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PCR for monitoring of minimal residual disease in hematologic malignancy
Clinica Chimica Acta 413 (2012) 74–80
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Invited critical review
PCR for monitoring of minimal residual disease in hematologic malignancy Kazuyuki Matsuda ⁎, Mitsutoshi Sugano, Takayuki Honda Department of Laboratory Medicine, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan
a r t i c l e
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Article history: Received 9 August 2011 Received in revised form 18 October 2011 Accepted 19 October 2011 Available online 25 October 2011 Keywords: Minimal residual disease Allele-specific quantitative PCR Single nucleotide mutation Hematopoietic stem cell transplantation
a b s t r a c t Monitoring minimal residual disease (MRD) is useful to evaluate therapeutic response and risk of relapse in patients with hematologic malignancy. Currently available quantitative MRD assays are fluorescence in situ hybridization of chromosomal aberrations; multiparameter flow cytometry of leukemia-associated immunophenotypes; and quantitative polymerase chain reaction (qPCR) analysis of fusion genes, immunoglobulin/ T-cell receptor gene rearrangements, genetic alterations, or over-expressed genes. Among the PCR-based markers, genetic alterations are found in acute myelogenous leukemia patients with cytogenetically normal karyotype and can be considered as applicable targets for monitoring of MRD. Screening, confirmation and quantification procedures are important to develop the patient- or tumor-specific MRD assays using the PCR-based markers. Wild-type blocking PCR or coamplification at lower denaturing temperature-PCR is suited for screening of low-abundant genetic alterations, and allele-specific qPCR using primers including mismatched base and locked nucleic acids can quantify not only insertion and duplication of several nucleotides but also single nucleotide mutation in the presence of an excess amount of wild-type nucleotides. In addition to the well-established MRD markers, such as immunoglobulin/T-cell receptor gene rearrangements and fusion genes, utilizing potential MRD markers such as genetic alterations may expand the spectrum of patients in whom MRD can be monitored. © 2011 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical significance of the monitoring of MRD . . . . . . . . . . . . . . . . . . . . . . . . . PCR-based procedures and markers for monitoring of MRD . . . . . . . . . . . . . . . . . . . 3.1. Ig and TCR gene rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Fusion gene transcripts (RNA level) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Fusion gene (DNA level) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Genetic alterations including single nucleotide mutation, deletion, insertion, and duplication 3.4.1. Genetic alterations in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Screening and confirmation of genetic alterations . . . . . . . . . . . . . . . . 3.4.3. Direct detection of mutant DNA among an excess amount of wild-type DNA. . . . 3.4.4. Our data on application of AS-qPCRs for the evaluation of mutation-based MRD . . 3.5. Over-expressed genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: MRD, minimal residual disease; HSCT, hematopoietic stem cell transplantation; MFC, multiparameter flow cytometry; qPCR, quantitative polymerase chain reaction; Ig, immunoglobulin; TCR, T-cell receptor; STR-PCR, short tandem repeat-PCR; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; CN-AML, cytogenetically normal AML; WTB-PCR, wild-type blocking PCR; COLD-PCR, coamplification at lower denaturing temperature-PCR; LNA, locked nucleic acids; PNA, peptide nucleic acids; ARMS, amplification refractory mutation system; MAMA, mismatch amplification mutation assay; AS-qPCR, allele-specific qPCR. ⁎ Corresponding author. Tel.: + 81 263 37 2802; fax: + 81 263 34 5316. E-mail address: [email protected] (K. Matsuda). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.10.022
K. Matsuda et al. / Clinica Chimica Acta 413 (2012) 74–80
1. Introduction In patients with hematological malignancies, detection and quantification of minimal residual disease (MRD) during and after chemotherapy and post-hematopoietic stem cell transplantation (HSCT) are important in evaluating the patients' therapeutic response and risk of relapse. Currently available quantitative MRD assays are fluorescence in situ hybridization of numerical or structural aberrations at the chromosomal level; multiparameter flow cytometry (MFC) of aberrant immunophenotypes; and quantitative polymerase chain reaction (qPCR) analysis of fusion genes, immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements, genetic alterations, and over-expressed genes. Monitoring of disease status following transplantation can be performed by MRD detection and chimerism analyses by short tandem repeat-PCR (STR-PCR), which characterizes the origin of posttransplant hematopoiesis (Table 1). This review summarizes PCRbased procedures and markers including screening, confirmation, and quantification procedures used for MRD monitoring in hematological malignancies, with a focus on describing the methods for detecting single nucleotide mutations that can be used as potential MRD markers. 2. Clinical significance of the monitoring of MRD In large-scale studies of both acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patients, MRD detected by mainly MFC and/or PCR amplification of Ig or TCR genes has been used for risk stratification, and its prognostic significance has been confirmed. Analysis of aberrant immunophenotypes referred to as leukemia-associated immunophenotypes by flow cytometry is widely applicable to both ALL and AML [1–6]. For ALL patients, in addition to the leukemia-associated immunophenotypes, clonal rearrangements of Ig or TCR genes have been observed in more than 90% of patients [7] and monitoring of MRD has been accessible to many patients. Unlike ALL patients, however, AML patients lack widely applicable PCRbased MRD markers. The PCR-based markers frequently used for MRD detection in AML are fusion gene transcripts such as RUNX1-RUNX1T1, CBFB-MYH11, and PML-RARA resulting from t(8;21)(q22;q22), inv (16)(p13q22) or t(16;16)(p13;q22), and t(15;17)(q22;q12), respectively [1]. Although the application of fusion transcripts for MRD monitoring is limited to specific leukemia subtypes (approximately 25% of AML) [9,10], the clinical significance of quantification of these fusion transcripts has been established [11–14]. For patients with chronic myelogenous leukemia, quantitative monitoring of BCR-ABL1 transcripts has clinical utility not only for predicting relapse following stem cell transplantation, but also for evaluating the effectiveness of treatments such as donor leukocyte infusion, interferon alpha, and tyrosine kinase inhibitor, and it is also useful for providing information on the response to treatment in patients with Table 1 MRD markers for monitoring of MRD and chimerism.
Philadelphia chromosome-positive ALL (Ph + ALL) [15]. In addition to the recurrent reciprocal translocations associated with leukemogenesis, a number of genetic alterations, such as insertions, interstitial/partial tandem duplications, or single nucleotide mutations have been reported to be involved in the pathogenesis of leukemia and associated with the prognosis of the affected patients [16–18]. Approximately 40% of patients with AML display normal karyotype at diagnosis; these cases are defined as cytogenetically normal AML (CN-AML) [19]. CN-AML patients are discriminated into certain prognostic subgroups according to the different genetic alterations. In addition to their role as diagnostic or prognostic markers, the genetic alterations have been shown to be useful for MRD detection and quantification (Table 2). Recently, several groups have performed quantitative assessment of NPM1 or FLT3 mutations and used them as MRD markers in patients with AML [20–27]. In a large study of AML patients with mutated NPM1, the MRD level based on the level of mutated NPM1 during and after therapy was shown to allow for the identification of patients with high risk of relapse [28]. For patients without recurrent chromosomal and genetic abnormalities, molecular targets suitable for qPCR-based MRD monitoring include over-expressed genes such as WT1[29]. Quantification of WT1 expression has prognostic impact and has been evaluated as a marker for risk stratification [30–34]. 3. PCR-based procedures and markers for monitoring of MRD PCR procedures and targets including screening, confirmation, and quantification for monitoring of MRD are listed in Table 3. qPCR is the most widely available PCR-based quantification procedure and is regarded as a reliable method to evaluate MRD. qPCR can be performed with SYBR Green, hydrolysis (TaqMan) probes, or hybridization (LightCycler) probes [35]. We will focus on qPCR with TaqMan probes in this review. TaqMan-qPCR can reduce non-specific amplification due to the specificity of primers and probes. TaqMan-qPCR using RNA or DNA is broadly applicable to evaluate the quantity or expression level of target genes such as rearranged Ig or TCR genes, fusion genes, mutated genes, or aberrantly expressed genes (Table 3). 3.1. Ig and TCR gene rearrangements The leukemic cells in patients with ALL have a unique clonal rearrangement of Ig or TCR genes. PCR amplification of the rearranged Ig
Table 2 Genetic alterations with potential as markers for monitoring of MRD. Types of genetic alterations
Relevant genes
Insertion/duplication
FLT3-ITD NPM1 MLL-PTD WT1 FLT3-TKD KIT RAS (NRAS, KRAS) PTPN11 RUNX1 CEBPA IDH1 WT1 TP53 JAK2 MPL BRAF WT1 EVI1 PRAME BAALC ERG MN1
Single nucleotide mutation
Markers
Detection methods
For MRD Chromosomal abnormality Leukemia-associated immunophenotype Ig/TCR gene rearrangements Fusion genes Genetic alterations
FISH MFC PCR PCR PCR
For chimerism Sex chromosome Short tandem repeat (STR) Single nucleotide polymorphism (SNP)
FISH PCR PCR
MRD, minimal residual disease; FISH, fluorescence in situ hybridization; MFC, multiparameter flow cytometry; Ig, immunoglobulin; TCR, T-cell receptor; PCR, polymerase chain reaction.
75
Over-expression
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K. Matsuda et al. / Clinica Chimica Acta 413 (2012) 74–80
Table 3 PCR-based markers and procedures (screening, confirmation, quantification) for MRD monitoring. Markers
Screening
Ig/TCR rearrangements (DNA level) Fusion gene (RNA level) Fusion gene (DNA level) Genetic alterations (DNA or RNA levels)
BIOMED-2 multiplex direct PCR sequencing PCR or qPCR direct sequencing Long-distance PCR direct LDI-PCR sequencing Direct sequencing direct WTB-PCR sequencing HRM COLD-PCR qPCR
Over-expressed gene (RNA level)
Confirmation
Quantification qPCR, GeneScan qPCR qPCR qPCR (AS-qPCR)
qPCR
Ig, immunoglobulin gene; TCR, T-cell receptor gene; qPCR, quantitative polymerase chain reaction; LDI-PCR, long-distance inversed PCR; WTB-PCR, wild-type blocking PCR; HRM, high resolution melting analysis; AS-qPCR, allele-specific quantitative polymerase chain reaction; COLD-PCR, coamplification at lower denaturing temperaturePCR.
or TCR genes has been used as an MRD marker for patients with ALL. Ig/TCR gene rearrangements in stable DNA are patient-specific and the sensitivity of PCR for their detection is 10 − 4–10 − 5[35]. To use the specific rearrangements of Ig or TCR genes in individual patients as an MRD marker, identification of the rearranged sequences at diagnosis is needed. Recently, standardization of the multiplex PCR methods for detecting clonal Ig or TCR gene rearrangements was established by the BIOMED-2 Concerted Action BMH4-CT98-3936 study [36]. Combined use of BIOMED-2 multiplex PCRs with GeneScan analysis could become a highly sensitive and high-throughput method [36]. Using the BIOMED-2 multiplex PCR procedure, the detection rate of clonal rearrangement of TCRβ and TCRγ ranged from 94% to 98% in patients with T-cell malignancies and that of Ig from 91% to 96% in patients with B-cell malignancies [37,38]. Sandberg et al. [39] mentioned that BIOMED-2 multiplex PCR could replace Southern blot analysis in routine testing. For the screening and identification of the rearranged Ig/TCR genes, the BIOMED-2 multiplex PCR is useful. After BIOMED-2 multiplex PCR, the PCR products are subjected to agarose gel electrophoresis and then to direct sequencing. Then, the primers or TaqMan probes can be designed according to the sequence at the junction regions. Although the Ig/TCR gene rearrangements are useful MRD markers, the evaluation of clonality based on rearrangements is frequently hampered by the amplification of similar rearrangements from normal cells. Furthermore, rearrangement at diagnosis may be lost by secondary rearrangements during the clinical course [40]. To avoid false negative results, several Ig/TCR targets should be used for each patient.
3.2. Fusion gene transcripts (RNA level) Chromosomal structural abnormalities associated with malignancies can result in the formation of fusion genes. Fusion gene transcripts such as BCR-ABL1 in CML or ALL, PML-RARA, RUNX1-RUNX1T1 or CBFB-MYH11 in AML, and ETV6-RUNX1 in ALL patients are used as appropriate MRD markers by qPCR with a high sensitivity of 10 − 4– 10 − 6[35]. Designing primers or probes is less labor-intensive when the spliced exon of the two original genes constituting the fusion gene is known. Gabert et al. reported the primer/probe combinations for frequently detected fusion gene transcripts [TCF3 (E2A)-PBX1, MLL-AFF1 (AF4), ETV6-RUNX1, BCR-ABL1 major and minor type, STIL (SIL)-TAL1, PML-RARA, CBFB-MYH11, or RUNX1-RUNX1T1] [8]. Considering that some cases have variant fusions, qualitative PCR using primers that cover more spliced variants is important for screening at diagnosis [11]. Of note, MRD monitoring using RNA does not directly reflect the tumor load, because the transcripts themselves may be
affected by cytotoxic therapy, resulting in different transcript levels during therapy [35]. 3.3. Fusion gene (DNA level) For fusion gene-based MRD evaluation, the level of RNA transcripts is measured using qPCR as described above. In order to do this, it is desirable to use a patient-specific marker for evaluating MRD. Namely, in patients with a BCR-ABL1-, a PML-RARA-, or an MLL-related fusion gene, (semi) quantitative assessment of MRD by utilizing the patient-specific genomic DNA breakpoint has been reported [41–45]. Although long-range PCR and modified PCR using tagged primers or adoptor-ligation methods to detect genomic DNA fusion points are labor-intensive compared to the methods used to detect common exon–exon fusions at the RNA level [41–45], the identification of the genomic DNA breakpoint region could realize precise and stable monitoring of MRD because the RNA expression fluctuates during therapy and RNA is unstable [35]. Structural abnormalities involving the mixed-lineage leukemia (MLL) on 11q23 have been detected in a wide spectrum of hematological malignancies, such as AML, ALL, and myelodysplastic syndrome [46,47]. The rearrangement of MLL occurs during translocations, deletions, insertions, or partial tandem duplications involving a variety of genes on the partner chromosome [48]. A recently developed long-distance inverse PCR technique allows us to identify MLL-fusion partner genes generated by several mechanisms and to determine patient-specific breakpoint DNA sequences, which helps in the construct of the patient-specific primers or probes for MRD detection [45,49]. For patients with the MLL fusion gene, Burmeister et al. [50] developed a novel MRD assessment procedure using the genomic breakpoint region-related MLL gene as a patient-specific marker that could accurately and consistently reflect the quantity of leukemic cells. The sensitivity of the method based on the monitoring of patient-specific breakpoint DNA sequences was 10 − 4 to 10 − 5[50]. The genomic breakpoint sequence of the MLL fusion gene was not influenced by secondary rearrangement as in the case of the Ig/TCR genes [50]. In some cases with complex chromosomal structural abnormalities, fluorescence in situ hybridization capable of detecting the breakpoint region at the chromosomal level may provide information on the patient-specific aberrant gene configurations and help to set up the PCR-based MRD assays [51]. 3.4. Genetic alterations including single nucleotide mutation, deletion, insertion, and duplication 3.4.1. Genetic alterations in AML AML is a genetically heterogeneous disease in which genetic alterations as well as cytogenetic abnormalities constitute key events in the pathogenesis [19]. According to the review by Renneville et al. [17], the aberrant genes are clustered into three groups: genes that contribute to cell proliferation (KIT, FLT3, RAS, and PTPN11), genes involved in hematopoietic differentiation (RUNX1 and CEBPA), and genes implicated in cell cycle regulation and apoptosis (TP53 and NPM1). Among these genetic alterations, the NPM1 mutation characterized by tetranucleotide insertion and FLT3 internal tandem duplication occurs in about 50% and 40% of case of adult AML without chromosomal abnormalities, respectively. The quantitative assessments of NPM1 or FLT3 mutations as an MRD marker were performed by TaqMan qPCR or LightCycler assays using primers or probes specific for the duplicated nucleotides. The potential impact of MRD based on NPM1 or FLT3 has been demonstrated [20–28]. In addition to insertions/duplications, single nucleotide mutations in various genes are frequently detected. Single nucleotide mutation can be considered an applicable marker for monitoring of MRD; however, its application is limited in part because it is much more difficult
K. Matsuda et al. / Clinica Chimica Acta 413 (2012) 74–80
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3.4.2. Screening and confirmation of genetic alterations To use single nucleotide mutation as an MRD marker, the screening methods and specific detection methods are needed just as for other PCR-based markers (Table 3). The direct sequencing method is well established and is the most common and conventional method used for the screening and confirmation of mutations in tumor cells. When a diagnostic sample containing abundant leukemia cells is available, conventional direct sequencing analysis can be used to detect mutations; however, when only the samples following therapy are available, direct sequencing analysis does not have enough sensitivity to detect the reduced mutant DNA. Two recently developed methods, i.e., wild-type blocking PCR (WTB-PCR) and coamplification at lower denaturing temperaturePCR (COLD-PCR), are applicable as screening approaches to detect or amplify the known or unknown mutations among an excess amount of the wild-type DNA. Locked nucleic acid (LNA)- or peptide nucleic acid (PNA)-mediated WTB-PCR has been employed to block the wild-type sequence and preferentially amplify the mutant sequence. LNA- and PNA-substituted nucleotides increase the binding affinity compared to standard nucleotides [52–56]. In addition, PNA is resistant to the 5′ nuclease activity of Taq DNA polymerase, such that the nucleic acids are used for blocking the wild-type DNA, resulting in the preferential amplification of mutant sequences during PCR. Nagai et al. [57] have reported a PNA-LNA PCR clamp system for detection of EGFR gene mutations in the presence of a 100- to 1000-fold higher background level of wild-type EGFR. In the PNA-LNA clamp system, a PNA-containing oligonucleotide is used for blocking wild-type sequences and a fluorescent probe including LNA is used to detect the mutant amplicon. In addition to their use as probes for stable hybridization, LNA-containing oligonucleotides can also be used for blocking wild-type sequences [58]. Using a polymerase without intrinsic 5′–3′ exonuclease activity (Stoffel fragment), the stability of blocking by LNA-containing oligonucleotides can be enhanced [59]. Li et al. [60] have recently developed two COLD-PCR systems (full COLD-PCR and fast COLD-PCR) that are capable of enriching small quantities of known or unknown mutants. To detect tumorassociated mutations including both known and unknown mutations, direct sequencing analysis is commonly used; however, its sensitivity is low and not sufficient to detect mutations present in small quantities. In COLD-PCR utilizing critical a denaturation temperature (Tc) lower than the melting temperature (Tm), heteroduplexes of mutant and wild-type alleles preferentially denature over homoduplexes of wild-type alleles, which enables amplification of mutationcontaining alleles several-folds over WT alleles. Full COLD-PCR can enrich all possible mutations and fast COLD-PCR without a heteroduplexes formation step has a short reaction time. More recently, Milbury et al. [61] reported an Improved and Complete Enrichment (ice)-COLD-PCR utilizing a reference sequence which binds rapidly to the amplicon. Ice-COLD-PCR has advantages of full and fast COLD-PCR and allows for robust and efficient COLD-PCR. For COLDPCR, we have to use thermal cycler with high temperature precision and determine the Tc and Tm using high resolution melting analysis. In the routine mutation-screening test, COLD-PCR is a promising method that can be combined with subsequent molecular analyses, such as direct sequencing, high resolution melting analysis, and qPCR [62,63]. Combining the use of WTB-PCR or COLD-PCR and sequencing analyses allows us to detect unknown and/or known mutations and identify the precise gene alterations and their locations. Following the identification of the type of gene alterations by the procedures described above, the direct or allele-specific amplification using primers or probes specific to the mutant nucleotide can be performed.
3.4.3. Direct detection of mutant DNA among an excess amount of wild-type DNA For direct detection of mutant DNA, primers or probes specific to the mutant DNA are needed. The binding affinity of primers is affected by the modification of nucleotides around the 3′ terminal region of primers. PCR using primers including only a mutant-matched base in the 3′-end has been shown to result in non-specific amplification, so that additional modification is needed. Several allele-specific PCR (AS-PCR) methods based on an amplification refractory mutation system (ARMS) or mismatch amplification mutation assay (MAMA) have been developed for the detection of single nucleotide mutations [64,65]. The primers in AS-PCR included a mutant-matched nucleotide at the 3′-end and a template-mismatched nucleotide near the 3′-end. An AS-quantitative PCR (AS-qPCR) based on ARMS or MAMA was developed with a TaqMan probe to quantify low levels of mutant nucleotides in the presence of high levels of the counterpart wildtype nucleotides [66]. Recently, several studies have reported that single nucleotide mutations of JAK2 and MPL, or single nucleotide mutations of the ABL kinase domain in BCR-ABL1 were quantitatively monitored in myelofibrosis patients following transplantation [67–70] or in chronic myelogenous leukemia patients with imatinib resistance, respectively [71,72]. Pretreatment with restriction enzymes to digest residual wild-type nucleotides is performed to obtain high specificity and sensitivity of the AS-PCR; however, restriction enzymes that digested the wild-type sequence at various mutations are
-10 -15 -20 -25 Mutant AS-qPCR
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Fig. 1. Specificity (difference of threshold cycles, ΔCt) and sensitivity (the delay Ct) using constructed wild-type and mutant plasmid DNA by 3 types of AS-qPCRs. Three types of AS-qPCRs (black-bar, mutant AS-qPCR; gray-bar, mismatched AS-qPCR; open-bar, LNA-AS-qPCR) were compared in terms of the difference of threshold cycles (ΔCt) and delay in Ct (delay Ct) using equal copy numbers of constructed wild-type and mutant plasmid DNA. The ΔCt was calculated as follows: (Ct for mutant plasmid) − (Ct for wild-type plasmid) in each AS-qPCR assay. The delay Ct was calculated as follows: (Ct for mutant plasmid in mismatched AS-qPCR or LNA-AS-qPCR) − (Ct for mutant plasmid in mutant AS-qPCR). The dotted line indicates 3.33 cycles. The threshold cycles used for the present calculations were based on the data in our previously reported experiments [73,74]. Data are expressed as the means − S.D. for ΔCt and means + S.D. for ΔCt. 1, PTPN11 179G>A; 2, PTPN11 182A>T; 3, PTPN11 214G>A; 4, PTPN11 226G>A; 5, PTPN11 227A>C; 6, PTPN11 227A>G; 7, NRAS 34G>A; 8, NRAS 35G>A; 9, NRAS 38G>A; 10, KRAS 34G>A; 11, FLT3 2503G>T; 12, KIT 2446G>T; 13, KIT 2447A>T.
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not always available. Additional modification of primers or probes is needed. 3.4.4. Our data on application of AS-qPCRs for the evaluation of mutation-based MRD For single nucleotide mutations such as PTPN11, NRAS, KRAS, FLT3, c-KIT, or TP53, we developed AS-qPCRs using three different mutation-specific primers as follows: mutant AS-qPCR primers including only a mutant-matched nucleotide in the 3′-end; mismatched AS-qPCR primers, which are identical to ARMS or MAMA, including a mutant-matched nucleotide in the 3′-end and a templatemismatched nucleotide at the penultimate 3′-end; LNA-AS-qPCR primers including a mutant-matched nucleotide in the 3′-end, a template-mismatched nucleotide at the penultimate 3′-end, and LNA at the -2 position from the 3′-end. When compared with mutant AS-qPCR, both mismatched AS-qPCR and LNA-AS-qPCR exerted a more significant specificity for discrimination between wild-type and mutant DNA in all 13 types of mutations (6 types of PTPN11 mutations, 4 types of RAS mutations, 2 types of KIT mutations, and 1 type of FLT3 mutation) (Fig. 1A). Of note, the specificity of LNA-AS-qPCR was found to be greater than that of mismatched AS-qPCRs in all types of mutations (Fig. 1A). Additional primer modifications, such as the inclusion of a mismatched base or substitution with an LNA base, did not lead to a more than 10 (2 3.33)-fold reduction of sensitivity in 10 of 13 types of mutations (Fig. 1B). Using clinical samples, we developed specific mismatched or LNA-AS-qPCRs with a sensitivity of 10 − 3 to 10 − 4 for each mutation [73,74]. The AS-qPCRs for single nucleotide mutations had accuracy comparable to qPCR for the RUNX1RUNX1T1 fusion gene and WT1 and were applicable to monitoring of MRD throughout the clinical course, including prior to transplantation and post-transplantation [73]. Furthermore, the post-transplant changes of MRD levels assessed by LNA-AS-qPCR were well correlated with the percentage of autologous cells (chimerism) assessed by STRPCR, which characterizes the origin of post-transplant hematopoiesis [74]. The interpretation of conditions with mixed chimerism (MC) is difficult because of the variation of the interval between MC and relapse among patients and the long-lasting MC in some patients without relapse [75]. The post-transplant chimerism analysis at very short time intervals demonstrated a significant correlation between patients with increasing MC and relapse [76,77]. AS-qPCR analyses allow accurate assessment whether autologous cells belong to normal or malignant cell clones, which may lead to prediction of imminent relapse. 3.5. Over-expressed genes Other potential markers for qPCR-based MRD monitoring include the over-expressions of WT1, EVI1, PRAME, BAALC, ERG, or MN1 genes (Table 2). Among these markers, over-expression of the WT1 gene is most frequently used as a universal MRD marker applicable to patients without recurrent chromosomal or genetic abnormalities. To evaluate MRD, a comparison of the difference in expression levels between patients and healthy controls is needed, because WT1 transcripts have been detected not only in tumor cells but also in normal cells [34]. 4. Conclusion MRD monitoring is applicable for the early detection of impending relapse that is not clinically evident as well as for the evaluation of disease status based on tumor burden. Furthermore, the potent application of MRD monitoring is to assess the effectiveness of new antileukemic agents, with an aim to identifying risk-adaptive therapies and circumventing suboptimal therapies [78,79]. Therefore, monitoring of MRD provides useful information in various aspects of the clinical management of patient with hematological malignancies. WTB-PCR and
COLD-PCR analyses, which are promising procedures for screening known or unknown gene alterations, can be combined with the downstream procedures, including AS-qPCR targeting the specific gene mutations. Utilizing potential MRD markers, such as gene mutations, along with well-established MRD markers, such as Ig/TCR rearrangements and fusion gene transcripts will be important for expanding the spectrum of patients in whom MRD can be monitored. Conflict of interest The authors state that they have no conflict of interest. Funding Our study was supported in part by a Grant-in-Aid for Science research from the Japan Society for the Promotion of Science (No. 20930022, KM), the Charitable Trust Laboratory Medicine Research Foundation of Japan (2008, KM), and the Hokuto Foundation for Bioscience (2010, KM). Acknowledgements We gratefully acknowledge Dr. Nobuo Okumura (Laboratory of Clinical Chemistry and Immunology, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan) for his helpful review and comments in the preparation of the manuscript. References [1] Rubnitz JE, Inaba H, Dahl G, et al. Minimal residual disease-directed therapy for childhood acute myeloid leukaemia: results of the AML02 multicentre trial. Lancet Oncol 2010;11:543–52. [2] Conter V, Bartram CR, Valsecchi MG, et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. Blood 2010;115:3206–14. [3] Pui CH, Campana D, Pei D, et al. Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med 2009;360:2730–41. [4] Vidriales MB, Perez JJ, Lopez-Berges MC, et al. Minimal residual disease in adolescent (older than 14 years) and adult acute lymphoblastic leukemias: early immunophenotypic evaluation has high clinical value. Blood 2003;101:4695–700. [5] Feller N, van der Pol MA, van Stijn A, et al. MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia. Leukemia 2004;18:1380–90. [6] Kern W, Voskova D, Schoch C, et al. Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004;104:3078–85. [7] van der Velden VH, Cazzaniga G, Schrauder A, et al. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of realtime quantitative PCR data. Leukemia 2007;21:604–11. [8] Gabert J, Beillard E, van der Velden VH, et al. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia — a Europe Against Cancer program. Leukemia 2003;17:2318–57. [9] Estey E, Döhner H. Acute myeloid leukaemia. Lancet 2006;368:1894–907. [10] Byrd JC, Mrózek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325–36. [11] van Dongen JJM, Macintyre EA, Gabert J, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13: 1901–28. [12] Perea G, Lasa A, Aventín A, et al. Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)]. Leukemia 2006;20:87–94. [13] Corbacioglu A, Scholl C, Schlenk RF, et al. Prognostic impact of minimal residual disease in CBFB-MYH11-positive acute myeloid leukemia. J Clin Oncol 2010;28: 3724–9. [14] Grimwade D, Jovanovic JV, Hills RK, et al. Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy. J Clin Oncol 2009;27:3650–8.
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Invited critical review
PCR for monitoring of minimal residual disease in hematologic malignancy Kazuyuki Matsuda ⁎, Mitsutoshi Sugano, Takayuki Honda Department of Laboratory Medicine, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan
a r t i c l e
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Article history: Received 9 August 2011 Received in revised form 18 October 2011 Accepted 19 October 2011 Available online 25 October 2011 Keywords: Minimal residual disease Allele-specific quantitative PCR Single nucleotide mutation Hematopoietic stem cell transplantation
a b s t r a c t Monitoring minimal residual disease (MRD) is useful to evaluate therapeutic response and risk of relapse in patients with hematologic malignancy. Currently available quantitative MRD assays are fluorescence in situ hybridization of chromosomal aberrations; multiparameter flow cytometry of leukemia-associated immunophenotypes; and quantitative polymerase chain reaction (qPCR) analysis of fusion genes, immunoglobulin/ T-cell receptor gene rearrangements, genetic alterations, or over-expressed genes. Among the PCR-based markers, genetic alterations are found in acute myelogenous leukemia patients with cytogenetically normal karyotype and can be considered as applicable targets for monitoring of MRD. Screening, confirmation and quantification procedures are important to develop the patient- or tumor-specific MRD assays using the PCR-based markers. Wild-type blocking PCR or coamplification at lower denaturing temperature-PCR is suited for screening of low-abundant genetic alterations, and allele-specific qPCR using primers including mismatched base and locked nucleic acids can quantify not only insertion and duplication of several nucleotides but also single nucleotide mutation in the presence of an excess amount of wild-type nucleotides. In addition to the well-established MRD markers, such as immunoglobulin/T-cell receptor gene rearrangements and fusion genes, utilizing potential MRD markers such as genetic alterations may expand the spectrum of patients in whom MRD can be monitored. © 2011 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical significance of the monitoring of MRD . . . . . . . . . . . . . . . . . . . . . . . . . PCR-based procedures and markers for monitoring of MRD . . . . . . . . . . . . . . . . . . . 3.1. Ig and TCR gene rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Fusion gene transcripts (RNA level) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Fusion gene (DNA level) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Genetic alterations including single nucleotide mutation, deletion, insertion, and duplication 3.4.1. Genetic alterations in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Screening and confirmation of genetic alterations . . . . . . . . . . . . . . . . 3.4.3. Direct detection of mutant DNA among an excess amount of wild-type DNA. . . . 3.4.4. Our data on application of AS-qPCRs for the evaluation of mutation-based MRD . . 3.5. Over-expressed genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: MRD, minimal residual disease; HSCT, hematopoietic stem cell transplantation; MFC, multiparameter flow cytometry; qPCR, quantitative polymerase chain reaction; Ig, immunoglobulin; TCR, T-cell receptor; STR-PCR, short tandem repeat-PCR; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; CN-AML, cytogenetically normal AML; WTB-PCR, wild-type blocking PCR; COLD-PCR, coamplification at lower denaturing temperature-PCR; LNA, locked nucleic acids; PNA, peptide nucleic acids; ARMS, amplification refractory mutation system; MAMA, mismatch amplification mutation assay; AS-qPCR, allele-specific qPCR. ⁎ Corresponding author. Tel.: + 81 263 37 2802; fax: + 81 263 34 5316. E-mail address: [email protected] (K. Matsuda). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.10.022
K. Matsuda et al. / Clinica Chimica Acta 413 (2012) 74–80
1. Introduction In patients with hematological malignancies, detection and quantification of minimal residual disease (MRD) during and after chemotherapy and post-hematopoietic stem cell transplantation (HSCT) are important in evaluating the patients' therapeutic response and risk of relapse. Currently available quantitative MRD assays are fluorescence in situ hybridization of numerical or structural aberrations at the chromosomal level; multiparameter flow cytometry (MFC) of aberrant immunophenotypes; and quantitative polymerase chain reaction (qPCR) analysis of fusion genes, immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements, genetic alterations, and over-expressed genes. Monitoring of disease status following transplantation can be performed by MRD detection and chimerism analyses by short tandem repeat-PCR (STR-PCR), which characterizes the origin of posttransplant hematopoiesis (Table 1). This review summarizes PCRbased procedures and markers including screening, confirmation, and quantification procedures used for MRD monitoring in hematological malignancies, with a focus on describing the methods for detecting single nucleotide mutations that can be used as potential MRD markers. 2. Clinical significance of the monitoring of MRD In large-scale studies of both acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patients, MRD detected by mainly MFC and/or PCR amplification of Ig or TCR genes has been used for risk stratification, and its prognostic significance has been confirmed. Analysis of aberrant immunophenotypes referred to as leukemia-associated immunophenotypes by flow cytometry is widely applicable to both ALL and AML [1–6]. For ALL patients, in addition to the leukemia-associated immunophenotypes, clonal rearrangements of Ig or TCR genes have been observed in more than 90% of patients [7] and monitoring of MRD has been accessible to many patients. Unlike ALL patients, however, AML patients lack widely applicable PCRbased MRD markers. The PCR-based markers frequently used for MRD detection in AML are fusion gene transcripts such as RUNX1-RUNX1T1, CBFB-MYH11, and PML-RARA resulting from t(8;21)(q22;q22), inv (16)(p13q22) or t(16;16)(p13;q22), and t(15;17)(q22;q12), respectively [1]. Although the application of fusion transcripts for MRD monitoring is limited to specific leukemia subtypes (approximately 25% of AML) [9,10], the clinical significance of quantification of these fusion transcripts has been established [11–14]. For patients with chronic myelogenous leukemia, quantitative monitoring of BCR-ABL1 transcripts has clinical utility not only for predicting relapse following stem cell transplantation, but also for evaluating the effectiveness of treatments such as donor leukocyte infusion, interferon alpha, and tyrosine kinase inhibitor, and it is also useful for providing information on the response to treatment in patients with Table 1 MRD markers for monitoring of MRD and chimerism.
Philadelphia chromosome-positive ALL (Ph + ALL) [15]. In addition to the recurrent reciprocal translocations associated with leukemogenesis, a number of genetic alterations, such as insertions, interstitial/partial tandem duplications, or single nucleotide mutations have been reported to be involved in the pathogenesis of leukemia and associated with the prognosis of the affected patients [16–18]. Approximately 40% of patients with AML display normal karyotype at diagnosis; these cases are defined as cytogenetically normal AML (CN-AML) [19]. CN-AML patients are discriminated into certain prognostic subgroups according to the different genetic alterations. In addition to their role as diagnostic or prognostic markers, the genetic alterations have been shown to be useful for MRD detection and quantification (Table 2). Recently, several groups have performed quantitative assessment of NPM1 or FLT3 mutations and used them as MRD markers in patients with AML [20–27]. In a large study of AML patients with mutated NPM1, the MRD level based on the level of mutated NPM1 during and after therapy was shown to allow for the identification of patients with high risk of relapse [28]. For patients without recurrent chromosomal and genetic abnormalities, molecular targets suitable for qPCR-based MRD monitoring include over-expressed genes such as WT1[29]. Quantification of WT1 expression has prognostic impact and has been evaluated as a marker for risk stratification [30–34]. 3. PCR-based procedures and markers for monitoring of MRD PCR procedures and targets including screening, confirmation, and quantification for monitoring of MRD are listed in Table 3. qPCR is the most widely available PCR-based quantification procedure and is regarded as a reliable method to evaluate MRD. qPCR can be performed with SYBR Green, hydrolysis (TaqMan) probes, or hybridization (LightCycler) probes [35]. We will focus on qPCR with TaqMan probes in this review. TaqMan-qPCR can reduce non-specific amplification due to the specificity of primers and probes. TaqMan-qPCR using RNA or DNA is broadly applicable to evaluate the quantity or expression level of target genes such as rearranged Ig or TCR genes, fusion genes, mutated genes, or aberrantly expressed genes (Table 3). 3.1. Ig and TCR gene rearrangements The leukemic cells in patients with ALL have a unique clonal rearrangement of Ig or TCR genes. PCR amplification of the rearranged Ig
Table 2 Genetic alterations with potential as markers for monitoring of MRD. Types of genetic alterations
Relevant genes
Insertion/duplication
FLT3-ITD NPM1 MLL-PTD WT1 FLT3-TKD KIT RAS (NRAS, KRAS) PTPN11 RUNX1 CEBPA IDH1 WT1 TP53 JAK2 MPL BRAF WT1 EVI1 PRAME BAALC ERG MN1
Single nucleotide mutation
Markers
Detection methods
For MRD Chromosomal abnormality Leukemia-associated immunophenotype Ig/TCR gene rearrangements Fusion genes Genetic alterations
FISH MFC PCR PCR PCR
For chimerism Sex chromosome Short tandem repeat (STR) Single nucleotide polymorphism (SNP)
FISH PCR PCR
MRD, minimal residual disease; FISH, fluorescence in situ hybridization; MFC, multiparameter flow cytometry; Ig, immunoglobulin; TCR, T-cell receptor; PCR, polymerase chain reaction.
75
Over-expression
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Table 3 PCR-based markers and procedures (screening, confirmation, quantification) for MRD monitoring. Markers
Screening
Ig/TCR rearrangements (DNA level) Fusion gene (RNA level) Fusion gene (DNA level) Genetic alterations (DNA or RNA levels)
BIOMED-2 multiplex direct PCR sequencing PCR or qPCR direct sequencing Long-distance PCR direct LDI-PCR sequencing Direct sequencing direct WTB-PCR sequencing HRM COLD-PCR qPCR
Over-expressed gene (RNA level)
Confirmation
Quantification qPCR, GeneScan qPCR qPCR qPCR (AS-qPCR)
qPCR
Ig, immunoglobulin gene; TCR, T-cell receptor gene; qPCR, quantitative polymerase chain reaction; LDI-PCR, long-distance inversed PCR; WTB-PCR, wild-type blocking PCR; HRM, high resolution melting analysis; AS-qPCR, allele-specific quantitative polymerase chain reaction; COLD-PCR, coamplification at lower denaturing temperaturePCR.
or TCR genes has been used as an MRD marker for patients with ALL. Ig/TCR gene rearrangements in stable DNA are patient-specific and the sensitivity of PCR for their detection is 10 − 4–10 − 5[35]. To use the specific rearrangements of Ig or TCR genes in individual patients as an MRD marker, identification of the rearranged sequences at diagnosis is needed. Recently, standardization of the multiplex PCR methods for detecting clonal Ig or TCR gene rearrangements was established by the BIOMED-2 Concerted Action BMH4-CT98-3936 study [36]. Combined use of BIOMED-2 multiplex PCRs with GeneScan analysis could become a highly sensitive and high-throughput method [36]. Using the BIOMED-2 multiplex PCR procedure, the detection rate of clonal rearrangement of TCRβ and TCRγ ranged from 94% to 98% in patients with T-cell malignancies and that of Ig from 91% to 96% in patients with B-cell malignancies [37,38]. Sandberg et al. [39] mentioned that BIOMED-2 multiplex PCR could replace Southern blot analysis in routine testing. For the screening and identification of the rearranged Ig/TCR genes, the BIOMED-2 multiplex PCR is useful. After BIOMED-2 multiplex PCR, the PCR products are subjected to agarose gel electrophoresis and then to direct sequencing. Then, the primers or TaqMan probes can be designed according to the sequence at the junction regions. Although the Ig/TCR gene rearrangements are useful MRD markers, the evaluation of clonality based on rearrangements is frequently hampered by the amplification of similar rearrangements from normal cells. Furthermore, rearrangement at diagnosis may be lost by secondary rearrangements during the clinical course [40]. To avoid false negative results, several Ig/TCR targets should be used for each patient.
3.2. Fusion gene transcripts (RNA level) Chromosomal structural abnormalities associated with malignancies can result in the formation of fusion genes. Fusion gene transcripts such as BCR-ABL1 in CML or ALL, PML-RARA, RUNX1-RUNX1T1 or CBFB-MYH11 in AML, and ETV6-RUNX1 in ALL patients are used as appropriate MRD markers by qPCR with a high sensitivity of 10 − 4– 10 − 6[35]. Designing primers or probes is less labor-intensive when the spliced exon of the two original genes constituting the fusion gene is known. Gabert et al. reported the primer/probe combinations for frequently detected fusion gene transcripts [TCF3 (E2A)-PBX1, MLL-AFF1 (AF4), ETV6-RUNX1, BCR-ABL1 major and minor type, STIL (SIL)-TAL1, PML-RARA, CBFB-MYH11, or RUNX1-RUNX1T1] [8]. Considering that some cases have variant fusions, qualitative PCR using primers that cover more spliced variants is important for screening at diagnosis [11]. Of note, MRD monitoring using RNA does not directly reflect the tumor load, because the transcripts themselves may be
affected by cytotoxic therapy, resulting in different transcript levels during therapy [35]. 3.3. Fusion gene (DNA level) For fusion gene-based MRD evaluation, the level of RNA transcripts is measured using qPCR as described above. In order to do this, it is desirable to use a patient-specific marker for evaluating MRD. Namely, in patients with a BCR-ABL1-, a PML-RARA-, or an MLL-related fusion gene, (semi) quantitative assessment of MRD by utilizing the patient-specific genomic DNA breakpoint has been reported [41–45]. Although long-range PCR and modified PCR using tagged primers or adoptor-ligation methods to detect genomic DNA fusion points are labor-intensive compared to the methods used to detect common exon–exon fusions at the RNA level [41–45], the identification of the genomic DNA breakpoint region could realize precise and stable monitoring of MRD because the RNA expression fluctuates during therapy and RNA is unstable [35]. Structural abnormalities involving the mixed-lineage leukemia (MLL) on 11q23 have been detected in a wide spectrum of hematological malignancies, such as AML, ALL, and myelodysplastic syndrome [46,47]. The rearrangement of MLL occurs during translocations, deletions, insertions, or partial tandem duplications involving a variety of genes on the partner chromosome [48]. A recently developed long-distance inverse PCR technique allows us to identify MLL-fusion partner genes generated by several mechanisms and to determine patient-specific breakpoint DNA sequences, which helps in the construct of the patient-specific primers or probes for MRD detection [45,49]. For patients with the MLL fusion gene, Burmeister et al. [50] developed a novel MRD assessment procedure using the genomic breakpoint region-related MLL gene as a patient-specific marker that could accurately and consistently reflect the quantity of leukemic cells. The sensitivity of the method based on the monitoring of patient-specific breakpoint DNA sequences was 10 − 4 to 10 − 5[50]. The genomic breakpoint sequence of the MLL fusion gene was not influenced by secondary rearrangement as in the case of the Ig/TCR genes [50]. In some cases with complex chromosomal structural abnormalities, fluorescence in situ hybridization capable of detecting the breakpoint region at the chromosomal level may provide information on the patient-specific aberrant gene configurations and help to set up the PCR-based MRD assays [51]. 3.4. Genetic alterations including single nucleotide mutation, deletion, insertion, and duplication 3.4.1. Genetic alterations in AML AML is a genetically heterogeneous disease in which genetic alterations as well as cytogenetic abnormalities constitute key events in the pathogenesis [19]. According to the review by Renneville et al. [17], the aberrant genes are clustered into three groups: genes that contribute to cell proliferation (KIT, FLT3, RAS, and PTPN11), genes involved in hematopoietic differentiation (RUNX1 and CEBPA), and genes implicated in cell cycle regulation and apoptosis (TP53 and NPM1). Among these genetic alterations, the NPM1 mutation characterized by tetranucleotide insertion and FLT3 internal tandem duplication occurs in about 50% and 40% of case of adult AML without chromosomal abnormalities, respectively. The quantitative assessments of NPM1 or FLT3 mutations as an MRD marker were performed by TaqMan qPCR or LightCycler assays using primers or probes specific for the duplicated nucleotides. The potential impact of MRD based on NPM1 or FLT3 has been demonstrated [20–28]. In addition to insertions/duplications, single nucleotide mutations in various genes are frequently detected. Single nucleotide mutation can be considered an applicable marker for monitoring of MRD; however, its application is limited in part because it is much more difficult
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3.4.2. Screening and confirmation of genetic alterations To use single nucleotide mutation as an MRD marker, the screening methods and specific detection methods are needed just as for other PCR-based markers (Table 3). The direct sequencing method is well established and is the most common and conventional method used for the screening and confirmation of mutations in tumor cells. When a diagnostic sample containing abundant leukemia cells is available, conventional direct sequencing analysis can be used to detect mutations; however, when only the samples following therapy are available, direct sequencing analysis does not have enough sensitivity to detect the reduced mutant DNA. Two recently developed methods, i.e., wild-type blocking PCR (WTB-PCR) and coamplification at lower denaturing temperaturePCR (COLD-PCR), are applicable as screening approaches to detect or amplify the known or unknown mutations among an excess amount of the wild-type DNA. Locked nucleic acid (LNA)- or peptide nucleic acid (PNA)-mediated WTB-PCR has been employed to block the wild-type sequence and preferentially amplify the mutant sequence. LNA- and PNA-substituted nucleotides increase the binding affinity compared to standard nucleotides [52–56]. In addition, PNA is resistant to the 5′ nuclease activity of Taq DNA polymerase, such that the nucleic acids are used for blocking the wild-type DNA, resulting in the preferential amplification of mutant sequences during PCR. Nagai et al. [57] have reported a PNA-LNA PCR clamp system for detection of EGFR gene mutations in the presence of a 100- to 1000-fold higher background level of wild-type EGFR. In the PNA-LNA clamp system, a PNA-containing oligonucleotide is used for blocking wild-type sequences and a fluorescent probe including LNA is used to detect the mutant amplicon. In addition to their use as probes for stable hybridization, LNA-containing oligonucleotides can also be used for blocking wild-type sequences [58]. Using a polymerase without intrinsic 5′–3′ exonuclease activity (Stoffel fragment), the stability of blocking by LNA-containing oligonucleotides can be enhanced [59]. Li et al. [60] have recently developed two COLD-PCR systems (full COLD-PCR and fast COLD-PCR) that are capable of enriching small quantities of known or unknown mutants. To detect tumorassociated mutations including both known and unknown mutations, direct sequencing analysis is commonly used; however, its sensitivity is low and not sufficient to detect mutations present in small quantities. In COLD-PCR utilizing critical a denaturation temperature (Tc) lower than the melting temperature (Tm), heteroduplexes of mutant and wild-type alleles preferentially denature over homoduplexes of wild-type alleles, which enables amplification of mutationcontaining alleles several-folds over WT alleles. Full COLD-PCR can enrich all possible mutations and fast COLD-PCR without a heteroduplexes formation step has a short reaction time. More recently, Milbury et al. [61] reported an Improved and Complete Enrichment (ice)-COLD-PCR utilizing a reference sequence which binds rapidly to the amplicon. Ice-COLD-PCR has advantages of full and fast COLD-PCR and allows for robust and efficient COLD-PCR. For COLDPCR, we have to use thermal cycler with high temperature precision and determine the Tc and Tm using high resolution melting analysis. In the routine mutation-screening test, COLD-PCR is a promising method that can be combined with subsequent molecular analyses, such as direct sequencing, high resolution melting analysis, and qPCR [62,63]. Combining the use of WTB-PCR or COLD-PCR and sequencing analyses allows us to detect unknown and/or known mutations and identify the precise gene alterations and their locations. Following the identification of the type of gene alterations by the procedures described above, the direct or allele-specific amplification using primers or probes specific to the mutant nucleotide can be performed.
3.4.3. Direct detection of mutant DNA among an excess amount of wild-type DNA For direct detection of mutant DNA, primers or probes specific to the mutant DNA are needed. The binding affinity of primers is affected by the modification of nucleotides around the 3′ terminal region of primers. PCR using primers including only a mutant-matched base in the 3′-end has been shown to result in non-specific amplification, so that additional modification is needed. Several allele-specific PCR (AS-PCR) methods based on an amplification refractory mutation system (ARMS) or mismatch amplification mutation assay (MAMA) have been developed for the detection of single nucleotide mutations [64,65]. The primers in AS-PCR included a mutant-matched nucleotide at the 3′-end and a template-mismatched nucleotide near the 3′-end. An AS-quantitative PCR (AS-qPCR) based on ARMS or MAMA was developed with a TaqMan probe to quantify low levels of mutant nucleotides in the presence of high levels of the counterpart wildtype nucleotides [66]. Recently, several studies have reported that single nucleotide mutations of JAK2 and MPL, or single nucleotide mutations of the ABL kinase domain in BCR-ABL1 were quantitatively monitored in myelofibrosis patients following transplantation [67–70] or in chronic myelogenous leukemia patients with imatinib resistance, respectively [71,72]. Pretreatment with restriction enzymes to digest residual wild-type nucleotides is performed to obtain high specificity and sensitivity of the AS-PCR; however, restriction enzymes that digested the wild-type sequence at various mutations are
-10 -15 -20 -25 Mutant AS-qPCR
B: Sensitivity (delay Ct)
Mismatched AS-qPCR
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to detect single nucleotide mutations in the presence of an excess amount of wild-type nucleotides than to detect insertions and/or duplications involving several nucleotides.
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Fig. 1. Specificity (difference of threshold cycles, ΔCt) and sensitivity (the delay Ct) using constructed wild-type and mutant plasmid DNA by 3 types of AS-qPCRs. Three types of AS-qPCRs (black-bar, mutant AS-qPCR; gray-bar, mismatched AS-qPCR; open-bar, LNA-AS-qPCR) were compared in terms of the difference of threshold cycles (ΔCt) and delay in Ct (delay Ct) using equal copy numbers of constructed wild-type and mutant plasmid DNA. The ΔCt was calculated as follows: (Ct for mutant plasmid) − (Ct for wild-type plasmid) in each AS-qPCR assay. The delay Ct was calculated as follows: (Ct for mutant plasmid in mismatched AS-qPCR or LNA-AS-qPCR) − (Ct for mutant plasmid in mutant AS-qPCR). The dotted line indicates 3.33 cycles. The threshold cycles used for the present calculations were based on the data in our previously reported experiments [73,74]. Data are expressed as the means − S.D. for ΔCt and means + S.D. for ΔCt. 1, PTPN11 179G>A; 2, PTPN11 182A>T; 3, PTPN11 214G>A; 4, PTPN11 226G>A; 5, PTPN11 227A>C; 6, PTPN11 227A>G; 7, NRAS 34G>A; 8, NRAS 35G>A; 9, NRAS 38G>A; 10, KRAS 34G>A; 11, FLT3 2503G>T; 12, KIT 2446G>T; 13, KIT 2447A>T.
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not always available. Additional modification of primers or probes is needed. 3.4.4. Our data on application of AS-qPCRs for the evaluation of mutation-based MRD For single nucleotide mutations such as PTPN11, NRAS, KRAS, FLT3, c-KIT, or TP53, we developed AS-qPCRs using three different mutation-specific primers as follows: mutant AS-qPCR primers including only a mutant-matched nucleotide in the 3′-end; mismatched AS-qPCR primers, which are identical to ARMS or MAMA, including a mutant-matched nucleotide in the 3′-end and a templatemismatched nucleotide at the penultimate 3′-end; LNA-AS-qPCR primers including a mutant-matched nucleotide in the 3′-end, a template-mismatched nucleotide at the penultimate 3′-end, and LNA at the -2 position from the 3′-end. When compared with mutant AS-qPCR, both mismatched AS-qPCR and LNA-AS-qPCR exerted a more significant specificity for discrimination between wild-type and mutant DNA in all 13 types of mutations (6 types of PTPN11 mutations, 4 types of RAS mutations, 2 types of KIT mutations, and 1 type of FLT3 mutation) (Fig. 1A). Of note, the specificity of LNA-AS-qPCR was found to be greater than that of mismatched AS-qPCRs in all types of mutations (Fig. 1A). Additional primer modifications, such as the inclusion of a mismatched base or substitution with an LNA base, did not lead to a more than 10 (2 3.33)-fold reduction of sensitivity in 10 of 13 types of mutations (Fig. 1B). Using clinical samples, we developed specific mismatched or LNA-AS-qPCRs with a sensitivity of 10 − 3 to 10 − 4 for each mutation [73,74]. The AS-qPCRs for single nucleotide mutations had accuracy comparable to qPCR for the RUNX1RUNX1T1 fusion gene and WT1 and were applicable to monitoring of MRD throughout the clinical course, including prior to transplantation and post-transplantation [73]. Furthermore, the post-transplant changes of MRD levels assessed by LNA-AS-qPCR were well correlated with the percentage of autologous cells (chimerism) assessed by STRPCR, which characterizes the origin of post-transplant hematopoiesis [74]. The interpretation of conditions with mixed chimerism (MC) is difficult because of the variation of the interval between MC and relapse among patients and the long-lasting MC in some patients without relapse [75]. The post-transplant chimerism analysis at very short time intervals demonstrated a significant correlation between patients with increasing MC and relapse [76,77]. AS-qPCR analyses allow accurate assessment whether autologous cells belong to normal or malignant cell clones, which may lead to prediction of imminent relapse. 3.5. Over-expressed genes Other potential markers for qPCR-based MRD monitoring include the over-expressions of WT1, EVI1, PRAME, BAALC, ERG, or MN1 genes (Table 2). Among these markers, over-expression of the WT1 gene is most frequently used as a universal MRD marker applicable to patients without recurrent chromosomal or genetic abnormalities. To evaluate MRD, a comparison of the difference in expression levels between patients and healthy controls is needed, because WT1 transcripts have been detected not only in tumor cells but also in normal cells [34]. 4. Conclusion MRD monitoring is applicable for the early detection of impending relapse that is not clinically evident as well as for the evaluation of disease status based on tumor burden. Furthermore, the potent application of MRD monitoring is to assess the effectiveness of new antileukemic agents, with an aim to identifying risk-adaptive therapies and circumventing suboptimal therapies [78,79]. Therefore, monitoring of MRD provides useful information in various aspects of the clinical management of patient with hematological malignancies. WTB-PCR and
COLD-PCR analyses, which are promising procedures for screening known or unknown gene alterations, can be combined with the downstream procedures, including AS-qPCR targeting the specific gene mutations. Utilizing potential MRD markers, such as gene mutations, along with well-established MRD markers, such as Ig/TCR rearrangements and fusion gene transcripts will be important for expanding the spectrum of patients in whom MRD can be monitored. Conflict of interest The authors state that they have no conflict of interest. Funding Our study was supported in part by a Grant-in-Aid for Science research from the Japan Society for the Promotion of Science (No. 20930022, KM), the Charitable Trust Laboratory Medicine Research Foundation of Japan (2008, KM), and the Hokuto Foundation for Bioscience (2010, KM). Acknowledgements We gratefully acknowledge Dr. Nobuo Okumura (Laboratory of Clinical Chemistry and Immunology, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan) for his helpful review and comments in the preparation of the manuscript. References [1] Rubnitz JE, Inaba H, Dahl G, et al. Minimal residual disease-directed therapy for childhood acute myeloid leukaemia: results of the AML02 multicentre trial. Lancet Oncol 2010;11:543–52. [2] Conter V, Bartram CR, Valsecchi MG, et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. Blood 2010;115:3206–14. [3] Pui CH, Campana D, Pei D, et al. Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med 2009;360:2730–41. [4] Vidriales MB, Perez JJ, Lopez-Berges MC, et al. Minimal residual disease in adolescent (older than 14 years) and adult acute lymphoblastic leukemias: early immunophenotypic evaluation has high clinical value. Blood 2003;101:4695–700. [5] Feller N, van der Pol MA, van Stijn A, et al. MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia. Leukemia 2004;18:1380–90. [6] Kern W, Voskova D, Schoch C, et al. Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004;104:3078–85. [7] van der Velden VH, Cazzaniga G, Schrauder A, et al. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of realtime quantitative PCR data. Leukemia 2007;21:604–11. [8] Gabert J, Beillard E, van der Velden VH, et al. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia — a Europe Against Cancer program. Leukemia 2003;17:2318–57. [9] Estey E, Döhner H. Acute myeloid leukaemia. Lancet 2006;368:1894–907. [10] Byrd JC, Mrózek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325–36. [11] van Dongen JJM, Macintyre EA, Gabert J, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13: 1901–28. [12] Perea G, Lasa A, Aventín A, et al. Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)]. Leukemia 2006;20:87–94. [13] Corbacioglu A, Scholl C, Schlenk RF, et al. Prognostic impact of minimal residual disease in CBFB-MYH11-positive acute myeloid leukemia. J Clin Oncol 2010;28: 3724–9. [14] Grimwade D, Jovanovic JV, Hills RK, et al. Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy. J Clin Oncol 2009;27:3650–8.
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