BRAFV600E detection in melanoma is highly improved by COLD-PCR

Clinica Chimica Acta 412 (2011) 901–905

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

BRAFV600E detection in melanoma is highly improved by COLD-PCR Pamela Pinzani a,⁎, Claudio Santucci a, Irene Mancini a, Lisa Simi a, Francesca Salvianti a, Nicola Pratesi a, Daniela Massi b, Vincenzo De Giorgi c, Mario Pazzagli a, Claudio Orlando a a b c

Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy Department of Critical Care Medicine and Surgery, University of Florence, Viale G.B. Morgagni 85, 50134 Florence, Italy Department of Dermatological Sciences, University of Florence, Via della Pergola 60, Florence, Italy

a r t i c l e

i n f o

Article history: Received 30 December 2010 Received in revised form 11 January 2011 Accepted 12 January 2011 Available online 22 January 2011 Keywords: COLD-PCR Somatic mutation BRAFV600E Melanoma

a b s t r a c t Background: The BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues. Methods: COLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations. Results: We used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAFV600E were found positive after COLD-PCR. Conclusions: COLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Cancers are clonal proliferations due to mutations in a subset of genes that confer selective growth advantage to cells [1]. Somatic mutations occur in the genome of all dividing cells, both normal and neoplastic, as a result of misincorporation during DNA replication or through exposure to exogenous or endogenous mutagens. Cancer genomes carry somatic mutations arising from these various processes. ‘Driver’ mutations in cancer genes confer growth advantage to the cell in which they occur, and are causally implicated in cancer development, and therefore positively selected. Conversely, ‘passenger’ mutations have not been subject to selection [2]. BRAF protein kinase was reported as one of the genes that are most likely to carry driver mutations. It was reported as mutated in 66% of malignant melanomas [3–5] and, at a lower frequency, in a wide range of other human cancers, such as colorectal (5–12%) [6], serous ovarian (about 15%) [7], and thyroid carcinoma (20–45%) [8]. Over 40 different missense mutations in BRAF have been identified, most of

Abbreviations: FFPE, formalin fixed paraffin embedded; COLD-PCR, co-amplification at lower denaturation temperature-PCR; HRMA, high resolution melting analysis; Tm, melting temperature; Tc, critical denaturation temperature. ⁎ Corresponding author. Tel.: +39 0554271441; fax: +39 0554271371. E-mail address: [email protected]fi.it (P. Pinzani). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.01.014

them being extremely rare (0.1%–2% of all cases). However, the thymidine to adenosine transversion at nucleotide 1799, converting valine to glutamate (V600E), is predominant, accounting for over 90% of the mutations in melanoma and thyroid cancer, as well as a high proportion of those in colorectal cancer [7]. The BRAF gene was identified as an oncogene in human cancer and, in particular, the V600E mutation was shown to be associated with clinico pathological features of primary invasive melanomas [9]. Moreover, due the high incidence of mutations throughout melanoma progression, BRAF may be an attractive therapeutic target [10,11]. The recent development of a specific inhibitor of the mutant isoform of BRAF kinase (PLX-4032) [12,13] increases the value of BRAFV600E identification in melanoma patients who could benefit from this treatment. The identification of BRAF as a commonly mutated target in human cancer, and particularly in melanoma patients, leads us to the development of a sensitive method for the detection and accurate quantification of the amount of mutated DNA in a biological sample. PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low percentages of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes which can have a profound impact on clinical decision-making and outcome, or

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can serve as cancer biomarkers. The limited sensitivity of standard methods to detect DNA mutations in tissues highlights the need for highly sensitive assays which are able to work on degraded DNA. COLD-PCR (co-amplification at lower denaturation temperaturePCR) [16] is a novel form of PCR that selectively amplifies minority alleles from mixtures of wild-type and mutation-containing sequences, irrespective of the mutation type or position on the sequence. Since PCR constitutes an ubiquitous initial step for almost all genetic analyses, COLD-PCR provides a general platform for the improvement of the sensitivity of essentially all technologies for DNAvariation detection including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR consists of a particular PCR thermal cycling protocol in which a denaturation step at a lower temperature is applied. A single-nucleotide mismatch anywhere along a dsDNA generates a small but predictable change in the melting temperature (Tm). Depending on the sequence context and position of the mismatch, Tm changes by 0.2–1.5 °C. For each DNA sequence there is a critical denaturation temperature (Tc), below which PCR efficiency drops abruptly. DNA amplicons differing by a single nucleotide reproducibly have different amplification efficiencies, strongly dependent on the DNA sequence, when PCR denaturation temperature is set to Tc. In COLD-PCR, an intermediate annealing temperature is used during PCR cycling to allow heteroduplex formation through cross-hybridization of mutant to wild-type alleles. Heteroduplexes melt at a lower temperature than homoduplexes and are selectively denatured and amplified at Tc, whereas homoduplexes remain double-stranded and do not amplify efficiently. COLD-PCR can replace conventional PCR as the first step for mutation detection technologies to improve sensitivity. Our aim was to investigate COLD-PCR as a new approach to be used as a pre-analytical enrichment of BRAFV600E variant present in formalin fixed paraffin embedded (FFPE) melanoma tissues. The main issue for the development of a COLD-PCR procedure for the enrichment of the BRAFV600E variant was the design of a suitable primer pair and the accurate setting of the Tc. Furthermore, we have been working in order to demonstrate the enrichment of the low-represented variants by COLD PCR in comparison with a conventional PCR procedure. The post-PCR detection methods were represented by sequencing and high resolution melting analysis. Confirmatory results were also obtained by pyrosequencing. 2. Materials and methods 2.1. Patients and cell lines Forty-five patients treated at the Department of Dermatological Sciences, University of Florence, were evaluated for BRAFV600E mutation in DNA extracted from FFPE tissues. The series included patients undergoing surgery for in situ melanoma (n = 5) and invasive melanoma (n = 40). Moreover, three FFPE samples of normal epidermis were used as negative controls. The median age of patients with in situ melanoma was 66 years (age range 39–76 years). There were 4 males and 1 female. Tumor site was the trunk in all patients. These cases were all in situ superficial spreading melanomas. The median age of patients with invasive malignant melanoma (n = 40) was 67 years (age range 25–86 years). Twenty-three patients (57.5%) were males, and 17 patients (42.5%) were females. The tumor site distribution was as follows: head and neck (n = 1, 2.5%); upper and lower extremities (n = 12, 30%); trunk (n = 21, 51.5%); acral regions (n = 4, 10%), and genital area (n = 2, 5%). Superficial spreading melanoma was the most common histologic type (n = 29, 72.5%), followed by nodular melanoma (n = 5, 12.5%), acral-lentiginous melanoma (n = 4, 10%). Nine cases (22.5%) had a

Clark's level of II, and 11 cases (27.5%) and 17 cases (42.5%) had levels III and IV, respectively. Nineteen (47.5%) patients had melanomas ≤1.0 mm in Breslow thickness, 8 (20%) had lesions 1.0–2.0 mm in thickness, 7 (17.5%) patients had melanomas more than 2.0 mm but less than 4.0 mm in thickness and 3 patients (7.5%) had melanomas N4.0 mm in thickness. Ulceration was demonstrated in 11 (27.5%) of the invasive melanomas. DNA from the melanoma cell line SK-MEL-28, homozygote for the BRAFV600E mutation, used as positive control, and from the wild type breast cancer cell line MCF7, used as negative control, were also included in each run. Reconstituted samples were generated serially diluting SK-MEL-28 and MCF-7 DNA to obtain samples at known percentages of mutated alleles (from 100% to 0.8%) to be used as standards. 2.2. DNA extraction DNA was extracted from FFPE tissues with the Qiamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) in a 20 μl elution volume. DNA quantity was evaluated by absolute real time PCR method based on the measurement of a single copy gene (APP; Amyloid Precursor Protein) as previously reported [15]. 2.3. Conventional PCR protocol for BRAF amplification Amplification of the BRAF gene, exon 15, was performed using 10 ng of DNA in a final volume of 20 μl using 1× QuantiTect Probe PCR Master Mix (Qiagen, Milan, Italy), 300 nM of each primer and 1.5 μM of Syto9 (Invitrogen, Milan, Italy). The primers amplifying a 200 bp fragment were common to both conventional and COLD-PCR reactions: forward primer 5′-ACAGAATTATAGAAATTAGATCTCTTACC-3′ and reverse primer 5′-GACAACTGTTCAAACTGATGG-3′. Conventional PCR amplification protocol was composed of an initial activating step at 95 °C for 15 min, a cycling step (40 cycles) performed as follows: 95 °C for 30 s, 62 °C for 30 s and 72 °C for 30 s and of a final extension at 72 °C for 15 min. 2.4. COLD-PCR reaction setting and operative protocol Once the primers that allowed efficient amplification of our template had been chosen, the identification of the Tc which allows the enrichment of the BRAFV600E allele, had to be determined. The COLD-PCR reaction was performed in a 20 μl final volume, using 10 ng DNA per reaction. Amplification mix was composed by: 1× QuantiTect Probe PCR Master Mix (Qiagen, Milan, Italy), 300 nM of each primer and 1.5 μM of Syto9 Dye (Invitrogen, Milan, Italy). The COLD-PCR cycling protocol was assessed by varying the Tc in a range close to the amplicon Tm, as evaluated on the Corbett 6000 instrument by HRMA. The Tm values of the wild type and mutated amplicons resulted as 79.68 and 79.35 °C respectively, showing a difference of 0.33 °C, in agreement with that expected for a TNA transversion. We varied the Tc of 0.5 °C starting from 77.5 to 80.5 °C. Following this process, we determined the critical denaturation temperature (79.5 °C) for full COLD-PCR procedure. Samples were then submitted to the proper COLD-PCR cycling according to the following protocol: 95 °C for 15 min; 10 cycles at 95 °C for 15 s, 62 °C for 30 s, 72 °C for 1 min; then 35 cycles at 95 °C for 15 s, 70 °C for 7 min, 79.5 °C for 3 s, 62 °C for 30 s and 72 °C for 1 min. 2.5. High resolution melting and sequencing analysis HRMA of both conventional and COLD-PCR products was performed on RotorGene 6000 (Corbett Research, Sydney, Australia) submitting samples to the following denaturation profile: 5 min at

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95 °C, 1 min at 40 °C and a melting profile from 74 °C to 85 °C using a ramping degree of 0.05. To confirm the results, direct sequencing was performed for all samples. After HRMA, samples were purified using the PCR Purification Kit (Qiagen, Milan, Italy) and submitted to the cycle sequencing reaction containing, 2 μl of BigDye Terminator Ready Reaction Mix (Applied Biosystems, Monza, Italy) and 0.16 μmol/l of the same primers used for HRMA in a final volume of 10 μl. After purification with the DyeEx 2.0 Spin Kit (Qiagen, Milan, Italy), samples were analyzed with the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Monza, Italy). 2.6. Pyrosequencing The amplification reaction preceding sample analysis by pyrosequencing was performed by a previously described protocol [16] except for: i) the final reaction volume (50 μl), ii) the amount of DNA used in each reaction (10 ng), and iii) the reverse primer biotin modification at the 5′ end of the sequence used in the PCR reaction. Sample analysis by pyrosequencing was done by the PyroMark Q96 ID instrument (Biotage, Uppsala, Sweden). After PCR amplification, electrophoresis on 2% agarose gel was performed to verify that amplification took place. Thirty microliters of the amplification product was immobilized on the PSQ Vacuum Prep Tool plate (Biotage, Uppsala, Sweden) tank to the biotin binding to streptavidin-coated Sepharose high-performance beads (GE Healthcare, Chalfont St. Giles, UK). The PCR products immobilized on the plate were processed by PSQ Sample Preparation kit (Biotage) in order to obtain single strand DNA templates which were subsequently incubated with 2 μM sequencing primer (5′-GGT GAT TTT GGT CTA GCT AC-3′) [16] in a PSQ96 plate at 80 °C for 2 min. The sequencing reaction by synthesis was performed with PyroGold kit reagents using PyroMark Q96 ID instrument (Biotage).

Fig. 1. HRM analysis of a conventional PCR (left panels) and a COLD-PCR (right panels) amplification of samples at known percentage of mutated allele (range 100%–3.1% BRAFV600E mutated allele). Results are illustrated as difference plots of mutated samples normalized against the wild-type DNA (upper graphs) and as melting profiles (lower graphs).

3. Results 3.1. Effect of COLD-PCR on downstream methods for BRAFV600E detection To evaluate the theoretical sensitivity of our COLD-PCR assay, serial dilutions of BRAFV600E mutated (SK-MEL-28 cell line) and wild type (MCF7 cell line) DNA have been used, to obtain reconstituted samples containing 50, 25, 12.5, 6.2, 3.1%, 1.6% and 0.8% of mutated alleles. These samples were submitted to conventional PCR and COLDPCR protocols, separately. The sensitivity was then evaluated by HRM analysis, sequencing and pyrosequencing. As already reported [17], after conventional PCR, 12.5% BRAFV600E mutated alleles were detectable in HRMA differential graphs (Fig. 1, left panel). The sensitivity was improved using COLD-PCR amplification, which allowed the detection of 3.1% BRAFV600E mutated alleles by HRMA (Fig. 1, right panel). By direct sequencing, 12.5% BRAFV600E was detectable after conventional PCR and 6.2% after COLD-PCR amplification (Fig. 2, upper panel). Similar results were obtained by pyrosequencing, the detection limit for BRAFV600E mutated allele resulting 6.2% after conventional PCR and 3.1% after COLD-amplification (Fig. 2, lower panel). 3.2. Melanoma samples: COLD-PCR versus conventional PCR (sensitivity on clinical specimens) The analysis of 45 melanoma samples by direct sequencing revealed that 17/45 patients (38%) showed BRAFV600E mutation. The remaining 28 samples were classified as wild type (Table 1). In a second phase, all samples were re-submitted to a complete analysis to detect BRAFV600E mutation by using COLD-PCR as the amplification step.

Fig. 2. Sensitivity evaluation using samples at known percentage of mutated allele (12.5%, 6.2% and 3.1% BRAFV600E mutated allele) after conventional PCR and COLD-PCR amplification. Upper panel: electropherograms of sequencing analysis after the two different amplification procedures. The red arrows indicate the presence of a signal due to the presence of BRAFV600E allele. Lower panel: pyrosequencing analysis of the same samples containing 12.5, 6.2 and 3.1% of BRAFV600E mutated allele after the two different amplification procedures.

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Table 1 Number of BRAFV600E variants detected by direct sequencing following a conventional PCR amplification or a COLD-PCR procedure. Direct sequencing

HRMA

Genotype

Conventional PCR

COLD PCR

Conventional PCR

COLD PCR

Wild type BRAFV600E

28 (62%) 17 (38%)

22 (49%) 23 (51%)

31 (69%) 14 (31%)

22 (49%) 23 (51%)

by HRMA: the conventional procedure gave the 35% (14/40) mutation frequency, while by COLD-HRMA it was 52.5% (21/40). No statistically significant association could be evidenced when analyzing BRAFV600E mutation results on the basis of the most common clinical pathological parameters (histologic type, Clark's level, Breslow thickness, and ulceration).

4. Discussion Adopting the COLD-PCR protocol, we confirmed the presence of BRAF genetic variant in all the samples that were initially classified as positive after PCR and sequencing, but in 6 samples apparently negative for BRAFV600E variant, we detected this variant by COLD-PCR and sequencing method (Table 1). HRMA performed after COLD-PCR produced results that overlap the sequencing analysis. In fact, by HRMA-COLD 23/45 samples (51%) showed melting curves indicative of BRAFV600E mutation (Table 1). The results of four representative samples turning positive after COLD-PCR amplification are illustrated in Fig. 3. 3.3. Melanoma sample analysis When sorting the results on the basis of the class lesion, the percent of mutated samples varied on the basis of the utilized technique. Among invasive melanomas (n = 40), conventional sequencing detected BRAFV600E mutation in 40% (16/40) of patients, while using COLD-PCR as the amplification step the mutation could be detected in 52.5% (21/40) of patients. The same phenomenon could be evidenced

The identification of BRAF mutations in a high percentage of malignant melanomas has led way to optimism that therapy with specific RAF kinase inhibitors might be beneficial to this disease, at least in advanced stages [18]. Since the response to the RAF kinase inhibitors is dependent upon the presence of BRAF-activating mutations in the tumor, the detection of this genetic variant appears to be of great importance, also in view of a personalized therapy [19]. Here we reported the results of different techniques used to identify the presence of somatic mutations in tumor samples, working on formalin fixed-paraffin embedded tissues. The sequencing method which implies the preliminary amplification by conventional PCR will be considered the reference method, and all the other approaches will be compared to it. It is well known that the sensitivity of sequencing is not suited for the research of somatic mutations, unless they are present in a significant proportion of cells in the tissue (about 20%, see Ref. [20]). Pyrosequencing represents a sequencing strategy which is claimed to be more sensitive, but above all especially designed for quantitative evaluation of the mutated allele.

Fig. 3. HRMA melting profiles and electropherograms of sequencing analysis relative to four samples producing different results depending on the type of amplification procedure (conventional- and COLD-PCR).

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Recently, COLD-PCR was proposed as an amplification method able to increase the sensitivity of the detection of rare mutated allele by its selective enrichment by means of peculiar thermal cycling conditions [14,21,22]. In this manuscript, we report the comparison of the results obtained by both direct sequencing and HRMA following conventional and COLD-PCR amplification. Moreover, a pyrosequencing procedure was performed in order to complete the correlation study. It has been demonstrated that the use of COLD-PCR increases the theoretical sensitivity of different post-PCR methods, varying from sequencing to pyrosequencing and HRMA. As expected, the increase in sensitivity, at least in standard samples from cell lines, seems more evident for HRMA, due to its higher discriminating capacity, as previously described [17]. We clearly demonstrated that more than 20% of patients, initially classified as negative for BRAFV600E, turned out to be positive after COLD-PCR. The increase of COLD-PCR sensitivity is even more significant taking into account that this type of mutation (TNA) is the most difficult to identify by COLD-PCR, due to the limited thermal shift. COLD-PCR amplification procedure was confirmed as a suitable method for the enrichment of mutated alleles especially when they are present at very low concentrations, particularly for samples in which the percentage of tumor cells can be variably affected by contamination of non cancer cells. Acknowledgements The study was financed by Istituto Toscano Tumori (I.T.T.). References [1] Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes. Nat Rev Cancer 2004;4:177–83. [2] Greenman C, Stephens P, Smith R, et al. Patterns of somatic mutation in human cancer genomes. Nature 2007;446:153–8. [3] Shinozaki M, O'Day SJ, Kitago M, et al. Utility of circulating B-RAF DNA mutation in serum for monitoring melanoma patients receiving biochemotherapy. Clin Cancer Res 2007;13:2068–74. [4] Chang DZ, Panageas KS, Osman I, Polsky D, Busam K, Chapman PB. Clinical significance of BRAF mutations in metastatic melanoma. J Transl Med 2004;2:46.

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