEBPA-P2
c.199_200dupTA Mutation C
Results
C T
A C A
T
C
G
A C
C
C G
G C
C G
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Test Design and Assay Performance We searched the Catalogue of Somatic Mutations in Cancer Mutation Database for CEBPA mutations. Given the highly variable mutations that occurred throughout the coding region of the gene, including nonsense point mutations, we chose to design a sequencing-based assay rather than a fragment-length test. The high clinical significance of accurately distinguishing single- versus double-mutationepositive cases, including those with homozygous nonsense mutations, along with a relatively common length-affecting polymorphism (H195_P196dup), were other reasons for choosing a sequencing-based test.20,25 CEBPA is a single-exon gene with a 1077 bp coding region. Primers were designed such that two overlapping Q11 fragments spanning the entire coding region could be ½F1 amplified by PCR (Figure 1A). A two-amplicon design was chosen to minimize the number of amplicons requiring sequencing. Other published assays require four amplicons.11,19,23 Primer design and optimization of the PCR/ sequencing reactions was challenging because of the high GC content of the coding region (75%), the presence of a trinucleotide repeat (GCC), and problems with secondary structure. Optimization of the PCR required the addition of 8% dimethyl sulfoxide and the use of GC-rich PCR buffer to minimize production of nonspecific amplicons (Figure 1, B and C). Nested bidirectional sequencing primers were used to provide high-quality sequence data by minimizing the sequencing of nonspecific amplicons. The analytic sensitivity of the assay was determined by analyzing various dilutions of mutation-positive genomic DNA in wild-type DNA. Mutant alleles could be detected reliably down to a level of 10% using software-assisted sequence analysis (Figure 1D).
Testing Experience Test results were reviewed from 2420 consecutive specimens referred to our institution for assessment of CEBPA mutation status. PCR and sequencing results adequate for interpretation were obtained for 2393 (98.9%) specimens. Test failures occurred in 27 (1.1%) cases as a result of repeated PCR amplification failure and in most cases correlated with receipt
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20%
10%
5%
WT
Figure 1
Assay design and optimization. A: CEBPA is a single-exon gene with a 1077-bp coding region. Primers were designed such that two overlapping fragments spanning the entire coding region are amplified by PCR. B: Optimization of the PCR required the addition of 8% dimethyl sulfoxide (DMSO) and the use of GC-rich PCR buffer. Primer design and optimization of the PCR/sequencing reactions was challenging because of the high GC content of the coding region (75%), the presence of a trinucleotide repeat (GCC)7, and problems with secondary structure. C: Amplification products after optimization. D: Sensitivity of direct sequencing for CEBPA mutations. Mutation-positive genomic DNA was diluted into wildtype DNA. Mutations could be identified reliably down to a level of 10%. DBD/ZIP, DNA binding and dimerization domain (aa 278 to 358); TAD1, transactivation domain 1 (aa 70 to 97); TAD2, transactivation domain 2 (aa 127 to 200); WT, wild type.
of client-extracted DNA of low quantity. A review of successful test results showed that 160 cases (6.7%) harbored CEBPA mutations, including 86 cases (3.6%) with a single mutation (CEBPAsm) and 74 cases (3.1%) with double mutations (CEBPAdm). It is important to note that because the diagnosis was not known in the majority of the tested cases, the prevalence of mutations in our cohort may not be representative of the CEBPA mutation rate in AML and particularly that of CN-AML. Example sequence traces for N-terminal and C-terminal mutations are shown in Figure 2. A complete list of mutations is provided in Supplemental Table S1. Clinicopathologic information was available in 23 cases with CEBPA variants. Detailed information about these cases with diagnosis; karyotype; and CEBPA, NPM1, and FLT3 status is provided in Supplemental Table S2. N-terminal mutations (n Z 118) were small insertions or deletions (89%) or nonsense mutations (10%), which both
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373 N-Terminal Mutations C-Terminal Mutations Unknown Variant 374 c.68delC, p.P23fs c.937_939delAAG, p.K313del c.667G>A, p.G223S 375 G C A G C C C G G T C A C C C C C C C C G C A C G C G C C C G C A G A A G G T G C T G G A 376 377 * 378 379 c.245_246insGG, p.F82fs (homozygous) c.914_916dupAGC, p.Q305dup C A G C G C A A C G T G G A G T G T T C C A G C A C A G C C 380 381 H195_P196dup Polymorphism 382 383 c.584_589dup, p.H195_P196dup 384 c.962A>G, p.N321S c.21C>G, p.Y7X A G T G A C A A T G A C C G C G A C T T C T A C G A G G C G C A C C C G C C G C C C G C G 385 * 386 * 387 388 Figure 2 Sequence traces of N- and C-terminal CEBPA mutations, variants of unknown significance, and the c.584_589dup, p.H195_P196dup poly389 morphism. A variety of sequence alterations were detected, including both length-affecting mutations and nucleotide substitution mutations. The wild-type 390 CEBPA sequence is listed above the traces. The insertion mutation in the second panel of the left column (c.245_246insGG, p.F82fs) showed more than 90% 391 mutant allele frequency and therefore was interpreted as homozygous. Brackets in each panel indicate the deleted/inserted/duplicated nucleotide sequence; 392 asterisks, nucleotide substitutions. 393 394 mutations (21%), and a small number of frameshift (11%) 395 ½F3 predict truncation of the full-length p42 protein (Figure 3). and nonsense mutations (1%). A single case (1%) with a large 17eamino acid inframe 396 Mutations in CEBPAsm cases occurred mostly within the deletion in the N-terminal region was considered pathogenic 397 398 N-terminal region (52%), followed by the midregion (30%), through interpretative discretion. Midregion mutations 399 and then the C-terminal region (18%) (Table 2). Within the ½T2 (n Z 37) also mostly were small frameshift insertions or 400 deletions (86%) or nonsense mutations (14%); however, CEBPAdm cases, 88% harbored two distinct mutations. The 401 these predicted truncation of both p42 and p30 proteins. Cmajority of these cases (72%) harbored the classically 402 terminal region mutations (n Z 72) consisted mostly of described combination of a frameshift or nonsense mutation 403 small inframe insertions or deletions (67%) or missense in the N-terminal region and an inframe or missense 404 405 406 407 TAD1 TAD2 DBD/ZIP 358 408 1 120 278 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 Frameshift Nonsense Missense Inframe 427 Figure 3 Distribution of CEBPA mutations and variants identified in this study. The locations of the mutations are shown with respect to the CEBPA protein 428 and functional regions. Arrows depict the two translation initiation sites at amino acids (aa) 1 and 120. For interpretative purposes, the coding region of 429 CEBPA was divided into three regions: N-terminal (aa 1 to 120), midregion (aa 121 to 277), and C-terminal (aa 278 to 358). The majority of CEBPAdm cases 430 harbor a combination of a truncating frameshift or nonsense mutation in the N-terminal region and an inframe insertion/deletion or missense mutation in the 431 C-terminal region. Mutations in CEBPAsm cases are distributed in the entire coding region, with a greater portion in the midregion. Missense and inframe 432 mutations of the N-terminal and midregions were classified as variants of unknown significance. TAD1, transactivation domain 1 (aa 70 to 97); TAD2, 433 transactivation domain 2 (aa 127 to 200); DBD/ZIP, DNA binding and dimerization domain (aa 278 to 358). 434
Variants
Single Mutations
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Double Mutations
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Sanger Sequencing for CEBPA Mutation Testing Table 2
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Summary of CEBPA Mutations
CEBPA mutation status
Mutation 1
CEBPAsm
Frameshift Nonsense
N-terminal AA 1 to 120
Mutation 2 Middle AA 121 to 277
C-terminal AA 278 to 358
N-terminal AA 1 to 120
Middle AA 121 to 277
C-terminal AA 278 to 358
Frameshift Nonsense Frameshift Inframe Missense Total CEBPAdm
Frameshift Frameshift Nonsense Frameshift Frameshift Frameshift Nonsense Nonsense
Inframe Missense Inframe Frameshift Frameshift Frameshift Inframe Nonsense Frameshift
Missense Inframe Frameshift and missense* Inframe
Frameshift Frameshift Nonsense Frameshift and nonsense* Total CEBPAhm
Frameshift
Number of patients 38 7 23 3 4 2 9 86 42 4 1 2 3 6 1 1 1 1 1 1 1 65 6 1 1 1 9
Frameshift Nonsense Inframe Nonsense
Total *These patients harbored three mutations. AA, amino acid number; hm, homozygous-mutated.
mutation in the C-terminal region. A nonclassic combination such as one of the two mutations occurring in the midregion, both mutations occurring in the N- or C-terminal region, or a C-terminal frameshift was detected in the remaining (28%) cases. Two cases with a nonclassic mutation pattern harbored three distinct mutations: one case showed a frameshift mutation in the midregion and two mutations (frameshift and missense) in the C-terminal region; another case harbored two truncating mutations in the N-terminal region (frameshift and nonsense); and a third case showed a truncating mutation in the C-terminal region (frameshift). Six of 65 nonhomozygous CEBPAdm cases (9%) showed an obvious discrepancy in the frequency of their two mutant alleles based on the sequence chromatograms. Five of these six cases harbored a nonclassic combination of mutations. ½F4 An example of such a case is shown in Figure 4. Given that the finding of discrepant mutation levels may indicate the presence of mutations in two different subclones, we interpreted these results cautiously and included a comment to indicate that these two mutations may represent two clones
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with a single CEBPA mutation. In support of this we tested a relapse specimen from a case that originally displayed two N-terminal frameshift mutations at different levels. Only one of the two mutations remained after treatment. Nine CEBPAdm cases (12%) harbored single mutations that were scored as homozygous based on the presence of a higher mutant peak trace compared with the wild-type trace on the sequence chromatograms. These mutations included six frameshift mutations in N-terminal region, two nonsense substitutions (one N-terminal and one C-terminal), and an inframe duplication in the C-terminal region. Similar to Green et al,12 these cases were interpreted as harboring biallelic mutations and thus were classified as CEBPAdm. In addition, we identified 20 cases with variants of unknown significance, including 14 missense variants and 6 small inframe duplications within the N-terminal and midregions. The designation of variants of unknown significance was based on the nonclassic pattern and the unknown clinicopathologic significance as further described in the Discussion section. With the exception of one case in which the variants of unknown significance co-occurred with an
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621 Case 41 Case 79 622 c.125_155del, p.P42fs c.232delC, p.L78fs A G C C T C C C G C C C G T T C C T G G C C G A 623 624 * * * 625 * * * * * * * * * 626 c.937_939dupAAG, p.K313dup c.436delC, p.L146fs 627 A A G G T G C T G G A G C C C C T G T A C G A G 628 * * * 629 * * * * * * * * * * * 630 631 Figure 4 Examples of CEBPAdm cases with equal (case 41) and 632 discrepant (case 79) mutant allele frequencies. Brackets highlight the 633 deleted/duplicated sequence; asterisks, the mutant peak. 634 635 N-terminal frameshift mutation, the remainder of the vari636 ants were found in isolation. The evaluation of variant allele 637 frequency in most of these variants showed values close to 638 50%, which suggested that these may represent germline 639 rather than somatic variants. Remission specimens were 640 available for four cases. Three variants (D75E, G104dup, 641 and G103_G104dup) were determined to be germline 642 because they were present in the remission specimen, 643 644 whereas the fourth variant (H84Q) was determined to be 645 somatic. 646 647 Correlation of the CEBPA Results with NPM1 and FLT3 648 649 Molecular testing for FLT3 internal tandem duplication 650 (FLT3-ITD), FLT3-D835, and NPM1 mutations were 651 available in 68 (79%) CEBPAsm and 64 (86%) CEBPAdm 652 ½T3 cases. Table 3 shows the frequency of each of these muta653 tions among CEBPAsm and CEBPAdm cases. FLT3-ITD 654 was the most common concurrent mutation with CEBPA, 655 and was encountered in 28% and 16% of CEBPAsm and 656 CEBPAdm cases, respectively. In the subset of CEBPAdm 657 658 cases with homozygous CEBPA mutations, 1 of 9 harbored 659 a FLT3-ITD and none harbored an NPM1 mutation. 660 NPM1 mutations were seen mainly in CEBPAsm patients 661 (31%); however, three CEBPAdm cases (5%) also harbored 662 an NPM1 mutation. Interestingly, each of these three cases 663 showed discrepant CEBPA mutant allele frequency with 664 CEBPA mutations in a nonclassic pattern rather than the 665 classic combination of an N-terminal and a C-terminal 666 mutation. In one of these patients several mutations were 667 detected at the time of AML diagnosis including two 668 CEBPA mutations, FLT3-ITD, FLT3-D835, and NPM1. At 669 670 relapse only one of the CEBPA mutations was present in 671 addition to a homozygous FLT3-ITD and NPM1 mutation. 672 These findings show evidence of clonal heterogeneity at the 673 time of diagnosis and that the two CEBPA mutations were 674 present in two different clones. 675 676 Discussion 677 678 679 Here, we describe one of the largest series of CEBPA mu680 tations reported to date. Similar to previous reports, the 681 majority of CEBPA mutations in our series were frameshift 682
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mutations of N-terminus and inframe mutations of the Cterminus.10,12,26e29 We also report several novel and recurring variants, the significance of which has not been determined at this time. The description and cataloging of such variants provides an opportunity for future clarification. Two variants, G122E and A176V, have been reported Q12 by other investigators,30e32 however, their functional significance remains unclear. Sequencing of the CEBPA gene is hampered by a repetitive nucleotide sequence and a high GC-rich content, which can lead to technical difficulties in assay design and optimization. To overcome these difficulties we optimized our primer design and incorporated a titrated concentration of dimethyl sulfoxide into the amplification reactions. An analysis of Sanger sequencing data also can be challenging and laborious, however, software can greatly boost the efficiency and quality of sequence analysis. In particular, we found the mutation electropherogram feature of Mutation Surveyor software to be especially helpful for detecting lowlevel mutations. This feature also provided the ability to scan and review sequence data in a rapid fashion. Other useful software features include the ability to analyze multiple patients and PCR amplicons within a single session and the ability to output mutations according to HGVS Q13 Q14 nomenclature. Although the software reliably detected all types of mutations (substitutions, duplications, insertions, and deletions), we found that only single base substitutions were annotated reliably. Length mutations sometimes were called incorrectly and these always required manual anno- Q15 tation for accurate mutation reporting. Once annotated, the interpretation of detected variants required familiarization with polymorphisms and the expected pattern of mutations. The interpretive approach as described in Materials and Methods section has been updated over time and is the result of our collective experience and the current literature. Implementing all of these elements has provided a reliable assay with robust performance. Many laboratories use multiplex PCR-based assays with fragment-length analysis for screening, followed by Sanger sequencing for confirmation in positive cases. Although this strategy is more efficient and detects the majority of CEBPA mutations, it will miss cases harboring only base substitution mutations. The shortcoming of PCR fragment-length Table 3 Co-Occurrence of FLT3 and NPM1 Mutations in Cases with CEBPA Mutations CEBPA þ
FLT3-ITD FLT3-D835þ NPM1þ FLT3-ITDþ/NPM1þ
þ
Single
Double
19 2 21 12
10 3 3 2
(28%) (3%) (31%) (18%)
(16%) (5%) (5%) (3%)
FLT3 and NPM1 results were available for 68 single and 64 double CEBPA mutation cases. ITD, internal tandem duplication.
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analysis as a screening method has been emphasized by previous investigators comparing this technique with upfront sequencing-based assays.19 Our data confirm this concern because in our series there were 19 (22%) CEBPAsm cases and 3 (4%) CEBPAdm cases that harbored only nonelength-affecting mutations, which would not have been detected by the fragment-length analysis screening strategy. Other investigators have reported an even higher frequency of point mutations among CEBPAdm cases,19,23 further emphasizing the necessity of an up-front sequencing strategy. Sanger sequencingebased assays inherently have a lower analytic sensitivity compared with fragment analysis. Our assay reliably detected 10% of the mutant allele burden, which is adequate for the detection of CEBPA mutations in diagnostic specimens of CN-AML patients, which, by definition, have more than 20% leukemic blasts. The utility of CEBPA mutation analysis in specimens after treatment is still under investigation; if a CEBPA assay was needed to detect minimal residual disease or early recurrence, then certainly more sensitive techniques are needed. Interpretation of sequence variants remains a challenging aspect of CEBPA testing. Similar to previous studies, the majority of the CEBPAdm cases in our cohort harbored a combination of a truncating N-terminal mutation and an inframe C-terminal mutation. Assuming that the two mutations are on different alleles, they are predicted to cause a p30 isoform translation from the allele with a truncating N-terminal mutation and a nonfunctional protein from the C-terminal mutation. These two proteins are believed to explain the role of CEBPAdm in leukemogenesis. Although there is consensus that such N-terminaletruncating and C-terminaleinframe variants represent bona fide pathogenic mutations, whether homozygous mutations are considered double mutations is debated.14 A small subset of cases in our study had homozygous mutations and similar unexpected combinations of mutations such as two concurrent mutations within the N- or C-terminal region or a mutation in the midregion. This pattern of mutations will not generate both of the earlier-mentioned aberrant proteins and thus their role in leukemogenesis remains unclear. Other studies have included homozygous and similar nonclassic mutations among CEBPAdm cases.12 Despite their uncertain role in leukemogenesis, the prognostic impact of a nonclassic pattern of mutations has not been studied separately. We found that some cases in our series harbored two CEBPA mutations with different allelic levels. Interestingly, most of these cases were among those with a nonclassic pattern of mutations. The finding of discrepant mutation levels raises concern that the two mutations could belong to different subclones and therefore these cases may be better classified as CEBPAsm. This hypothesis is supported by our observation of a case in which one of the two mutations became undetectable at relapse. Consistent with the literature, we reported cases with a nonclassic pattern of mutations as CEBPAdm. However, in the case of an obvious
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discrepancy in the frequency between the two mutant alleles, we add a cautionary result and comment in our report that such findings may indicate two single mutations in different clones; thus, the prognostic impact of these mutations is unclear. In addition, the classification of missense and inframe mutations can be challenging because of concerns of distinguishing pathogenic mutations from benign polymorphisms. For instance, the midregion inframe variant p.H195_P196dup once was reported as a pathogenic mutation; however, subsequent studies that screened healthy populations determined that this represents a benign polymorphism.20 Similarly, we and others identified a number of Q17 missense variants that required careful interpretation. In this large series we found that missense variants in the N-terminal and midregions usually were present at an allelic frequency close to 50% and were never found in combi- Q18 nation with a mutation, suggesting that they may represent germline variants with unknown pathogenic significance. In contrast, we found that the missense variants located in the C-terminal region tended to be present at allelic levels below 50% and occasionally were found in combination with a classic N-terminaletruncating mutation and with similar allelic frequencies. Polyphen analysis supported a damaging Q19 effect to the protein in all C-terminal missense mutations and only a subset of those in the N-terminal and midregions (data not shown). Furthermore, we were able to determine that several of the N-terminal variants (D75E, G104dup, and G103_G104dup) were germline because they were present in remission specimens. However, it should be noted that it remains unclear if these are benign variants or if they contribute to germline susceptibility to myeloid neoplasia. One N-terminal variant (H84Q) was determined to be somatic because of its absence in a remission specimen. Q20 Collectively, these findings support a pathogenic role for many missense mutations in the C-terminal region. The significance of missense and inframe mutations in the N-terminal and midregions is less clear. The presence of co-occurring CEBPA mutations with other common mutations in AML such as FLT3-ITD and NPM1 mutations has been reported previously, but the prevalence of this co-occurrence is variable in different studies.12,13,33 Our large cohort provides an opportunity to address this issue further. In our study, FLT3-ITD mutations frequently were encountered in cases with both single and double CEBPA mutations (28% and 16%, respectively). CEBPAdm cases have no impact on prognosis when FLT3ITD mutations are present.12 Similar to the study by Dufour et al,33 we observed a high frequency of NPM1 mutations (31%) among CEBPAsm cases. An important question that cannot be addressed by our study is the prognostic impact of these co-occurring mutations. Co-occurring CEBPAdm and NPM1 are rare, and this is attributed to the fact that both mutations are thought to play a critical role in the initial stages of leukemogenesis. In our series, we encountered three such cases, all of which were among the nonclassic
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CEBPAdm cases. As discussed before and shown in one of our cases, these cases may represent two neoplastic clones with CEBPAsm. Dufour et al33 showed a survival advantage for the presence of mono-allelic CEBPA mutations in NPM1 mutated cytogenetically normal AML patients. This further emphasizes the necessity of screening for all CEBPA mutations, including single mutations that previously were thought to have no prognostic impact.12,13 Further clarification of the clinical impact of various co-existing genes may require larger studies. Given the rapidly growing list of prognostic and therapeutically relevant genetic alterations in AML, wide genomic profiling strategies using technologies such as next-generation sequencing may be beneficial.34 Assays with comprehensive mutational analysis panels currently are offered by a few clinical laboratories and likely will replace many single-gene tests. Importantly, in our experience and that of others, the detection of CEBPA mutations by nextgeneration sequencing techniques appears challenging. This is because of the high GC content of the gene as well as the complex nature of the mutations, including frequent expansions or contractions of homopolymer sequences and the large number of different insertions and deletions. Thus, even in the light of emerging technologies capable of panelbased testing, reliable clinical-grade testing of some genes, including CEBPA, likely still will require alternate singlegene assays for some time. Altogether, the Sanger-sequencingebased assay described herein represents a robust testing solution for the detection of all CEBPA mutations. In addition, we describe one of the largest series of published CEBPA mutations, characterizing the type and distribution of such mutations, and their correlation with other prognostically relevant mutations that can help strategize molecular profiling of AML.
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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2014.09.007.
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References 15. 1. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J, Hann I, Stevens R, Burnett A, Goldstone A: The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998, 92:2322e2333 2. Klaus M, Haferlach T, Schnittger S, Kern W, Hiddemann W, Schoch C: Cytogenetic profile in de novo acute myeloid leukemia with FAB subtypes M0, M1, and M2: a study based on 652 cases analyzed with morphology, cytogenetics, and fluorescence in situ hybridization. Cancer Genet Cytogenet 2004, 155:47e56 3. Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, Pettenati MJ, Patil SR, Rao KW, Watson MS, Koduru PR, Moore JO, Stone RM, Mayer RJ, Feldman EJ, Davey FR, Schiffer CA, Larson RA, Bloomfield CD: Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse,
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and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002, 100:4325e4336 Grimwade D, Walker H, Harrison G, Oliver F, Chatters S, Harrison CJ, Wheatley K, Burnett AK, Goldstone AH: The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood 2001, 98: 1312e1320 Arthur DC, Berger R, Golomb HM, Swansbury GJ, Reeves BR, Alimena G, Van Den Berghe H, Bloomfield CD, de la Chapelle A, Dewald GW, et al: The clinical significance of karyotype in acute myelogenous leukemia. Cancer Genet Cytogenet 1989, 40:203e216 Mrozek K, Heerema NA, Bloomfield CD: Cytogenetics in acute leukemia. Blood Rev 2004, 18:115e136 Schlenk RF, Dohner K, Krauter J, Frohling S, Corbacioglu A, Bullinger L, Habdank M, Spath D, Morgan M, Benner A, Schlegelberger B, Heil G, Ganser A, Dohner H: Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008, 358:1909e1918 Tenen DG, Hromas R, Licht JD, Zhang DE: Transcription factors, normal myeloid development, and leukemia. Blood 1997, 90:489e519 Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG: Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci U S A 1997, 94:569e574 Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S, Behre G, Hiddemann W, Tenen DG: Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet 2001, 27:263e270 Frohling S, Schlenk RF, Stolze I, Bihlmayr J, Benner A, Kreitmeier S, Tobis K, Dohner H, Dohner K: CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations. J Clin Oncol 2004, 22:624e633 Green CL, Koo KK, Hills RK, Burnett AK, Linch DC, Gale RE: Prognostic significance of CEBPA mutations in a large cohort of younger adult patients with acute myeloid leukemia: impact of double CEBPA mutations and the interaction with FLT3 and NPM1 mutations. J Clin Oncol 2010, 28:2739e2747 Dufour A, Schneider F, Metzeler KH, Hoster E, Schneider S, Zellmeier E, Benthaus T, Sauerland MC, Berdel WE, Buchner T, Wormann B, Braess J, Hiddemann W, Bohlander SK, Spiekermann K: Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol 2010, 28:570e577 Fasan A, Haferlach C, Alpermann T, Jeromin S, Grossmann V, Eder C, Weissmann S, Dicker F, Kohlmann A, Schindela S, Kern W, Haferlach T, Schnittger S: The role of different genetic subtypes of CEBPA mutated AML. Leukemia 2014, 28:794e803 Benthaus T, Schneider F, Mellert G, Zellmeier E, Schneider S, Kakadia PM, Hiddemann W, Bohlander SK, Feuring-Buske M, Braess J, Spiekermann K, Dufour A: Rapid and sensitive screening for CEBPA mutations in acute myeloid leukaemia. Br J Haematol 2008, 143:230e239 Lin LI, Lin TC, Chou WC, Tang JL, Lin DT, Tien HF: A novel fluorescence-based multiplex PCR assay for rapid simultaneous detection of CEBPA mutations and NPM mutations in patients with acute myeloid leukemias. Leukemia 2006, 20:1899e1903 Lin LI, Chen CY, Lin DT, Tsay W, Tang JL, Yeh YC, Shen HL, Su FH, Yao M, Huang SY, Tien HF: Characterization of CEBPA mutations in acute myeloid leukemia: most patients with CEBPA mutations have biallelic mutations and show a distinct immunophenotype of the leukemic cells. Clin Cancer Res 2005, 11:1372e1379 Fuster O, Barragan E, Bolufer P, Such E, Valencia A, Ibanez M, Dolz S, de Juan I, Jimenez A, Gomez MT, Buno I, Martinez J, Cervera J, Montesinos P, Moscardo F, Sanz MA: Fragment length
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993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032
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analysis screening for detection of CEBPA mutations in intermediaterisk karyotype acute myeloid leukemia. Ann Hematol 2012, 91:1e7 Ahn JY, Seo K, Weinberg O, Boyd SD, Arber DA: A comparison of two methods for screening CEBPA mutations in patients with acute myeloid leukemia. J Mol Diagn 2009, 11:319e323 Wouters BJ, Louwers I, Valk PJ, Lowenberg B, Delwel R: A recurrent in-frame insertion in a CEBPA transactivation domain is a polymorphism rather than a mutation that does not affect gene expression profiling-based clustering of AML. Blood 2007, 109:389e390 Pabst T, Eyholzer M, Fos J, Mueller BU: Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favourable prognosis. Br J Cancer 2009, 100:1343e1346 Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R: Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood 2009, 113:3088e3091 Grossmann V, Schnittger S, Schindela S, Klein HU, Eder C, Dugas M, Kern W, Haferlach T, Haferlach C, Kohlmann A: Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology. J Mol Diagn 2011, 13:129e136 Tizzani L, Wesolowski-Louvel M, Forte V, Romitelli F, Salani F, Lemaire M, Neil H, Bianchi MM: Mutations of the RAG3 gene encoding a regulator of fermentation in Kluyveromyces lactis are suppressed by a mutation of the transcription factor gene KlGCR1. FEMS Yeast Res 2007, 7:675e682 Biggio V, Renneville A, Nibourel O, Philippe N, Terriou L, Roumier C, Amouyel P, Cottel D, Castaigne S, Dombret H, Thomas X, Fenaux P, Preudhomme C: Recurrent in-frame insertion in C/EBPalpha TAD2 region is a polymorphism without prognostic value in AML. Leukemia 2008, 22:655e657 Gombart AF, Hofmann WK, Kawano S, Takeuchi S, Krug U, Kwok SH, Larsen RJ, Asou H, Miller CW, Hoelzer D, Koeffler HP: Mutations in the gene encoding the transcription factor CCAAT/ enhancer binding protein alpha in myelodysplastic syndromes and acute myeloid leukemias. Blood 2002, 99:1332e1340 Preudhomme C, Sagot C, Boissel N, Cayuela JM, Tigaud I, de Botton S, Thomas X, Raffoux E, Lamandin C, Castaigne S,
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Fenaux P, Dombret H: Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood 2002, 100:2717e2723 Barjesteh van Waalwijk van Doorn-Khosrovani S, Erpelinck C, Meijer J, van Oosterhoud S, van Putten WL, Valk PJ, Berna Beverloo H, Tenen DG, Lowenberg B, Delwel R: Biallelic mutations in the CEBPA gene and low CEBPA expression levels as prognostic markers in intermediate-risk AML. Hematol J 2003, 4:31e40 Snaddon J, Smith ML, Neat M, Cambal-Parrales M, Dixon-McIver A, Arch R, Amess JA, Rohatiner AZ, Lister TA, Fitzgibbon J: Mutations of CEBPA in acute myeloid leukemia FAB types M1 and M2. Genes Chromosomes Cancer 2003, 37:72e78 Shih LY, Huang CF, Lin TL, Wu JH, Wang PN, Dunn P, Kuo MC, Tang TC: Heterogeneous patterns of CEBPalpha mutation status in the progression of myelodysplastic syndrome and chronic myelomonocytic leukemia to acute myelogenous leukemia. Clin Cancer Res 2005, 11:1821e1826 Pabst T, Eyholzer M, Haefliger S, Schardt J, Mueller BU: Somatic CEBPA mutations are a frequent second event in families with germline CEBPA mutations and familial acute myeloid leukemia. J Clin Oncol 2008, 26:5088e5093 Bienz M, Ludwig M, Leibundgut EO, Mueller BU, Ratschiller D, Solenthaler M, Fey MF, Pabst T: Risk assessment in patients with acute myeloid leukemia and a normal karyotype. Clin Cancer Res 2005, 11:1416e1424 Dufour A, Schneider F, Hoster E, Benthaus T, Ksienzyk B, Schneider S, Kakadia PM, Sauerland MC, Berdel WE, Buchner T, Wormann B, Braess J, Subklewe M, Hiddemann W, Bohlander SK, Spiekermann K: Monoallelic CEBPA mutations in normal karyotype acute myeloid leukemia: independent favorable prognostic factor within NPM1 mutated patients. Ann Hematol 2012, 91:1051e1063 Patel JP, Gonen M, Figueroa ME, Fernandez H, Sun Z, Racevskis J, Van Vlierberghe P, Dolgalev I, Thomas S, Aminova O, Huberman K, Cheng J, Viale A, Socci ND, Heguy A, Cherry A, Vance G, Higgins RR, Ketterling RP, Gallagher RE, Litzow M, van den Brink MR, Lazarus HM, Rowe JM, Luger S, Ferrando A, Paietta E, Tallman MS, Melnick A, Abdel-Wahab O, Levine RL: Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med 2012, 366:1079e1089
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A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing: Report of a Large Series of Myeloid Neoplasms Q29
Amir Behdad, Helmut C. Weigelin, Kojo S.J. Elenitoba-Johnson, and Bryan L. Betz From the Department of Pathology, University of Michigan, Ann Arbor, Michigan Accepted for publication September 29, 2014. Address correspondence to Bryan L. Betz, Ph.D., Department of Pathology, University of Michigan, Traverwood II, 2910 Huron Pkwy., Ste. C, Ann Arbor, MI 48105. E-mail: [email protected].
Q4
Diagnostic testing for CEBPA mutations is the standard of care for cytogenetically normal acute myeloid leukemia. Widespread implementation of this testing is hampered by technical challenges associated with the GC content of the gene, the variability of the mutations, and the complexity of the sequence analysis and variant interpretation. We developed a robust Sanger-sequencing test to detect CEBPA mutations in diagnostic acute myeloid leukemia specimens. Our experience with testing 2393 cases of suspected myeloid neoplasms is presented. NPM1, FLT3einternal tandem duplication (ITD), and FLT3D835 mutation status were determined in parallel; 160 (6.7%) cases harbored CEBPA mutations, including 86 with a single mutation and 74 with double mutations. Nineteen single-mutant cases and 3 double-mutant cases showed only nucleotide substitutions that could not be detected by fragmentanalysisebased tests. A subset of cases harbored double mutations with uneven mutant allele frequency and required careful interpretation because of possible leukemic heterogeneity. NPM1 and FLT3-ITD mutations were more frequent in single- compared with double-mutation cases (31% versus 5% for NPM1, and 28% versus 16% for FLT3-ITD). This sequencing-based assay provides an efficient and reliable CEBPA mutation testing platform, permitting detection of all mutations with immediate distinction of single- and double-mutation cases. Given the technical challenges, robust Sangersequencing assays continue to maintain an important role in clinical CEBPA testing despite the development of multigene next-generation sequencing assays. (J Mol Diagn 2014, -: 1e9; http:// dx.doi.org/10.1016/j.jmoldx.2014.09.007)
Acute myeloid leukemia (AML) is a heterogeneous group of diseases with variable survival rates, disease course, and response to therapy. Inclusion of cytogenetic information in the classification of AML has helped tremendously to improve risk stratification of this disease,1e5 which can guide therapeutic decisions. However, 40% to 50% of AMLs are cytogenetically normal (CN-AML),1,6 and molecular markers are becoming increasingly important for further classification of this subgroup.7 Among the molecular alterations in AML, mutations of the CEBPA, NPM1, and FLT3 genes are among the most common and bear important prognostic significance. CEBPA, a single-exon gene, encodes a leucine zipper transcription factor with an important role in myeloid differentiation.8,9 CEBPA mutations are encountered in
approximately 8% to 15% of AML and are one of the most common mutations in CN-AML.7,10,11 Patients with CEBPAmutated AML (CEBPA-AML) may carry one (single-mutated CEPBA) or two mutated alleles (double-mutated CEPBA). The majority of double-mutated CEPBA cases harbor a truncating mutation in the N-terminal region of the protein combined with an inframe mutation in the C-terminal region. The N-terminal mutations abolish translation of full-length p42 protein, leading to production of only a smaller p30 isoform coded from an internal ATG start codon at amino acid 120. The p30 protein is thought to have a dominant-negative effect Supported by the Department of Pathology, University of Michigan, Ann Q2 Arbor, MI. Disclosure: None declared. Q3
Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.09.007
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Behdad et al and play a crucial role in leukemogenesis.10 In contrast, C-terminal inframe mutations lead to proteins with impaired DNA binding and dimerization. Recent studies have shown that the favorable outcomes associated with CEBPA mutations are limited to double-mutated CEPBA cases.12e14 Given its prognostic importance, AML with mutated CEBPA represents a provisional entity in the 2008 World Health Organization classification of tumors of the hematopoietic and lymphoid tissues. Consequently, a reliable and accurate testing method is necessary to assess CEBPA mutations in the work-up of all CN-AML patients. CEBPA mutations are highly variable and can occur across the entire coding region of the gene. The development of robust assays for CEBPA mutation testing is hampered by the high GC content of the gene (75% in the coding region), the presence of a trinucleotide repeat region, the complexity of the mutations, and the frequent occurrence of mutations in mononucleotide repeats. Similarly, inclusion of CEBPA as part of next-generation sequencing panels has been impeded by poor amplicon coverage and technical challenges associated with identification and calling of variants. Techniques that have been used successfully to screen for CEBPA mutations include Sanger sequencing or PCR-based fragment-length assays.11,15e18 Although efficient and sensitive, the fragmentlength assays only detect mutations that result in a net insertion or deletion, and not substitution mutations.19 Fragment-length analysis-based tests also cannot distinguish a common 6-bp duplication polymorphism (p.H195_P196dup) from an actual insertion or duplication mutation.20 Sequencing-based assays, despite their ability to detect all mutations, are more labor-intensive and expensive. Expertise in sequence annotation and interpretation also is required, which can be difficult in cases with unusual variants or complex length-affecting mutations. Consequently, many laboratories struggle with implementing a reliable and sensitive sequencing-based CEBPA assay for routine diagnostic purposes. Here, we describe a clinical-grade, Sanger-sequencinge based test to detect CEBPA mutations in diagnostic AML specimens. We evaluated the performance of this test over a duration of 4 years and describe our experience with interpreting one of the largest series of reported CEBPA mutations. In addition, we correlated the co-occurrence of these mutations with FLT3 internal tandem duplication (FLT3-ITD), FLT3D835, and NPM1 mutations.
other diagnostic data were available for only a limited number of patients and thus were not included for correlation.
DNA Extraction Genomic DNA was extracted from peripheral blood or bone marrow using the Qiagen Biorobot EZ1 (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions.
CEBPA Mutation Assay
The entire coding sequence of the CEBPA gene was amplified by PCR in two overlapping fragments, using the primers with sequences listed in Table 1. Each 30 mL PCR reaction ½T1 contained 50 ng DNA, 300 nmol/L of each primer, 1X Phusion GC mastermix (Finnzymes), and 8% dimethyl Q6 sulfoxide. Cycling conditions were as follows: 98 C for 30 seconds, 35 cycles of 99 C for 5 seconds, 68 C for 20 seconds, 72 C for 30 seconds, and final elongation at 72 C for 2 minutes. An aliquot of each PCR product was confirmed by gel electrophoresis. The rest was purified by the QIAquick PCR purification kit (Qiagen) and subjected to bidirectional sequencing using nested sequencing primers (Table 1) with ABI BigDye v1.1 terminators on an ABI 3130xl genetic analyzer (Applied Biosystems). Sequences spanning the Q7 entire CEBPA coding region were analyzed for mutations with software-assisted review (Mutation Surveyor, SoftGe- Q8 netics) in comparison with a GenBank reference sequence (https://www.ncbi.nlm.nih.gov/nuccore; GenBank accession Q9 number NM_004364). For interpretative purposes the coding region of CEBPA was divided into three regions: N-terminal (amino acids 1 to 120), midregion (amino acids 121 to 277), and C-terminal (amino acids 278 to 358), as previously described.14 Lengthaffecting frameshift mutations and nonsense point mutations in any region of the gene were considered pathogenic. Inframe length-affecting mutations and missense point mutations in the C-terminal region also were considered pathogenic. Inframe and missense mutations in the N-terminal and midregions were reported as variants of uncertain significance. Pathogenic mutations in which the mutant sequence trace was present at a higher level than the wildtype trace were considered homozygous and interpreted as
After approval by the University of Michigan Institutional Review Board, we conducted a retrospective study of the results of our clinical diagnostic testing for CEBPA mutations. Bone marrow or blood specimens in EDTA from 2420 patients with suspected myeloid neoplasm were submitted to our laboratory for routine diagnostic purposes. Clinical and
Table 1 CEBPA Assay Primers PCR primers CEBPA P1 Forward: 50 -GAGCAGGGTCTCCGGGTGG-30 Reverse: 50 -CCAGCTGCTTGGCTTCATCC-30 CEBPA P2 Forward: 50 -GCAGGCTGGAGCCCCTGTAC-30 Reverse: 50 -GACCCCAAACCACTCCCTGG-30 Sequencing primers CEBPA P1-SEQ Forward: 50 -AGGCTGGAGGCCGCCGAG-30 Reverse: 50 -GCTGCTTGGCTTCATCCTCC-30 CEBPA P2-SEQ Forward: 50 -GGCTGGAGCCCCTGTACGAG-30 Reverse: 50 -AACCACTCCCTGGGTCCCCGC-30
2
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Materials and Methods Patients
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double mutations.12,21,22 Synonymous variants and polymorphisms including c.402G>A (p.A134A), c.573C>T (p.H191H), c.690G>T (p.T230T), and c.584_589dup (p.H195_P196dup)20 were scored as wild type.
CEBPA-P2 721 bp CEBPA-P1 609 bp TAD1
TAD2
1
DBD/ZIP 1077 bp
(GCC) 7
FLT3 and NPM1 Mutation Testing Q10
When available, results of FLT3-ITD, FLT3-D835, and NPM1 mutations were retrieved for cases that tested positive for a CEBPA mutation. FLT3 and NPM1 testing was performed as previously described.23,24
3% DMSO 8% DMSO GC Buffer
CEBPA-P1
CEBPA-P2
c.199_200dupTA Mutation C
Results
C T
A C A
T
C
G
A C
C
C G
G C
C G
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Test Design and Assay Performance We searched the Catalogue of Somatic Mutations in Cancer Mutation Database for CEBPA mutations. Given the highly variable mutations that occurred throughout the coding region of the gene, including nonsense point mutations, we chose to design a sequencing-based assay rather than a fragment-length test. The high clinical significance of accurately distinguishing single- versus double-mutationepositive cases, including those with homozygous nonsense mutations, along with a relatively common length-affecting polymorphism (H195_P196dup), were other reasons for choosing a sequencing-based test.20,25 CEBPA is a single-exon gene with a 1077 bp coding region. Primers were designed such that two overlapping Q11 fragments spanning the entire coding region could be ½F1 amplified by PCR (Figure 1A). A two-amplicon design was chosen to minimize the number of amplicons requiring sequencing. Other published assays require four amplicons.11,19,23 Primer design and optimization of the PCR/ sequencing reactions was challenging because of the high GC content of the coding region (75%), the presence of a trinucleotide repeat (GCC), and problems with secondary structure. Optimization of the PCR required the addition of 8% dimethyl sulfoxide and the use of GC-rich PCR buffer to minimize production of nonspecific amplicons (Figure 1, B and C). Nested bidirectional sequencing primers were used to provide high-quality sequence data by minimizing the sequencing of nonspecific amplicons. The analytic sensitivity of the assay was determined by analyzing various dilutions of mutation-positive genomic DNA in wild-type DNA. Mutant alleles could be detected reliably down to a level of 10% using software-assisted sequence analysis (Figure 1D).
Testing Experience Test results were reviewed from 2420 consecutive specimens referred to our institution for assessment of CEBPA mutation status. PCR and sequencing results adequate for interpretation were obtained for 2393 (98.9%) specimens. Test failures occurred in 27 (1.1%) cases as a result of repeated PCR amplification failure and in most cases correlated with receipt
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20%
10%
5%
WT
Figure 1
Assay design and optimization. A: CEBPA is a single-exon gene with a 1077-bp coding region. Primers were designed such that two overlapping fragments spanning the entire coding region are amplified by PCR. B: Optimization of the PCR required the addition of 8% dimethyl sulfoxide (DMSO) and the use of GC-rich PCR buffer. Primer design and optimization of the PCR/sequencing reactions was challenging because of the high GC content of the coding region (75%), the presence of a trinucleotide repeat (GCC)7, and problems with secondary structure. C: Amplification products after optimization. D: Sensitivity of direct sequencing for CEBPA mutations. Mutation-positive genomic DNA was diluted into wildtype DNA. Mutations could be identified reliably down to a level of 10%. DBD/ZIP, DNA binding and dimerization domain (aa 278 to 358); TAD1, transactivation domain 1 (aa 70 to 97); TAD2, transactivation domain 2 (aa 127 to 200); WT, wild type.
of client-extracted DNA of low quantity. A review of successful test results showed that 160 cases (6.7%) harbored CEBPA mutations, including 86 cases (3.6%) with a single mutation (CEBPAsm) and 74 cases (3.1%) with double mutations (CEBPAdm). It is important to note that because the diagnosis was not known in the majority of the tested cases, the prevalence of mutations in our cohort may not be representative of the CEBPA mutation rate in AML and particularly that of CN-AML. Example sequence traces for N-terminal and C-terminal mutations are shown in Figure 2. A complete list of mutations is provided in Supplemental Table S1. Clinicopathologic information was available in 23 cases with CEBPA variants. Detailed information about these cases with diagnosis; karyotype; and CEBPA, NPM1, and FLT3 status is provided in Supplemental Table S2. N-terminal mutations (n Z 118) were small insertions or deletions (89%) or nonsense mutations (10%), which both
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373 N-Terminal Mutations C-Terminal Mutations Unknown Variant 374 c.68delC, p.P23fs c.937_939delAAG, p.K313del c.667G>A, p.G223S 375 G C A G C C C G G T C A C C C C C C C C G C A C G C G C C C G C A G A A G G T G C T G G A 376 377 * 378 379 c.245_246insGG, p.F82fs (homozygous) c.914_916dupAGC, p.Q305dup C A G C G C A A C G T G G A G T G T T C C A G C A C A G C C 380 381 H195_P196dup Polymorphism 382 383 c.584_589dup, p.H195_P196dup 384 c.962A>G, p.N321S c.21C>G, p.Y7X A G T G A C A A T G A C C G C G A C T T C T A C G A G G C G C A C C C G C C G C C C G C G 385 * 386 * 387 388 Figure 2 Sequence traces of N- and C-terminal CEBPA mutations, variants of unknown significance, and the c.584_589dup, p.H195_P196dup poly389 morphism. A variety of sequence alterations were detected, including both length-affecting mutations and nucleotide substitution mutations. The wild-type 390 CEBPA sequence is listed above the traces. The insertion mutation in the second panel of the left column (c.245_246insGG, p.F82fs) showed more than 90% 391 mutant allele frequency and therefore was interpreted as homozygous. Brackets in each panel indicate the deleted/inserted/duplicated nucleotide sequence; 392 asterisks, nucleotide substitutions. 393 394 mutations (21%), and a small number of frameshift (11%) 395 ½F3 predict truncation of the full-length p42 protein (Figure 3). and nonsense mutations (1%). A single case (1%) with a large 17eamino acid inframe 396 Mutations in CEBPAsm cases occurred mostly within the deletion in the N-terminal region was considered pathogenic 397 398 N-terminal region (52%), followed by the midregion (30%), through interpretative discretion. Midregion mutations 399 and then the C-terminal region (18%) (Table 2). Within the ½T2 (n Z 37) also mostly were small frameshift insertions or 400 deletions (86%) or nonsense mutations (14%); however, CEBPAdm cases, 88% harbored two distinct mutations. The 401 these predicted truncation of both p42 and p30 proteins. Cmajority of these cases (72%) harbored the classically 402 terminal region mutations (n Z 72) consisted mostly of described combination of a frameshift or nonsense mutation 403 small inframe insertions or deletions (67%) or missense in the N-terminal region and an inframe or missense 404 405 406 407 TAD1 TAD2 DBD/ZIP 358 408 1 120 278 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 Frameshift Nonsense Missense Inframe 427 Figure 3 Distribution of CEBPA mutations and variants identified in this study. The locations of the mutations are shown with respect to the CEBPA protein 428 and functional regions. Arrows depict the two translation initiation sites at amino acids (aa) 1 and 120. For interpretative purposes, the coding region of 429 CEBPA was divided into three regions: N-terminal (aa 1 to 120), midregion (aa 121 to 277), and C-terminal (aa 278 to 358). The majority of CEBPAdm cases 430 harbor a combination of a truncating frameshift or nonsense mutation in the N-terminal region and an inframe insertion/deletion or missense mutation in the 431 C-terminal region. Mutations in CEBPAsm cases are distributed in the entire coding region, with a greater portion in the midregion. Missense and inframe 432 mutations of the N-terminal and midregions were classified as variants of unknown significance. TAD1, transactivation domain 1 (aa 70 to 97); TAD2, 433 transactivation domain 2 (aa 127 to 200); DBD/ZIP, DNA binding and dimerization domain (aa 278 to 358). 434
Variants
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435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
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Summary of CEBPA Mutations
CEBPA mutation status
Mutation 1
CEBPAsm
Frameshift Nonsense
N-terminal AA 1 to 120
Mutation 2 Middle AA 121 to 277
C-terminal AA 278 to 358
N-terminal AA 1 to 120
Middle AA 121 to 277
C-terminal AA 278 to 358
Frameshift Nonsense Frameshift Inframe Missense Total CEBPAdm
Frameshift Frameshift Nonsense Frameshift Frameshift Frameshift Nonsense Nonsense
Inframe Missense Inframe Frameshift Frameshift Frameshift Inframe Nonsense Frameshift
Missense Inframe Frameshift and missense* Inframe
Frameshift Frameshift Nonsense Frameshift and nonsense* Total CEBPAhm
Frameshift
Number of patients 38 7 23 3 4 2 9 86 42 4 1 2 3 6 1 1 1 1 1 1 1 65 6 1 1 1 9
Frameshift Nonsense Inframe Nonsense
Total *These patients harbored three mutations. AA, amino acid number; hm, homozygous-mutated.
mutation in the C-terminal region. A nonclassic combination such as one of the two mutations occurring in the midregion, both mutations occurring in the N- or C-terminal region, or a C-terminal frameshift was detected in the remaining (28%) cases. Two cases with a nonclassic mutation pattern harbored three distinct mutations: one case showed a frameshift mutation in the midregion and two mutations (frameshift and missense) in the C-terminal region; another case harbored two truncating mutations in the N-terminal region (frameshift and nonsense); and a third case showed a truncating mutation in the C-terminal region (frameshift). Six of 65 nonhomozygous CEBPAdm cases (9%) showed an obvious discrepancy in the frequency of their two mutant alleles based on the sequence chromatograms. Five of these six cases harbored a nonclassic combination of mutations. ½F4 An example of such a case is shown in Figure 4. Given that the finding of discrepant mutation levels may indicate the presence of mutations in two different subclones, we interpreted these results cautiously and included a comment to indicate that these two mutations may represent two clones
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with a single CEBPA mutation. In support of this we tested a relapse specimen from a case that originally displayed two N-terminal frameshift mutations at different levels. Only one of the two mutations remained after treatment. Nine CEBPAdm cases (12%) harbored single mutations that were scored as homozygous based on the presence of a higher mutant peak trace compared with the wild-type trace on the sequence chromatograms. These mutations included six frameshift mutations in N-terminal region, two nonsense substitutions (one N-terminal and one C-terminal), and an inframe duplication in the C-terminal region. Similar to Green et al,12 these cases were interpreted as harboring biallelic mutations and thus were classified as CEBPAdm. In addition, we identified 20 cases with variants of unknown significance, including 14 missense variants and 6 small inframe duplications within the N-terminal and midregions. The designation of variants of unknown significance was based on the nonclassic pattern and the unknown clinicopathologic significance as further described in the Discussion section. With the exception of one case in which the variants of unknown significance co-occurred with an
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621 Case 41 Case 79 622 c.125_155del, p.P42fs c.232delC, p.L78fs A G C C T C C C G C C C G T T C C T G G C C G A 623 624 * * * 625 * * * * * * * * * 626 c.937_939dupAAG, p.K313dup c.436delC, p.L146fs 627 A A G G T G C T G G A G C C C C T G T A C G A G 628 * * * 629 * * * * * * * * * * * 630 631 Figure 4 Examples of CEBPAdm cases with equal (case 41) and 632 discrepant (case 79) mutant allele frequencies. Brackets highlight the 633 deleted/duplicated sequence; asterisks, the mutant peak. 634 635 N-terminal frameshift mutation, the remainder of the vari636 ants were found in isolation. The evaluation of variant allele 637 frequency in most of these variants showed values close to 638 50%, which suggested that these may represent germline 639 rather than somatic variants. Remission specimens were 640 available for four cases. Three variants (D75E, G104dup, 641 and G103_G104dup) were determined to be germline 642 because they were present in the remission specimen, 643 644 whereas the fourth variant (H84Q) was determined to be 645 somatic. 646 647 Correlation of the CEBPA Results with NPM1 and FLT3 648 649 Molecular testing for FLT3 internal tandem duplication 650 (FLT3-ITD), FLT3-D835, and NPM1 mutations were 651 available in 68 (79%) CEBPAsm and 64 (86%) CEBPAdm 652 ½T3 cases. Table 3 shows the frequency of each of these muta653 tions among CEBPAsm and CEBPAdm cases. FLT3-ITD 654 was the most common concurrent mutation with CEBPA, 655 and was encountered in 28% and 16% of CEBPAsm and 656 CEBPAdm cases, respectively. In the subset of CEBPAdm 657 658 cases with homozygous CEBPA mutations, 1 of 9 harbored 659 a FLT3-ITD and none harbored an NPM1 mutation. 660 NPM1 mutations were seen mainly in CEBPAsm patients 661 (31%); however, three CEBPAdm cases (5%) also harbored 662 an NPM1 mutation. Interestingly, each of these three cases 663 showed discrepant CEBPA mutant allele frequency with 664 CEBPA mutations in a nonclassic pattern rather than the 665 classic combination of an N-terminal and a C-terminal 666 mutation. In one of these patients several mutations were 667 detected at the time of AML diagnosis including two 668 CEBPA mutations, FLT3-ITD, FLT3-D835, and NPM1. At 669 670 relapse only one of the CEBPA mutations was present in 671 addition to a homozygous FLT3-ITD and NPM1 mutation. 672 These findings show evidence of clonal heterogeneity at the 673 time of diagnosis and that the two CEBPA mutations were 674 present in two different clones. 675 676 Discussion 677 678 679 Here, we describe one of the largest series of CEBPA mu680 tations reported to date. Similar to previous reports, the 681 majority of CEBPA mutations in our series were frameshift 682
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mutations of N-terminus and inframe mutations of the Cterminus.10,12,26e29 We also report several novel and recurring variants, the significance of which has not been determined at this time. The description and cataloging of such variants provides an opportunity for future clarification. Two variants, G122E and A176V, have been reported Q12 by other investigators,30e32 however, their functional significance remains unclear. Sequencing of the CEBPA gene is hampered by a repetitive nucleotide sequence and a high GC-rich content, which can lead to technical difficulties in assay design and optimization. To overcome these difficulties we optimized our primer design and incorporated a titrated concentration of dimethyl sulfoxide into the amplification reactions. An analysis of Sanger sequencing data also can be challenging and laborious, however, software can greatly boost the efficiency and quality of sequence analysis. In particular, we found the mutation electropherogram feature of Mutation Surveyor software to be especially helpful for detecting lowlevel mutations. This feature also provided the ability to scan and review sequence data in a rapid fashion. Other useful software features include the ability to analyze multiple patients and PCR amplicons within a single session and the ability to output mutations according to HGVS Q13 Q14 nomenclature. Although the software reliably detected all types of mutations (substitutions, duplications, insertions, and deletions), we found that only single base substitutions were annotated reliably. Length mutations sometimes were called incorrectly and these always required manual anno- Q15 tation for accurate mutation reporting. Once annotated, the interpretation of detected variants required familiarization with polymorphisms and the expected pattern of mutations. The interpretive approach as described in Materials and Methods section has been updated over time and is the result of our collective experience and the current literature. Implementing all of these elements has provided a reliable assay with robust performance. Many laboratories use multiplex PCR-based assays with fragment-length analysis for screening, followed by Sanger sequencing for confirmation in positive cases. Although this strategy is more efficient and detects the majority of CEBPA mutations, it will miss cases harboring only base substitution mutations. The shortcoming of PCR fragment-length Table 3 Co-Occurrence of FLT3 and NPM1 Mutations in Cases with CEBPA Mutations CEBPA þ
FLT3-ITD FLT3-D835þ NPM1þ FLT3-ITDþ/NPM1þ
þ
Single
Double
19 2 21 12
10 3 3 2
(28%) (3%) (31%) (18%)
(16%) (5%) (5%) (3%)
FLT3 and NPM1 results were available for 68 single and 64 double CEBPA mutation cases. ITD, internal tandem duplication.
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683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
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analysis as a screening method has been emphasized by previous investigators comparing this technique with upfront sequencing-based assays.19 Our data confirm this concern because in our series there were 19 (22%) CEBPAsm cases and 3 (4%) CEBPAdm cases that harbored only nonelength-affecting mutations, which would not have been detected by the fragment-length analysis screening strategy. Other investigators have reported an even higher frequency of point mutations among CEBPAdm cases,19,23 further emphasizing the necessity of an up-front sequencing strategy. Sanger sequencingebased assays inherently have a lower analytic sensitivity compared with fragment analysis. Our assay reliably detected 10% of the mutant allele burden, which is adequate for the detection of CEBPA mutations in diagnostic specimens of CN-AML patients, which, by definition, have more than 20% leukemic blasts. The utility of CEBPA mutation analysis in specimens after treatment is still under investigation; if a CEBPA assay was needed to detect minimal residual disease or early recurrence, then certainly more sensitive techniques are needed. Interpretation of sequence variants remains a challenging aspect of CEBPA testing. Similar to previous studies, the majority of the CEBPAdm cases in our cohort harbored a combination of a truncating N-terminal mutation and an inframe C-terminal mutation. Assuming that the two mutations are on different alleles, they are predicted to cause a p30 isoform translation from the allele with a truncating N-terminal mutation and a nonfunctional protein from the C-terminal mutation. These two proteins are believed to explain the role of CEBPAdm in leukemogenesis. Although there is consensus that such N-terminaletruncating and C-terminaleinframe variants represent bona fide pathogenic mutations, whether homozygous mutations are considered double mutations is debated.14 A small subset of cases in our study had homozygous mutations and similar unexpected combinations of mutations such as two concurrent mutations within the N- or C-terminal region or a mutation in the midregion. This pattern of mutations will not generate both of the earlier-mentioned aberrant proteins and thus their role in leukemogenesis remains unclear. Other studies have included homozygous and similar nonclassic mutations among CEBPAdm cases.12 Despite their uncertain role in leukemogenesis, the prognostic impact of a nonclassic pattern of mutations has not been studied separately. We found that some cases in our series harbored two CEBPA mutations with different allelic levels. Interestingly, most of these cases were among those with a nonclassic pattern of mutations. The finding of discrepant mutation levels raises concern that the two mutations could belong to different subclones and therefore these cases may be better classified as CEBPAsm. This hypothesis is supported by our observation of a case in which one of the two mutations became undetectable at relapse. Consistent with the literature, we reported cases with a nonclassic pattern of mutations as CEBPAdm. However, in the case of an obvious
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discrepancy in the frequency between the two mutant alleles, we add a cautionary result and comment in our report that such findings may indicate two single mutations in different clones; thus, the prognostic impact of these mutations is unclear. In addition, the classification of missense and inframe mutations can be challenging because of concerns of distinguishing pathogenic mutations from benign polymorphisms. For instance, the midregion inframe variant p.H195_P196dup once was reported as a pathogenic mutation; however, subsequent studies that screened healthy populations determined that this represents a benign polymorphism.20 Similarly, we and others identified a number of Q17 missense variants that required careful interpretation. In this large series we found that missense variants in the N-terminal and midregions usually were present at an allelic frequency close to 50% and were never found in combi- Q18 nation with a mutation, suggesting that they may represent germline variants with unknown pathogenic significance. In contrast, we found that the missense variants located in the C-terminal region tended to be present at allelic levels below 50% and occasionally were found in combination with a classic N-terminaletruncating mutation and with similar allelic frequencies. Polyphen analysis supported a damaging Q19 effect to the protein in all C-terminal missense mutations and only a subset of those in the N-terminal and midregions (data not shown). Furthermore, we were able to determine that several of the N-terminal variants (D75E, G104dup, and G103_G104dup) were germline because they were present in remission specimens. However, it should be noted that it remains unclear if these are benign variants or if they contribute to germline susceptibility to myeloid neoplasia. One N-terminal variant (H84Q) was determined to be somatic because of its absence in a remission specimen. Q20 Collectively, these findings support a pathogenic role for many missense mutations in the C-terminal region. The significance of missense and inframe mutations in the N-terminal and midregions is less clear. The presence of co-occurring CEBPA mutations with other common mutations in AML such as FLT3-ITD and NPM1 mutations has been reported previously, but the prevalence of this co-occurrence is variable in different studies.12,13,33 Our large cohort provides an opportunity to address this issue further. In our study, FLT3-ITD mutations frequently were encountered in cases with both single and double CEBPA mutations (28% and 16%, respectively). CEBPAdm cases have no impact on prognosis when FLT3ITD mutations are present.12 Similar to the study by Dufour et al,33 we observed a high frequency of NPM1 mutations (31%) among CEBPAsm cases. An important question that cannot be addressed by our study is the prognostic impact of these co-occurring mutations. Co-occurring CEBPAdm and NPM1 are rare, and this is attributed to the fact that both mutations are thought to play a critical role in the initial stages of leukemogenesis. In our series, we encountered three such cases, all of which were among the nonclassic
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CEBPAdm cases. As discussed before and shown in one of our cases, these cases may represent two neoplastic clones with CEBPAsm. Dufour et al33 showed a survival advantage for the presence of mono-allelic CEBPA mutations in NPM1 mutated cytogenetically normal AML patients. This further emphasizes the necessity of screening for all CEBPA mutations, including single mutations that previously were thought to have no prognostic impact.12,13 Further clarification of the clinical impact of various co-existing genes may require larger studies. Given the rapidly growing list of prognostic and therapeutically relevant genetic alterations in AML, wide genomic profiling strategies using technologies such as next-generation sequencing may be beneficial.34 Assays with comprehensive mutational analysis panels currently are offered by a few clinical laboratories and likely will replace many single-gene tests. Importantly, in our experience and that of others, the detection of CEBPA mutations by nextgeneration sequencing techniques appears challenging. This is because of the high GC content of the gene as well as the complex nature of the mutations, including frequent expansions or contractions of homopolymer sequences and the large number of different insertions and deletions. Thus, even in the light of emerging technologies capable of panelbased testing, reliable clinical-grade testing of some genes, including CEBPA, likely still will require alternate singlegene assays for some time. Altogether, the Sanger-sequencingebased assay described herein represents a robust testing solution for the detection of all CEBPA mutations. In addition, we describe one of the largest series of published CEBPA mutations, characterizing the type and distribution of such mutations, and their correlation with other prognostically relevant mutations that can help strategize molecular profiling of AML.
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References 15. 1. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J, Hann I, Stevens R, Burnett A, Goldstone A: The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998, 92:2322e2333 2. Klaus M, Haferlach T, Schnittger S, Kern W, Hiddemann W, Schoch C: Cytogenetic profile in de novo acute myeloid leukemia with FAB subtypes M0, M1, and M2: a study based on 652 cases analyzed with morphology, cytogenetics, and fluorescence in situ hybridization. Cancer Genet Cytogenet 2004, 155:47e56 3. Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, Pettenati MJ, Patil SR, Rao KW, Watson MS, Koduru PR, Moore JO, Stone RM, Mayer RJ, Feldman EJ, Davey FR, Schiffer CA, Larson RA, Bloomfield CD: Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse,
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