CLB-08921; No. of pages: 4; 4C: Clinical Biochemistry xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem

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Anna L. Reid a, James B. Freeman a, Michael Millward b, Melanie Ziman a,c, Elin S. Gray a,⁎

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Article history: Received 21 September 2014 Received in revised form 3 December 2014 Accepted 6 December 2014 Available online xxxx

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Keywords: Circulating tumour cells Melanoma BRAF Droplet digital PCR castPCR WGA

School of Medical Sciences, Edith Cowan University, Perth, WA, Australia School of Medicine and Pharmacology, University of Western Australia, Crawley, WA, Australia School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, WA, Australia

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Objectives: Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. Design and methods: Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. Results: WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. Conclusions: Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR

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Short Communication

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Introduction

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In the last few years, substantial advances have been made in the treatment of metastatic melanoma with the advent of therapies, such as vemurafenib and dabrafenib, targeting somatic mutations in position V600 of the v-Raf murine sarcoma viral oncogene homolog B (BRAF) [1,2]. BRAF mutations mediate tumour proliferation and survival via activation of the RAF–MEK–ERK pathway [3]. Around 43–66% of diagnosed melanomas carry BRAF mutations, with the most common being V600E (80%), with other variants found at lower frequencies; V600K (12%), M (4%), R (5%) and D (b5%) [4,5].

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Abbreviations: BRAF, v-Raf murine sarcoma viral oncogene homolog B; CTCs, circulating tumourcells;ddPCR, droplet digitalPCR; castPCR, competitive allele-specific TaqMan; WGA, whole genome amplification. ⁎ Corresponding author at: Edith Cowan University (ECU), 270 Joondalup Drive, Joondalup, Perth, WA 6027, Australia. E-mail addresses: [email protected] (A.L. Reid), [email protected] (J.B. Freeman), [email protected] (M. Millward), [email protected] (M. Ziman), [email protected] (E.S. Gray).

Heterogeneity in BRAF mutation within a patient has been described between primary tumours and metastases, and between different metastases [6–8]. Testing BRAF mutation status is usually performed on the most recently resected or biopsy tumour. A recent case study pointed to misclassification using this strategy with the patient missing critical treatment [9]. CTCs provide a sample with which to test for mutations even when the tumour biopsy is too old, not available or difficult to obtain being inaccessible by percutaneous biopsy. We previously described a method for enrichment of melanoma CTCs from patient blood using immunomagnetic beads [10]. However, after enrichment, CTCs remain in a large background of white blood cells (1000–10,000 cells), which affects our capacity to detect cancer mutations. Two potential technologies to overcome this background and accurately detect the few copies of mutant DNA are competitive allele-specific TaqMan PCR (castPCR) or droplet digital PCR (ddPCR) [11]. Typically only 1–10 CTCs can be found in 4 mL of blood of metastatic melanoma patients [10]. Partitioning of the sample as required for

http://dx.doi.org/10.1016/j.clinbiochem.2014.12.007 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Please cite this article as: Reid AL, et al, Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.12.007

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Materials and methods

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DNA extraction and serial dilution curve construction

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Genomic DNA was isolated from peripheral blood cells of a healthy donor and the melanoma cell line SK-MEL-28 (homozygous for the BRAF-V600E mutation) using an AllPrep DNA/RNA Mini kit (Qiagen).

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Patient recruitment

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Metastatic melanoma patients were enrolled in the study at Sir Charles Gardner Hospital (SCGH) in Perth, Western Australia, based on having confirmed BRAF-V600E or V600K mutations by molecular analysis of the most recent tumour biopsy. Written informed consent was obtained from all patients. The study was approved by the Human Research Ethics Committees of Edith Cowan University (No. 2932) and Sir Charles Gairdner Hospital (No. 2007-123).

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CTC enrichment and nucleic acid extraction

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Patient peripheral blood samples were collected in 4 mL EDTA tubes, stored at 4 °C, and processed within 24 h of collection. CTCs were enriched as previously described [10]. In summary, whole blood was treated with red blood cell lysing buffer and remaining cells were incubated with immunomagnetic beads coated with antibodies against MCSP (clone 9.2.27, BD Biosciences), ABCB5 (clone 3C2-1D12, kindly provided by Prof. Markus Frank) [12], MAGEA3 (rabbit polyclonal, Thermo Scientific) or RANK (clone 80704, R&D systems) or a combination of these cell surface antigens to target CTCs. After washing, DNA and RNA were extracted from these CTC fractions using an adapted version of AllPrep DNA/RNA Mini kit, using RNeasy MinElute Spin Columns for RNA extraction (Qiagen). RNA was eluted in 12 μL and DNA is 20 μL of RNAse-free water. Extracted RNA (6 μL) was used for cDNA preparation using the SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies) followed by a TaqMan® PreAmp Master Mix Kit (Life Technologies). The presence of transcripts for melanoma genes MLANA, MAGEA3, Tyrosinase, ABCB5 and PAX3 was determined using TaqMan® probes in a ABI ViiA 7 Real-time instrument (Life Technologies). Detection of 18S was used as a positive control.

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Whole genome amplification (WGA) reaction

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DNA isolated from enriched CTC preparations as well as mutant/ wild-type DNA dilutions were whole genome amplified using the Repli-g Midi Kit (Qiagen). For CTC-DNA, WGA was adapted as follows; 18 μL of DNA was incubated with 2.2 μL of DLB buffer for 3 min at RT, after which 3 μL of stop solution was added. The denatured DNA was then combined with a master mix of 29 μL of reaction buffer and 1 μL of Repli-g DNA polymerase. The reaction was incubated for 16 h at 30 °C followed by 3 min at 65 °C. WGA DNA was stored at −80 °C.

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CAST PCR analysis

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Competitive Allele-Specific TaqMan PCR (castPCR) was performed using TaqMan® Mutation Detection Assays (Life Technologies) for BRAF-V600E. A reference assay is used as a control. Each 20 μL reaction contained: 1× Genotyping master mix, 1× TaqMan BRAF 476 mutant detection assay and 9 μL of template, either 1:20 diluted WGA DNA or 100 ng of genomic DNA. Amplifications were carried out in a ViiA 7 system (Applied Biosystems) under the following cycling conditions: 1 cycle of 95 °C for 10 min, 5 cycles of 92 °C for 15 s and 58 °C for 1 min, then 60 cycles of 92 °C for 10 min and 60 °C for 1 min. Results were analysed using the Mutation Detector software (Life Technologies).

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Results

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Effect of WGA on the sensitivity of castPCR and ddPCR

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Serial dilutions from 5 to 0.005% of mutant BRAF-V600E DNA were prepared in a constant background of homologous wild-type DNA and tested using both ddPCR and castPCR, with and without previous WGA. Wild-type DNA was included as a negative control in each assay. Analysis of BRAF-V600E using gDNA as input template produced comparable results in both castPCR and ddPCR (Fig. 1A). CastPCR overestimated the fractional abundance at 0.005% input frequency. This is consistent with the reported limit of the detection for the assay at 0.1% (www.lifetechnologies.com). Analysis of the results using WGA DNA as input template showed that castPCR yielded consistently lower than expected fractional abundances. This could have been interpreted as a bias amplification of the wild-type gene by the WGA reactions. However, ddPCRs using WGA DNA as template produced the results comparable to gDNA, with highly concordant fractional abundances. Moreover ddPCR was the only analysis which provided accurate fractional abundance estimations at 0.005% of V600E mutant (Fig. 1A). To further evaluate the sensitivity of the ddPCR using WGA DNA as template, a serial dilution curve extended to 0.00005% was evaluated, revealing a limit of sensitivity at 0.0005% (Fig. 1B).

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Either 1 μg genomic DNA or 4 μL of each WGA CTC-DNA sample was digested using 20 U HaeIII (New England Bio) and 1× NE Buffer 4 for 60 min at 37 °C followed by 20 min at 80 °C for enzyme inactivation. Each droplet of a PCR supermix (Bio-Rad) reaction contained: 1× droplet PCR supermix, 250 nM of each probe, 900 nM primers and 100 ng of digested genomic DNA or 1 uL digested WGA DNA, in a total reaction volume of 20 μL. Samples were analysed for BRAF-V600E or V600K mutations depending on the mutation identified in the patient biopsy. The following probes were used: T1799-VIC WT (VIC-CTAGCT ACAGTGAAATC-MGBNFQ) and A1799-FAM V600E (6FAM-TAGCTACA GAGAAATC-MGBNFQ) or A1799-FAM V600K (6FAM-TAGCTACAAAGA AATC-MGBNFQ). The following primers were used for both assays: 5 ′-CTACTGTTTTCCTTTACTTACTACTACACCTCAGA-3′ (forward) and 5′-ATCCAGACAACTGTTCAAACTGATG-3′ (reverse). Probes and primers were custom synthesised by Life Technologies. Droplets were generated and analysed using the QX100 system (Bio-Rad). Amplifications were performed using the following conditions: 1 cycle of 95 °C for 10 min, 40 cycles of 94 °C for 30 s and 55 °C for 1 min, and 1 cycle of 98 °C for 10 min. All samples, including no template controls, were run in eight replicates. QuantaSoft analysis software (Bio-Rad) enabled fractional abundance to be calculated for each sample.

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Droplet digital PCR

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droplet digital PCR might miss those rare copies of mutated DNA. Thus, pre-amplification of the input DNA is imperative for sample analysis. However, no previous analysis has been done on the suitability of whole genome amplified (WGA) DNA as a template for castPCR or ddPCR. In this study we compared the sensitivity of ddPCR to a competitive allele-specific TaqMan (castPCR) assay (Life Technologies) for the detection of BRAF-V600E and V600K mutations using gDNA or WGA DNA as template. Furthermore, we evaluated ddPCR for the detection of BRAF mutations in a CTC enriched fraction from melanoma patients undergoing targeted therapies.

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Analysis of BRAF mutation in a CTC enriched fraction from melanoma 181 patients 182 We next analysed DNA from 30 enriched CTC fractions from 15 183 metastatic melanoma patients with recorded BRAF-V600E mutated 184

Please cite this article as: Reid AL, et al, Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.12.007

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these markers in isolation [10,13]. The remaining samples were captured with a mix of bead types. The DNAs from CTCs isolated from patients with BRAF-V600K tumours were also analysed using a BRAF-V600K specific probe. As experimental control we utilised DNA extracted from an FFPE tumour tissue previously shown to be BRAF-V600K (Fig. 2C). A total of 18 CTC-enriched samples isolated from 5 patients were tested. Of those, 8 had detectable BRAF-V600K mutations (≥ 0.0005), ranging from 0.000475 to 0.01% (Fig. 2B). Of the positive samples, 2 were isolated with beads targeting MCSP, 2 with ABCB5, 1 with MAGE-A3 and the remainder with a mix of bead types.

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tumours. Samples were selected based on positivity of RNA from the CTC fraction, by RT-PCR for melanoma markers, confirming the presence of CTCs. The CTC-DNA was subjected to WGA prior to ddPCR analysis. We detected BRAF-V600E mutated DNA at a fractional abundance ≥0.0005% in 23 samples, ranging from 0.000514 to 0.0286% (Fig. 2A). Of note, not all the replicates exhibited mutated DNA, with 5 samples presenting mutant droplets in only one of the 8 replicates. Of the positive samples, 8 were isolated with beads targeting MCSP, 8 by targeting ABCB5, 4 by targeting MAGE-A3 and 1 by targeting RANK, further confirming that melanoma CTCs can be captured by targeting

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Fig. 1. Detection of BRAF-V600E mutant DNA in the presence of wild-type DNA. Serial dilutions of DNA from SK-MEL-28 cells were prepared in a constant amount of wild-type DNA from the blood of a healthy donor. (A) Comparison of ddPCR and castPCR for detection of mutant BRAF-V600E using gDNA and WGA-DNA. (B) Analysis by ddPCR of WGA DNA with serial dilutions extended from 5 to 0.00005%, demonstrated a limit of detection as a fractional abundance of 0.0005%. The mean and SD are shown for each dilution.

Fig. 2. Analysis of BRAF mutations in melanoma patients by ddPCR. (A) Quantification of BRAF-V600E (A) and BRAF-V600K (B) mutations in WGA DNA isolated from CTC enriched fractions of metastatic melanoma patients with mutated tumours. Dotted lines indicate the limit of detection of the assay. Asterisks indicate samples obtained from patients prior to therapy initiation. (C) DNA derived from a FFPE tumor sample positive for BRAF-V600K was analysed by ddPCR as control for the in-house BRAF-V600K assay.

Please cite this article as: Reid AL, et al, Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.12.007

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The authors thank all the participants for their assistance with the study. We would like to thank Dr. Dave Barryman from the Western Australian State Agricultural Biotechnology Centre for the use of the QX100 system. This study was funded by a NHMRC Grant 1013349 to MZ and MM, an Edith Cowan University Early Career Research Grant

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[1] Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012;380(9839):358–65. [2] McArthur GA, Chapman PB, Robert C, Larkin J, Haanen JB, Dummer R, et al. Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study. Lancet Oncol 2014;15(3):323–32. [3] Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, et al. Mechanism of activation of the RAF–ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116(6):855–67. [4] Rubinstein JC, Sznol M, Pavlick AC, Ariyan S, Cheng E, Bacchiocchi A, et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med 2010; 8:67. [5] Lovly CM, Dahlman KB, Fohn LE, Su Z, Dias-Santagata D, Hicks DJ, et al. Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS One 2012;7(4):e35309. [6] Houben R, Becker JC, Kappel A, Terheyden P, Brocker EB, Goetz R, et al. Constitutive activation of the Ras–Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J Carcinog 2004;3(1):6. [7] Lin J, Goto Y, Murata H, Sakaizawa K, Uchiyama A, Saida T, et al. Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression. Br J Cancer 2011;104(3):464–8. [8] Yancovitz M, Litterman A, Yoon J, Ng E, Shapiro RL, Berman RS, et al. Intra- and inter-tumor heterogeneity of BRAF(V600E) mutations in primary and metastatic melanoma. PLoS One 2012;7(1):e29336. [9] Richtig E, Schrama D, Ugurel S, Fried I, Niederkorn A, Massone C, et al. BRAF mutation analysis of only one metastatic lesion can restrict the treatment of melanoma: a case report. Br J Dermatol 2013;168(2):428–30. [10] Freeman JB, Gray ES, Millward M, Pearce R, Ziman M. Evaluation of a multi-marker immunomagnetic enrichment assay for the quantification of circulating melanoma cells. J Transl Med 2012;10:192. [11] Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 2011;83(22):8604–10. [12] Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, et al. Identification of cells initiating human melanomas. Nature 2008;451(7176): 345–9. [13] Kupas V, Weishaupt C, Siepmann D, Kaserer ML, Eickelmann M, Metze D, et al. RANK is expressed in metastatic melanoma and highly upregulated on melanomainitiating cells. J Invest Dermatol 2011;131(4):944–55. [14] Klinac D, Gray ES, Millward M, Ziman M. Advances in personalized targeted treatment of metastatic melanoma and non-invasive tumor monitoring. Front Oncol 2013;3:54.

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Our results demonstrate that ddPCR is more robust and more sensitive than castPCR, allowing the detection of BRAF-V600E/K mutations down to frequencies of 0.0005%, 200 times more sensitive than the defined 0.1% sensitivity of castPCR. This study confirmed and expanded the findings by Hindson et al. which identified ddPCR analysis as capable of detecting down to 0.001% BRAF mutant fraction in dilutions involving wild-type BRAF [11]. Furthermore we demonstrated that WGA combined with ddPCR allows the detection of cancer mutations in CTCs after partial isolation by enrichment. Moreover, given its high sensitivity, similar methods could be used to identify mutated tumour DNA in plasma derived cfDNA, providing a simpler and more cost effective method to evaluate cancer mutations in the blood samples. Furthermore, this method could be used to identify escape mutations that mediate melanoma resistance to BRAF inhibitors, such as NRAS-Q61R/H/K and MEK-C121S mutations, previously found in the tumour of patients undergoing targeted therapy [14]. Identifying relapse early will allow appropriate management of patients by transferring them to new treatment regimens while their melanoma tumour burden is still manageable.

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to EG and Scott Kirkbride Melanoma Research Centre Grant to EG, MZ 243 and MM. EG is supported by a fellowship from the Cancer Research 244 Trust. 245 Q6

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Two CTC-enriched samples, positive by RT-PCR for melanoma markers, from patients whose tumours were wild-type for BRAF were also analysed as negative controls. Both samples were negative in both BRAF-V600E and V600K assays. Most of the samples evaluated were derived from patients already undergoing BRAF targeted therapies such as vemurafenib or dabrafenib/trametinib combination. Only 4 samples were derived from the blood samples taken prior to therapy and all 4 of these were found positive for BRAF mutations (indicated by an asterisk in Fig. 2A and B).

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Please cite this article as: Reid AL, et al, Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.12.007

Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.

Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies...
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