Degree project, 30 ECTS
Detection of hotspot mutations in IDH1/2 in patients with
acute myeloid leukemia using Droplet Digital PCR
Version 2
Author: Johanna Wågberg, Bachelor of Medicine Örebro University Örebro, Sweden Supervisor: Tatjana Pandzic, PhD Department of Clinical Genetics, Uppsala University Uppsala, Sweden
Word count
Abstract: 250 Manuscript: 2631
Abstract
Introduction
Acute myeloid leukemia (AML) is caused by a wide range of genetic aberrations, including mutations within the genes that encode the enzymes isocitrate dehydrogenase 1 and 2
(IDH1/2). Drugs that target mutant IDH1/2 are now available, which makes assessment of the mutational status of IDH1/2 important in clinical diagnostics of AML. A promising method to detect these mutations is the droplet digital polymerase chain reaction (ddPCR), which shows advantages of a high sensitivity and a simple workflow.
Aim
To evaluate ddPCR as method of choice to detect hotspot mutations in IDH1 (codon R132) and IDH2 (codon R140 and R172) in patients with AML.
Methods
Fifteen AML patients known to be positive for IDH1/2 diagnosed by a previously performed next generation sequencing (NGS) were selected for evaluation of ddPCR. Diagnostic samples were tested for 14 patients, whereas follow-up samples were tested for one patient. ddPCR was performed using QX200™ Droplet Digital PCR system and data were presented as fractional abundance of mutant allele.
Results
The amount of mutant IDH1/2 in samples reported by ddPCR correlated well with the results from NGS when using probes that target their specific mutation. The detection limit for mutant allele in the background of wild type IDH1/2 was 0,5% for IDH2 p.R140Q and 0.1% for IDH1 p.R132C/H.
Conclusion
ddPCR that target specific mutations shows a great potential in measuring minimal residual disease during follow-up. However, its use in screening for mutant IDH1/2 at the time of diagnosis is limited and alternative approaches should be considered.
Abbreviations
AML: Acute myeloid leukemia
ddPCR: Droplet digital polymerase chain reaction FISH: Fluorescence in Situ Hybridization
(RT)-PCR: Reverse Transcriptase Polymerase Chain Reaction NGS: Next generation sequencing
DNA: Deoxyribonucleic acid
IDH1/2: Isocitrate dehydrogenase 1/2
R132C: Arginine to Cysteine at position 132 R132H: Arginine to Histidine at position 132 R140Q: Arginine to Glutamine at position 140 R172K: Arginine to Lysine at position 172 WT: Wild type
MT: Mutant
2-HG: D-2-hydroxyglutarate HEX: Hexachloro fluroescein FAM: Fluroescein amidite MRD: Minimal residual disease
Introduction
Acute myeloid leukemia (AML) is the most common leukemia among adults, with an yearly incidence of 3.5 cases per 100,000 in Sweden [1]. It is a heterogenous disease caused by genetic aberrations, which lead to clonal expansion of hematopoietic progenitor cells of the myeloid lineage [2,3]. Resulting anemia, thrombocytopenia and defective immunity rapidly leads to death if left untreated. Current classifications of the disease are made according to phenotypic and genetic profiles, guiding diagnostics and prognostication as well as being used in monitoring minimal residual disease and for identification of therapeutic targets [4–6]. In order to set these profiles, a wide range of methods are used. Commonly performed methods are immunophenotyping, chromosome analysis (karyotyping), fluorescence in situ
hybridization (FISH), reverse transcriptase (RT)-PCR and sequencing analysis [7].
Knowledge of the molecular landscape of AML has expanded greatly with the development of new sequencing techniques. Mutations within the genes that encode the isocitrate
dehydrogenase (IDH) enzymes IDH1 and IDH2 are seen in a diverse array of cancer; including gliomas, chondrosarcoma, cholangiocarcinoma and AML [8–11]. Approximately 20% of patients diagnosed with AML exhibit mutant IDH1 or IDH2 [12]. The enzymes play a key role in metabolic and epigenetic cellular pathways essential for normal cellular
function[13]. In their mutated form, the oncometabolite D-2-hydroxyglutarate (2-HG) is generated, leading to epigenetic dysregulation and blockage of cellular differentiation [14– 16]. Oncogenic mutations occur selectively to key residues within the active sites, specifically codon R132 in IDH1, and the two codons R172 and R140 in IDH2 [17]. Missense
substitutions vary within these codons, the most common in AML being IDH1 p.R132C/H,
IDH2 p.R140Q and IDH2 p.R172K [5,18].
IDH-mutated AML patients are associated with specific clinicopathological characteristics
such as older age, intermediate-risk cytogenetics and increased blast-count in the bone marrow at diagnosis [19]. IDH1/2 have also been shown to be reliable markers monitoring minimal residual disease (MRD) in order to predict disease relapse and to monitor response to treatment [20,21]. Moreover, mutant IDH1/2 are now targets for therapeutic agents. The mutIDH-inhibitors Enasidenib and Ivosedinib which target IDH2 and IDH1 respectively, prevent build-up of oncogenic 2-HG and stimulate myeloid differentiation with good clinical response [22,23]. The drugs can now be prescribed to patients with relapsed or refractory AML, as the Food and Drug Administration recently committed their approval. The use may expand as ongoing clinical trials currently investigate their use in both solid tumors and in
other subgroups of AML, including newly diagnosed AML [24,25]. There are also a host of other promising mutIDH-inhibitors under development, some of which have reached clinical testing [26].
Genetic aberrations, including mutant IDH1/2, are today detected by next generation
sequencing (NGS) during the diagnostic procedure of AML [7]. Frequent occurring mutations are targeted and sequenced in a massively parallel fashion, which enables investigation of several genes at the same time [27]. However, the workflow is time consuming and results require a bioinformatics to be analyzed. Low variant allele frequencies may also be missed as the current error rates sets the detection limit to about 1% [28]. In situations where rapid results of high accuracy are of major importance, such as screening for mutant IDH1/2 in order to start treatment with targeted drugs, the limitations of NGS demands another method to be established.
A promising tool for detection is the droplet digital polymerase chain reaction (ddPCR). In similarity with quantitative PCR (qPCR) , fluorescently-labeled probes targeting mutant (MT) and wild type (WT) alleles together with target-specific primers are used [29]. However, as qPCR relies on standard curves to perform an absolute quantification of the target molecule, no standard curve is needed in ddPCR. This is possible due to the partitioning of DNA prior to amplification, generating a high number of independent replicates encapsulated in oil droplets. PCR amplification will only occur in droplets with target DNA molecules (positive reactions), whereas droplets without targets will not be amplified (negative reactions). The fraction of positive reactions can be used to calculate the concentration with Poisson’s statistics, enabling absolute quantification of target DNA molecules in a sample [29]. Sorting out and enriching rare targets also decreases noise of background DNA, which results in high accuracy of acquired results. The major advantages of ddPCR are independency of controls and high precision with ability to identify low amount of target [30]. Furthermore, the
workflow is simple and results do not require a bioinformatics expert to be analyzed, making ddPCR useful in clinical diagnostic routines [31].
Finally, assessment of the mutational status of IDH1/2 in patients with AML is of great interest in clinical diagnostics of AML. As targeted drugs for patients with mutant IDH1/2 are soon available, results of the mutational status will become a diagnostic urgency for
clinicians. The benefits of ddPCR with simplicity, reliability and rapid results makes this method an eligible approach to detect these mutations.
Aim
The aim of this study was to evaluate ddPCR as method of choice for detection of hotspots mutations in IDH1 (codon R132) and IDH2 (codons R140 and R172) in patients with AML.
Material and Methods
Patients
A cohort of 166 newly diagnosed AML cases had previously been sequenced with NGS-Trusight Myeloid panel at Uppsala University Hospital, of which 48 patients were shown to be positive for IDH1 and/or IDH2. Out of those, 15 patients were selected for analysis with ddPCR. Selection was based on the specific mutations in IDH1/2, so that the most common mutations as well as other rare mutations in IDH1/2 in various variant allele frequencies were included. A diagnostic sample was tested for 14 patients, whereas follow-up samples were tested for one patient. Patients DNA were extracted from bone marrow or peripheral blood samples with EZ1 DNA Blood kit (Qiagen, Hilden, Germany) according to instructions.
Ethical considerations
The study is part of a larger study with approval by the ethical review board in Stockholm (2017/2085-31/2). Informed written consent was obtained from all patients included in accordance with Swedish law concerning ethics approval of research on human subjects. Subjects were pseudonymized and results were labeled with an ID number.
Droplet digital PCR
PCR reagents, including prime/probe mix for variants of IDH1 p.R132C and p.R132H as well as IDH2 p.R140Q and p.R172K, were purchased from Bio-Rad™ (Bio-Rad Laboratories, Inc., California, U.S.A.). Probes covering designated mutations were designed using Bio-Rad’s online tool, with HEX-labeled wild type allele and FAM-labeled mutant alleles. Due to the fact that IDH1 p.R132C and p.R132H mutations are located in the same codon, there is a possibility that p.R132C-probe can interact with p.R132H and vice versa. Therefore, all samples with mutant IDH1 were tested with both IDH1-probes to evaluate their cross-reactivity.
ddPCR was performed using QX200™ Droplet Digital PCR system (Bio-Rad) according to the manufacturer’s instructions. A PCR-mix containing 1XddPCR mix, 900 nM of each primer, 250 nM of each probe and 60 ng sample DNA (20 ng/µL) was mixed and transferred to a 96-well plate designed to target samples with matching probes. Negative controls and non-template controls (H2O) were used for each of the different probes. All reactions
(including negative controls and no template control) were performed in duplicates. Droplets were generated on QX200 Droplet Generator and PCR was performed with following cycling conditions: 95°C for 10 min, 40 cycles of: 94°C for 10 s, 53°C for 60 s; finished by 98°C for 10 min (ramp rate 2°C/sec). PCR products were analyzed with QX200 Droplet Reader and thereafter processed with QuantaSoft Pro software according to instructions.
Assay sensitivity
To establish the performance of assays, the false-positive rate by using negative control DNA (DNA known to have only WT sequence) was measured. Furthermore, dilution of known concentration of mutant allele in the background of WT IDH1 or IDH2 was performed. DNA from cell lines with IDH1/2 mutations were used. The four most common mutations in
IDH1/2 were tested: IDH1 p.R132H, IDH1 p.R132C, IDH2 p.R140Q, IDH2 p.R172K. Both IDH-mutant and WT cell lines were purchased from Horizon Discover. Genomic DNA
derived from mutation-positive cells were mixed with DNA from WT cells so that the following fractional abundance of mutant alleles were obtained: 10%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.003125%.
Results
Fractional abundance of mutant IDH1/2 detected by ddPCR
All selected samples could be analyzed with ddPCR. As ddPCR makes absolute
quantification by counting and comparing the number of positive and negative droplets, results can be presented as fractional abundance of mutant DNA detected among WT sequence. After manual thresholding of positive and negative droplets, all samples were analyzed, and fractional abundance were plotted (table 1). Figure 2 displays a representative 2-D plot from one of the samples, in which manual thresholding was performed.
The fractional abundance of mutant IDH1/2 in each sample detected by ddPCR was compared to results from NGS as a validation step. When testing samples with ddPCR using probes specific for their mutation, the fractional abundance detected by ddPCR correlated well with the results of mutant amount reported by NGS (table 1).
Figure 2. 2-D plot from sample ID 1 with mutant isocitrate dehydrogenase 1 (IDH1) p.R132H (arginine to histidine at position 132) and a detected fractional abundance of 46.6%. Each dot represents one droplet. The x-axis represents the amplitude of fluorescence emitted by the hydrolysis-probe HEX, corresponding to the amount of wild type IDH1. The y-axis represents the amplitude of fluorescence detected from the hydrolysis-probe FAM,
corresponding to the amount of mutant IDH1. By manual thresholding of channels, four clusters can be identified. The green cluster (FAM-negative, HEX-positive) represents droplets containing wild type IDH1, while the orange cluster (FAM-positive, HEX-positive) represents droplets encompassing both wild type and mutant IDH1. The blue cluster (FAM-positive, HEX-negative) represents droplets with mutant-only, while the black cluster represents double negative droplets, i.e. droplets without DNA.
Table 1. Fractional abundances of mutant IDH1/2 detected in samples by NGS versus ddPCR.
Sample
ID Target Fa NGS Fa ddPCR using probe IDH1 R132C Fa ddPCR using probe IDH1 R132H Fa ddPCR using probe IDH2 R140Q Fa ddPCR using probe IDH2 R172K 1 IDH1 c.395G>A p.R132H 47 0.03 46.06 2 IDH1 c.394C>T p.R132C 45 45.52 0.08 3 IDH1 c.394C>T p.R132C 22 23.08 0.04 4 IDH1 c.394C>T p.R132C 49 47.57 0.07 5 IDH1 c.394C>G p.R132G 46 39.83 0.03 6 IDH1 c.394C>A p.R132S 47 0.07 0.02 7 IDH1 c.394C>A p.R132S 44 0.11 8 IDH2 c.419G>A p.R140Q 2 2.62 9 IDH2 c.419G>A p.R140Q 5 4.87 10 IDH2 c.419G>A p.R140Q 46 45.62 11 IDH2 c.515G>A p.R172K 75 77.92 12 IDH2 c.515G>A p.R172K 42 41.77 13 IDH2 c.515G>A p.R172K 44 46.6 14 IDH2 c.514A>T p.R172W 35 0.03
Samples were tested with ddPCR using matching probes to targets. All samples with mutant IDH1 were tested by both IDH1-probes due to the possibility of cross-reactivity, except sample ID 7 owing to a deficit in sample material.
All values are given as percentages of total IDH1/2 represented by the mutated forms.
Fa: Fractional abundance; NGS: Next generation sequencing; ddPCR: Droplet digital polymerase chain reaction; IDH1/2: Isocitrate dehydrogenase 1/2
Assay sensitivity
Figure 3 displays the results from the serial dilutions performed and the detection limit for three assays. The background signal (false positive results) of each assay was determined by analyzing wild type DNA with IDH1/2 assays: 0.018% for IDH1 p.R132C, 0.04% for IDH1 p.R132H, 0.04% for IDH2 p.R140Q, 0.04% for IDH2 p.R172K.
When analyzing IDH1 p.R172K, an additional cluster was found adjacent to wild type droplets. Owing to difficulties of thresholding and classifying positive and negative droplets (Figure 4), this assay was excluded as measurements of fractional abundance would be of questionable accuracy.
Figure 3. Serial dilutions of isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations in order to evaluate assay sensitivity. Blue dots represent the percentages of mutant alleles among wild type IDH1/2 detected by digital polymerase chain reaction (ddPCR), and the blue dotted lines illustrate trendlines. A correlationcoefficient (R2) between observed and expected values are reported. A: Serial dilution of IDH1 p.R132H with a detection
limit of 0.1% of mutant allele. B: Serial dilution of IDH1 p.132C with a detection limit of 0.1% of mutant allele. C: Serial dilution of IDH2 p.R140Q with a detection limit of 0.5% of mutant allele.
The performance of ddPCR in minimal residual disease analysis
The role of ddPCR in disease monitoring was evaluated by testing follow-up samples from one patient under treatment for AML. A retrospective analysis of all available samples (including the diagnostic sample) was performed with ddPCR using probes targeting IDH1 p.R132H. The first three samples were negative for IDH1 p.R132H, but positive samples could be detected thereafter (Figure 5). This correlated with the clinical status as disease relapse was reported at this time.
Figure 5. Burden of mutant isocitrate dehydrogenase 1 (IDH1) detected by ddPCR when testing follow-up samples from one patient under treatment for acute myeloid leukemia.
Figure 4.2-D plot from mutant isocitrate dehydrogenase 2 (IDH2) p.R172K (arginine to lysine at position 172) in the dilutional series experiment. Each dot represents one droplet. The x-axis represents the amplitude of fluorescence emitted by the hydrolysis-probe HEX, corresponding to the amount of wild type IDH2. The y-axis represents the amplitude of fluorescence detected from the hydrolysis-probe FAM, corresponding to the amount of mutant IDH2. Between the double-negative cluster (black) and wild type-only cluster (green), droplets encompassing an additional cluster can be identified (encircled in red).
Discussion and Conclusion
In this study, we have evaluated the performance of ddPCR in detection of mutant IDH1/2 in 15 patients with AML. We found that samples with mutant IDH1/2 need to be tested with matching probes in order to get accurate results, as no cross-reactivity was seen between the different probes. The method showed a high sensitivity with a detection limit of 0.1% for
IDH1 p.R132C/H and 0.5% for IDH2 p.R140Q.
When evaluating the sensitivity by dilutional series, a high discrepancy between observed and expected values was seen for 0.5% for assay IDH2 p.R140Q. Linearity was however
preserved for lower values, which may indicate a poor management of reagents and
performance. This could be evaluated by performing the dilutional series repeatedly, which would also give information about the precision of our method. This was however not
performed in our study and constitutes a limitation. Furthermore, the assay of IDH2 p.R172K was excluded as an additional cluster was identified and made it difficult to separate droplets. The detection limit could therefore not be determined for this assay. Due to its clear
separation from the other two clusters (double-negative droplets and WT-positive droplets), it can be hypothesized that this cluster represents an additional mutation in IDH2 present in these cells, as shown in figure 4.
The high sensitivity of ddPCR could be suitable to measure small amount of target. This is especially attractive during follow-up of patients when aiming to prevent disease relapse[31]. The role of ddPCR in MRD-analysis was demonstrated in our study by testing follow-up samples from one patient under treatment for AML. Mutant IDH1 p.R132H was detected in two follow-up samples, which indicates that the mutation was acquired during the course of disease. The increase in IDH1 p.R132H also correlated well with the clinical outcome, which suggests the idea of mutant IDH1/2 as a stable disease marker. This is supported by other studies that have shown mutant IDH1/2 as reliable predictors of disease relapse [19,20].
A negative ddPCR result or a lower fluorescence amplitude ddPCR efficacy could be seen in samples having a mismatch between probes and targets in the DNA sequence. At the time of diagnosis when the mutational status is unknown, ddPCR could thereby fail to detect a mutant
IDH1/2 if the wrong probe is used. However, to screen for mutant IDH1/2 at the time of
diagnosis, it is more important to investigate if the patient harbors a mutation or not, rather than to calculate the specific amount of mutant burden. Nonetheless, when cross-reactivity was evaluated with the use of nonmatching probes to targets, the detected fractional
abundances were below the detection limit. This means that every patient sample would need to be tested with all probes in several single assays to cover all IDH1/2 mutations. This is not an option considering how time consuming and expensive it would be.
Multiplex assays allow to test for presence of several mutations in a single assay. As in qPCR, probes labeled with different fluorescent dyes can be used to detect several targets at once. However, this technique requires systems enabled to read multiple emission spectra, where the ddPCR system restricted to two fluorescence filters fails [32]. An easier and less technical approach is an amplitude-based multiplex assay, where single dye probes are used at different final concentrations. This is illustrated by Zhong et al. (2011), showing that five different targets can easily be detected in a 5-plex assay using only two fluorophores [33]. As probe fluorescent intensities varies in proportion to probe concentration, it was found that solid clusters representing the targets forms at different lengths along the y- and x-axis in 2-D plots. Designing multiplex assays targeting IDH1 and IDH2 respectively, the most common
mutations could be included as a screening method. However, despite the relatively limited number of mutations occurring in IDH1/2, it is possible that a patient could exhibit a genetic aberration occurring at a different position in the genes. In such cases, a mutational IDH1/2 could still be missed with a multiplex assay.
A different approach to scan for multiple mutations is by using drop-off assays. This is demonstrated by Bidshahri et al. (2016), in which multiple BRAF mutations are detected with a novel WT-negative strategy [34]. In this study, assays were designed to target only the WT
BRAF and a highly conserved region within the gene. Independently of the specific mutations,
the absence of signaling from the WT probe indicated the presence of mutant alleles with a limit of detection of 0.05%. The requirement of a perfect match between probes and targets are turned into an advantage instead of an obstacle, which overcomes the problem of missing mutations by using the wrong probe. This could be well applicable in testing for mutant
IDH1/2 at the time of diagnosis when specific mutations are unknown. It is however not
suitable during follow-up as knowledge of the specific mutation of IDH1/2 is required when designing individual assays.
Our results showed that ddPCR has a highly sequence specific sensitivity when targeting mutant IHD1/2. This is suitable during follow-up when designing individual assays, but not for testing unknown mutations at the time of diagnosis. For this purpose, the NGS still conquers ddPCR. Future work needs to investigate the possibility of multiplex assays and
drop-off assays that target mutant IDH1/2 before the method can be implemented as a screening method.
In conclusion, ddPCR targeting specific mutations is a precise and sensitive method which shows a great potential for efficient measurements of minimal residual disease during follow-up of patients. However, its usefulness in screening for mutant IDH1/2 at the time of
diagnosis is limited and alternative approaches, such as multiplexing or drop-off assays, should be considered and evaluated in future studies.
Acknowledgements
I would like to express my very great appreciation to supervisor Tatjana Pandzic for your guidance and useful critique at all stages during this work.
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Etisk reflektion
I denna studie har genetiskt material (DNA) använts för att utvärdera metoden droplet digital PCR (ddPCR) i syftet att identifiera mutationer i IDH1/2 hos patienter med AML. De prover som användes utgjordes av blod- eller benmärgsprov som patienterna lämnat vid diagnos och/eller uppföljning av sin sjukdom och som sparats i Uppsalas biobank. Enligt
biobankslagen har patienterna lämnat skriftligt samtycke till att deras prover sparas med ändamålet forskning och/eller klinisk prövning.
Till skillnad från helgenomsekvensering inriktar sig denna metod på sekvensering av kända mutationer i patientens DNA som är av betydelse för blodsjukdomen. Då andra potentiella mutationer förblir okända undviker man problemet av att finna till exempel riskgener för andra sjukdomar. Det kan dock argumenteras för att gener som är relaterade till
blodsjukdomen fortfarande kan vara associerade till andra sjukdomar. I ett vidare perspektiv skulle det kunna forskas fram att en viss genetisk förändring som sekvenserats för
blodsjukdomen visar sig vara en riskfaktor för en annan sjukdom. Det kan då tänkas bli ett etiskt dilemma för den som ansvarar för informationen huruvida patienten bör få reda på detta. Nyttan med att ta fram genetisk information och att utveckla nya metoder för
effektivare diagnostik och behandling får dock anses vara betydligt större än riskerna för den enskilda individen såväl som för samhället i stort.
Cover letter
Dear editor of the Blood Journal,
Please consider the enclosed manuscript entitled ”Droplet Digital PCR in detection of hotspot mutations in IDH1/2 in patients with acute myeloid leukemia” for publication in your journal. We have evaluated the capacity of ddPCR in detection of hotspot mutations in the genes
isocitrate dehydrogenase 1 and 2 (IDH1/2) in patients with acute myeloid leukemia (AML),
both at the time of diagnosis and during follow-up. Due to the fact that mutant IDH1/2 are of great interest in AML, both as targets for newly released drugs and as markers for minimal residual disease, we believe that our work is of great interest to your readers. We found that ddPCR shows a great potential in measuring minimal residual disease during follow-up, but is of limited value in screening for mutant IDH1/2 at the time of diagnosis. Alternative
approaches, such as multiplexing and drop-off assays, should be evaluated in future studies. We believe that our results are important contributions to the field of genetic diagnostics and may be useful in upcoming research.
Our work has not been published elsewhere and is not under consideration by any other journal. All authors have approved the final version of the manuscript and declare that there is no conflict of interest. The publishing ethics of the journal is agreed.
Yours sincerely,
Johanna Wågberg
Dept. of Medical Science Örebro University
Örebro, Sweden
Contact:
Johanna.wagberg@gmail.com +46(0)70 360 01 96
En ny metod för att snabbt upptäcka om
patienter med akut leukemi kan behandlas
med målinriktade cancerläkemedel
När en patient med akut leukemi får sin diagnos, utförs en rad olika analyser på arvsmassan. Detta görs dels för att hjälpa läkaren att uttala sig om hur svår sjukdomen är, men också vilken behandling som passar. Till vissa patienter med akut leukemi har forskare nu kommit med nya effektiva mediciner. För att kunna erbjudas en sådan medicin krävs dock att de canceromvandlade blodcellerna har en skada på en viss plats i deras arvsmassa (DNA).
De analyser som nu finns för att undersöka om dessa förändringar finns tar lång tid och kräver mycket arbete på laboratoriet. Det finns dock en ny lovande metod som ger säkra resultat på kort tid, vilken kallas droplet digital PCR (ddPCR). I vår studie har vi utvärderat om denna metod är tillräckligt bra på att
upptäcka de specifika förändringarna i arvsmassan.
Vi kom fram till att metoden är mycket bra på att mäta förändringar i arvsmassan som är kända sedan tidigare. Vid diagnos, när inga analyser gjorts och förändringar i arvsmassan är okända, är metoden däremot inte tillräckligt bra. Det finns dock
