RAF mutations by multiplex digital PCR

    Screening for Circulating RAS/RAF Mutations by Multiplex Digital PCR Rikke Fredslund Andersen, Anders Jakobsen PII: DOI: Reference:

S0009-8981(16)30211-X doi: 10.1016/j.cca.2016.05.007 CCA 14364

To appear in:

Clinica Chimica Acta

Received date: Revised date: Accepted date:

31 March 2016 10 May 2016 10 May 2016

Please cite this article as: Andersen Rikke Fredslund, Jakobsen Anders, Screening for Circulating RAS/RAF Mutations by Multiplex Digital PCR, Clinica Chimica Acta (2016), doi: 10.1016/j.cca.2016.05.007

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ACCEPTED MANUSCRIPT Screening for Circulating RAS/RAF Mutations by Multiplex Digital PCR

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Rikke Fredslund Andersen1, Anders Jakobsen2

Department of Clinical Immunology and Biochemistry, Vejle Hospital, Vejle, Denmark.

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Department of Oncology, Vejle Hospital, Vejle, Denmark.

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Corresponding author:

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Rikke Fredslund Andersen

Dept. Clinical Immunology and Biochemistry, Vejle Hospital,

+45 79406529

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Kabbeltoft 25, DK-7100 Vejle, Denmark.

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[email protected]

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ACCEPTED MANUSCRIPT Abstract:

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Recent years have shown a large interest in the application of liquid biopsies in cancer management.

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Circulating tumor DNA (ctDNA) has been investigated for potential use in treatment selection,

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monitoring of treatment response, and early detection of recurrence. Advances have been hampered by technical challenges primarily due to the low levels of ctDNA in patients with localized disease

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and in patients responding to therapy.

The approach presented here is a multiplex digital PCR method of screening for 31 mutations in the

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KRAS, NRAS, BRAF, and PIK3CA genes in the plasma. The upper level of the limit of blank, which defines the specificity of the multiplexes, was 0.006%-0.06%. Mutations found by multiplex

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analyses were identified and quantified by duplex analyses. The method was tested on samples from

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cholangiocarcinoma patients with known tumor mutational status. Mutations found in the tumor were also found in plasma samples in all cases with analyses for all other mutations being negative.

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There was a perfect agreement as to wild type status in tumor and plasma. The method combines a high sensitivity with the ability to analyze for several mutations at a time

Keywords:

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and could be a step towards routine clinical application of liquid biopsies.

ctDNA, multiplex screening, digital PCR, plasma

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ACCEPTED MANUSCRIPT 1. Introduction

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Most malignant tumors harbor somatic mutations as an important biological characteristic. The

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development of malignancy is an ongoing process with continuous development of new mutations,

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which increases their number in advanced tumors. Somatic mutations occur at a negligible rate in normal cell populations and therefore exquisitely represent specific biomarkers of malignancy [1]. Some of the mutations are bystanders with no or minimal influence on tumor behavior, but a small

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fraction of them is of crucial importance to proliferation, invasion, and metastatic potential [2].

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The analysis of mutations has witnessed an enormous progress during the recent years, especially based on Next Generation Sequencing (NGS). The improved technology, however, has not been

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reflected as a proportional progress in the clinical application. One reason is tumor heterogeneity.

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Different parts of the tumor harbor different mutations and it is difficult to identify an overall picture of the clinical relevance. Furthermore, the spectrum of mutations may differ between

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primary tumor and metastases [3;4]. Another obstacle is the time factor. Analysis of the primary tumor does not necessarily reflect the picture in local or distant recurrence years later. The same

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problem arises during therapy. Treatment may lead to clonal selection and/or development of new mutations as demonstrated in the treatment of patients harboring a RAS wildtype tumor with monoclonal antibodies against the epidermal growth factor receptor (EGFR) [5-8]. Analysis of plasma or serum may reflect the overall mutational status at the time of therapeutic interest. Most malignant tumors shed mutated DNA into the circulation [5], and the "liquid biopsy" has recently seen considerable scientific and clinical interest. However, there are a number of obstacles on the way to clinical routine use. Mutated DNA in the circulation is present at a low frequency and valid methods for detection of rare events are required. A practical method should be able to analyze a spectrum of mutations in the same run with a minimum of resources. Two

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ACCEPTED MANUSCRIPT methods are generally used - NGS and digital polymerase chain reaction (dPCR), both of which have advantages and disadvantages. NGS is ideal for screening a large number of hotspots for

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mutations, but the sensitivity is around 2% [9], the costs are still relatively high, and data analysis is

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rather complex. Digital PCR has the necessary sensitivity (<0.01%) [9], but requires selection of

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specific mutations for analysis. In principle, PCR is dedicated to analyze one mutation at a time, but it holds potential for several analyses in the same run. The aim of the present study was to

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investigate an extension of digital PCR for screening of plasma using multiplex technology.

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2. Materials and methods

2.1 Materials: A large pool of residual whole blood genomic DNA (gDNA) from individuals

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genetically screened for lactose intolerance was used as wild type donor DNA. Positive controls

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were generated by site-directed mutagenesis as previously described [10]. Mutated PCR fragments were mixed with gDNA with a concentration of approximately 1600 copies/µl (5,3 ng/µl). Plasma

testing the method.

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samples from cholangiocarcinoma patients with known tumor mutational status were used for

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2.2 Plasma preparation: Plasma was isolated from 9 ml EDTA-blood samples by centrifugation at 2.000g for 10 minutes within 2 hours of collection. Samples were stored at -80°C until use. 2.3 DNA purification: DNA from 300 µl of whole blood was purified on a Maxwell 16 purification robot using the Maxwell 16 Blood DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. DNA was eluted in 300 µl of the supplied buffer and stored at 20°C. Prior to purification plasma was centrifuged at 10,000g for 10 minutes and the supernatant transferred to clean tubes. 5 µl of internal exogenous spike-in DNA fragment (CPP1) was added to each 1.2 ml of plasma [11]. DNA was purified from 4x 1.0 ml plasma on the QIASymphony SP 4

ACCEPTED MANUSCRIPT instrument using the QIAsymphony DSP Virus/Pathogen midi kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was eluted in 4x 110 µl of the supplied buffer

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and pooled.

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2.4 qPCR: 20 µl of pooled DNA from plasma was mixed with 30 µl of water and analyzed by qPCR for the number of gB2M and CPP1 alleles as described previously [11].

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2.5 Pre-amplification: The remaining 420 µl of pooled DNA from plasma was concentrated to 20 µl on a Millipore centrifugal filter unit (Millipore, Billerica, MA, USA). The DNA was pre-amplified

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10 cycles with Q5 mastermix (New England Biolabs, Ipswich, MA, USA) and Pre-amp primer mix (Primers from DNA Technology, Aarhus, Denmark, Suppl. table 1) in 50 µL reaction volumes

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according to the manufacturer’s recommendations. Cycling conditions were 98°C for 10 min, 10

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cycles of 98°C for 15 sec, 55°C for 1 min, and 72°C for 1 min followed by 99.9°C for 10 min.

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2.6 Selection of mutations for multiplex panel: Mutations included in the panel were selected based on publications with data from large series of colorectal tumors sequenced by NGS or Pyrosequencing to obtain reliable frequencies of mutations in the KRAS and NRAS genes [12;13].

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Mutations found in more than 0.2% of colorectal tumors were included in the panel. Mutations found at a lower frequency (i.e. mutations found in only one or two patients) were not included. On this basis, 17 mutations in KRAS, 9 mutations in NRAS, and 1 mutation in BRAF were selected. As the significance of PIK3CA mutations is still debated, the four most common mutations in this gene were included in the panel. 2.7 Multiplex set-up: Standard 96-well plate is the format used for droplet digital PCR. Assays were combined in eight multiplex reactions with assays detecting 3-5 mutations in each multiplex (Table 1). All assays detecting mutations in the same or adjacent codons were included in the same multiplex except for KRAS codons 12/13. The specific mutation present in a sample cannot be

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ACCEPTED MANUSCRIPT identified from multiplex analysis. Positive reactions must be split into single reactions to identify the one present.

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2.8 Droplet digital PCR – multiplex analysis: Samples were diluted 50 times after pre-amplification.

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12 µl of DNA was combined with 5 µl (multiplex 3 and 8) or 12 µl (multiplex 1, 2, 4, 5, 6 and 7) of multiplexes of PrimePCR ddPCR assays for mutations (Bio-Rad®, Hercules, CA, USA; Suppl. table 2) and Bio-Rad® 2x ddPCR supermix for probes in a total volume of 48 µl. Twenty-two µl

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samples were added to two wells of an Eppendorf Twin-tec 96-well plate. Droplets were generated from 20 µl of sample in an Auto Droplet Generator (Bio-Rad®) and 40 cycles of PCR amplification

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were carried out (95°C for 10 min, 40 cycles of 94°C for 30 sec, and 55°C for 60 sec followed by 98°C for 10 min). Samples were analyzed in a Droplet Reader QX100 (Bio-Rad®) and data

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analyzed with Quantasoft ddPCR Software ver. 1.7. 2.9 Droplet digital PCR – duplex analysis: Samples identified as positive by multiplex dPCR were

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analyzed by duplex dPCR for assays included in that multiplex. The procedure was as described for multiplex dPCR and the same dPCR assays used (Supplementary table 2). Five µl of duplex assay

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was used for each 48 ul reaction volume. Twenty µl of sample was analyzed per well. 2.10 Controls: Negative controls (water and gDNA) were pre-amplified and analyzed simultaneously when samples were pre-amplified. Positive controls were included in digital PCR analysis at every mastermix preparation. 2.11 Data analysis: The threshold settings for each of the eight multiplexes were determined from gDNA and positive controls, and the same thresholds applied to all samples. Data from two wells were merged for data analysis of controls and samples. Wells with less than 10,000 droplets were excluded from analysis. Based on the number of FAM and HEX positive droplets and the Poisson distribution the Quantasoft software calculated the number of wild type and mutated copies of DNA

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ACCEPTED MANUSCRIPT per µl of reaction. The fractional abundance was calculated as copies of mutated DNA/(copies of mutated DNA + copies of wild type DNA). Samples were classified as positive, if the lower limit of

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the 95% confidence interval for the fractional abundance of a multiplex reaction was above the

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upper limit of the 95% confidence interval of the corresponding multiplex for gDNA. In case of

3. Results

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3.1 Multiplex dPCR compared to duplex dPCR:

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overlapping confidence intervals, the sample was classified as negative.

Positive controls for all mutations mixed with wild type genomic DNA were pre-amplified and

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analyzed by multiplex and duplex dPCR in parallel. The fractional abundance of mutated DNA was

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calculated and compared (Suppl fig 1). Identical percentages were found if all assays in a multiplex

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covered the same or adjacent codons. Multiplexes with assays from two different genes (multiplex 6) or different regions of the same gene (multiplex 2 and 8) result in two wild type regions being amplified and more than one HEX positive cluster in the digital PCR. Different double positive

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clusters will appear, since droplets at a certain frequency (depending on the concentration of DNA) contain more than one template molecule (Suppl figure 2). Calculations of fractional abundance of mutated DNA from multiplexes with more than one wild type cluster are therefore not accurate but will always be calculated as being lower in the multiplex reaction. The number of FAM positive droplets per reaction when analyzing gDNA by duplex and multiplex dPCR was compared (Suppl figure 3). For multiplex 1, 2, 3, 5, and 8 the number of positive droplets was the same or lower than in the duplex reactions, whereas in multiplex 4, 6, and 7 the number was similar to the sum of positives in the individual duplex reactions. The numbers are low

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ACCEPTED MANUSCRIPT and hence the variation high, but there does not appear to be a significantly higher level of

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background in the multiplex reactions compared to duplex.

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3.2 Limit of blank

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3.2.1 Dilutions of gDNA: To investigate if the limit of blank (LOB) was dependent on the amount of wild type DNA in the reaction four-fold dilutions of a pool of gDNA was prepared and each dilution pre-amplified as described above. Each dilution was analyzed with the eight multiplexes

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and the number of FAM positive droplets per 20 µl reaction was counted (Figure 1). For multiplex 1, 2, 5, and 7 the LOB was not dependent on the amount of DNA in the reaction, whereas multiplex

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3, 4, 6, and 8 had increasing amounts of positive droplets with increasing amounts of wild type DNA.

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3.2.2 Pool of donor gDNA: A pool of healthy donor gDNA was analyzed several times. Genomic

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DNA was pre-amplified and analyzed by multiplex dPCR every time to determine the variation of

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the entire process and not just the dPCR step. The outcome is presented in Suppl figure 4 and shows reproducible results among the analyses. For subsequent analyses the highest level of the upper

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value of the fractional abundance found in all the replicates was used to determine whether samples were positive or negative. These values are shown together in Figure 2. 3.3 Patient samples: Plasma samples from five patients with known tumor mutation were analyzed by multiplex screening. All samples were positive for the expected multiplex and negative for the seven other multiplexes when compared to the limit of blank (Figure 3). After analyzing for specific mutations present in the positive multiplex all samples were clearly positive for the expected mutation and negative for the other mutations analyzed (Figure 3 and Suppl figure 5). Six plasma samples from patients with tumors previously determined wild type as to the 31 mutations included in the multiplex panel were screened for mutations. The samples were negative for all multiplexes with fractional abundances similar to or lower than the limit of blank (Figure 4). 8

ACCEPTED MANUSCRIPT 4. Discussion

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Circulating tumor specific DNA is of major clinical interest as a possible prognostic marker

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allowing for rational selection of patients, e.g. for adjuvant chemotherapy, but it may also play a

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role as a marker for early detection of recurrence. A further aspect is its promise as predictive marker with application for treatment monitoring. Consequently, development of new

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methodological approaches has high priority.

Analysis of circulating mutations seems preferable to analysis of the primary tumor, since a liquid

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biopsy reflects the mutational pattern in the specific therapeutic situation. A screening for relevant mutations seems to be a good first step towards rational utility of mutational status. Screening,

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however, faces a number of challenges. First of all, the rate of mutated alleles in the circulation is

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very low, especially when dealing with localized tumors or when mutations appear in the

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circulation as an early predictor of recurrence. The ideal screening method should have a very high sensitivity but at the same time be able to analyze for a large number of mutations in the same run. The cost needs to be relatively low with

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little hands-on time. A high throughput of samples and a quick result are also important. Digital PCR meets most of these requirements and is currently the method with the highest sensitivity. The application of multiplex technology allows for analysis of a sufficient number of mutations appropriate for screening. In a clinical setting it is important to focus on relevant mutations. In many situations it is sufficient to obtain information on a focused panel of mutations with relevance to a specific treatment. The method presented here consists of a panel of RAS/RAF mutations representing key mutations in many malignant tumors. With approximately 50% of colorectal tumors harboring these mutations they have obvious clinical relevance and would provide a rational basis for selection of anti-EGFR treatment, which is standard of care in tumor wild type colorectal

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ACCEPTED MANUSCRIPT cancer. The panel, moreover, can be adapted to match other tumor types and different clinical

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settings.

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Multiplex dPCR for mutation screening has only been sparsely investigated. Taly et al. [14]

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developed a KRAS multiplex panel for screening plasma from colorectal cancer patients consisting of seven KRAS mutations in two multiplex reactions. The dPCR was optimized to differentiate mutations by using different amounts of probes, which represents a further development of the

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method. The results were similar to those presented here except for the number of different mutations, which in our study covers almost the full spectrum of RAS/RAF mutations.

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Furthermore, our approach can easily be extended to comprise other mutations. Pender et al. [15] designed three multiplex reactions to screen for the nine most common KRAS mutations in lung

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cancer formalin fixed paraffin embedded tissue, where tumor tissue can be sparse and a high

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sensitivity is needed. They compared the method with Sanger sequencing and NGS. The results

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demonstrated a sensitivity by multiplex digital PCR of 0.5% compared to 2% by NGS and 10% by Sanger sequencing. The method presented here allows for detection of mutations with a rate of

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0.006% to 0.06%.

Targeted pre-amplification of cfDNA before dPCR analysis has recently been published [16]. The authors amplify regions from three different genes and analyze by dPCR. The study shows that preamplification can be used to improve robustness of the analyses. For broader screening more assays need to be included in the pre-amplification as presented here, where 10 sets of primers are included. Pre-amplification has also been used for qPCR analysis of cfDNA [17;18] but only for amplification of one specific region, i.e. no simultaneous amplification of several regions was performed.

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ACCEPTED MANUSCRIPT The targeted pre-amplification step ensures sufficient material to verify findings from the multiplex analysis by duplex analysis identifying the specific mutation present. It cannot be guaranteed that

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mutated and wild type targets are amplified equally in the pre-amplification, but the results are

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reproducible, and a relevant measure of the fractional abundance of mutated DNA is obtained. In conclusion, the present study shows that multiplex dPCR is a suitable method of screening plasma for RAS/RAF mutations. In the clinical setting multiplex screening should be followed by

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duplex PCR for identification of the mutation. This approach holds high promise of useful clinical

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application.

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Acknowledgements:

The authors wish to thank laboratory technicians Tina Brandt Christensen, Lone Hartmann Hansen

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and Pia Nielsen, Department of Clinical Immunology and Biochemistry, Vejle Hospital, for excellent technical assistance and Karin Larsen, Department of Oncology, Vejle Hospital, for

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linguistic editing.

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ACCEPTED MANUSCRIPT References

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1. Martincorena I, Campbell PJ. Somatic mutation in cancer and normal cells. Science 2015;

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349:1483-9.

2. Stratton MR, Campbell PJ, Futreal PA. The cancer genome. Nature 2009; 458:719-24.

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3. Goswami RS, Patel KP, Singh RR et al. Hotspot mutation panel testing reveals clonal evolution in a study of 265 paired primary and metastatic tumors. Clin Cancer Res 2015;

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21:2644-51.

4. Zhao ZM, Zhao B, Bai Y et al. Early and multiple origins of metastatic lineages within

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primary tumors. Proc Natl Acad Sci U S A 2016; 113:2140-5. 5. Bettegowda C, Sausen M, Leary RJ et al. Detection of circulating tumor DNA in early- and

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late-stage human malignancies. Sci Transl Med 2014; 6:224ra24. 6. Diaz LA, Jr., Williams RT, Wu J et al. The molecular evolution of acquired resistance to

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targeted EGFR blockade in colorectal cancers. Nature 2012; 486:537-40. 7. Misale S, Yaeger R, Hobor S et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012; 486:532-6. 8. Siravegna G, Mussolin B, Buscarino M et al. Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. Nat Med 2015; 21:827. 9. Diaz LA, Jr., Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol 2014; 32:579-86.

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ACCEPTED MANUSCRIPT 10. Spindler KL, Pallisgaard N, Vogelius I, Jakobsen A. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment

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with cetuximab anda irinotecan. Clin Cancer Res 2012; 18:1177-85.

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11. Pallisgaard N, Spindler KL, Andersen RF, Brandslund I, Jakobsen A. Controls to validate plasma samples for cell free DNA quantification. Clin Chim Acta 2015; 446:141-6.

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12. Douillard JY, Oliner KS, Siena S et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 2013; 369:1023-34.

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13. Vaughn CP, Zobell SD, Furtado LV, Baker CL, Samowitz WS. Frequency of KRAS, BRAF,

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and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer 2011; 50:307-12.

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14. Taly V, Pekin D, Benhaim L et al. Multiplex picodroplet digital PCR to detect KRAS

59:1722-31.

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mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem 2013;

15. Pender A, Garcia-Murillas I, Rana S et al. Efficient Genotyping of KRAS Mutant Non-Small

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Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach. PLoS One 2015; 10:e0139074.

16. Jackson JB, Choi DS, Luketich JD, Pennathur A, Stahlberg A, Godfrey TE. Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR. J Mol Diagn 2016; 18:235-43. 17. Castellanos-Rizaldos E, Paweletz C, Song C et al. Enhanced ratio of signals enables digital mutation scanning for rare allele detection. J Mol Diagn 2015; 17:284-92.

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ACCEPTED MANUSCRIPT 18. Diehl F, Li M, Dressman D et al. Detection and quantification of mutations in the plasma of

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patients with colorectal tumors. Proc Natl Acad Sci U S A 2005; 102:16368-73.

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ACCEPTED MANUSCRIPT Figure 1

90

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70 60

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50 40 30 10 0 5000

Multiplex 2 Multiplex 3 Multiplex 4 Multiplex 5 Multiplex 6 Multiplex 7 Multiplex 8

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Multiplex 1

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# FAM positive droplets / 20ul reaction

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0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

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% mutated DNA

Figure 2

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Max 0.007 0.006 0.06 0.07 0.04 0.06 0.05 0.05

Min 0.0003 0 0.02 0.03 0.01 0.03 0.02 0.03

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Multiplex 1 Multiplex 2 Multiplex 3 Multiplex 4 Multiplex 5 Multiplex 6 Multiplex 7 Multiplex 8

Fractional Abundance 0.004 0.003 0.04 0.05 0.02 0.04 0.04 0.04

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Multiplex 1 Multiplex 2 Multiplex 3 Multiplex 4 Multiplex 5 Multiplex 6 Multiplex 7 Multiplex 8

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ACCEPTED MANUSCRIPT Figure 3 2

0.2

Patient 1 Donor

0.1 0

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1 0.5

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E542K

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Patient 2

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% mutated DNA

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Patient 3 Donor

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0.5

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% mutated DNA

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% mutated DNA

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H1047R

H1047L

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% mutated DNA

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Patient 2 Donor

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0 KRAS G12D

KRAS G12V

KRAS G13D

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Patient 3 Donor

0.5 0 Q61H A>C Q61H A>T

Q61R

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Patient 4 Donor

% mutated DNA

% mutated DNA

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Patient 1 Donor

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0.4 % mutated DNA

% mutated DNA

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0.4 0.3 Patient 4

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Patient 5 Donor

% mutated DNA

% mutated DNA

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Q61L

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0.5 0 KRAS G12D KRAS G12V KRAS G13D

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ACCEPTED MANUSCRIPT Figure 4

Patient 6

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Patient 8

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Patient 10 Donor

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Patient 7

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ACCEPTED MANUSCRIPT Figure legends:

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Figure 1: Number of FAM positive droplets counted in 20 µl reactions with varying amounts of

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genomic wild type DNA tested with eight different multiplexes.

Figure 2: Limit of blank for the eight multiplexes expressed as fractional abundance of mutated

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DNA tested on wild type genomic DNA from healthy donors.

Figure 3: The fractional abundance of mutated DNA in patient samples tested by eight multiplexes

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(left). Samples were further tested by duplex analyses for all assays included in positive multiplexes

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(right). Samples were classified as positive if the lower level of the sample fractional abundance

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was higher than the higher level of the donor fractional abundance.

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Figure 4: The fractional abundance of mutated DNA in patient samples tested by eight multiplexes. Samples were classified as positive if the lower level of the sample fractional abundance was higher than the higher level of the donor fractional abundance.

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ACCEPTED MANUSCRIPT

Multiplex 4

Multiplex 5

Multiplex 6

Multiplex 7

Exon 4

KRAS

Exon 4

KRAS KRAS KRAS

Exon 4 Exon 4 Exon 4

A146T A146V A146P

NRAS NRAS NRAS NRAS NRAS

Exon 2 Exon 2 Exon 2 Exon 2 Exon 2

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KRAS

G12R G13C K117N A>C K117N A>T

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Exon 2 Exon 2

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KRAS KRAS

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Q61H A>C Q61H A>T Q61R Q61L

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Multiplex 3

Exon 3 Exon 3 Exon 3 Exon 3

G12D G12C G12V G13D G13R

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Multiplex 2

KRAS KRAS KRAS KRAS

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Multiplex 1

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Table 1: Mutations included in multiplex 1-8.

NRAS NRAS NRAS NRAS

Exon 3 Exon 3 Exon 3 Exon 3

Q61K Q61R Q61H Q61L

KRAS KRAS KRAS

G12D G12V G13D

BRAF

Exon 2 Exon 2 Exon 2 Exon 15

KRAS KRAS

Exon 2 Exon 2

G12A G12C

V600E

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E542K E545K H1047R H1047L

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PIK3CA Exon 9 PIK3CA Exon 9 Exon PIK3CA 20 Exon PIK3CA 20

G12S

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Multiplex 8

Exon 2

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KRAS

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ACCEPTED MANUSCRIPT Highlights

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Multiplex digital PCR screening for mutations in circulating cell-free DNA Simultaneous analyses of 31 somatic mutations The specificity of the multiplex digital PCR was 0.006%-0.06%

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  

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