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Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients
Clinica Chimica Acta 411 (2010) 1319–1324
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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
Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients Pamela Pinzani a,⁎,1, Francesca Salvianti a,1, Roberta Cascella 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 Human Pathology, University of Florence, Viale G.B. Morgagni 85, 50139 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 13 January 2010 Received in revised form 13 May 2010 Accepted 13 May 2010 Available online 1 June 2010 Keywords: Cell-free DNA Allele specific real-time PCR BRAFV600E mutation Melanoma Molecular marker Noninvasive diagnosis
a b s t r a c t Background: BRAF is the most frequently mutated oncogene in melanoma with BRAFV600E mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods: We propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAFV600E concentration in patients affected by cutaneous melanoma (n = 55) and non-melanoma skin cancers (n = 13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma. Results: A significant difference between the control group and invasive melanomas (p b 0.01) was evidenced in BRAFV600E concentration, either as relative percentage or absolute values. ROC curve indicated that BRAFV600E absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance. Conclusions: The allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAFV600E mutated DNA in plasma. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Somatic mutations of BRAF oncogene are the most frequent molecular variants in cutaneous melanoma [1–3]. BRAF mutants lead to a constitutive activation of the RAF/MEK pathway, a membrane-to nucleus signaling system that controls proliferation, differentiation and apoptosis in mammalian cells [1,4]. The activation of this pathway has been associated with proliferation of melanocytes and melanoma cells [5,6]. BRAF mutations are likely to be an early event in melanoma. Previous studies suggested that BRAF-mutated melanomas are dependent upon BRAF for proliferation and survival [7,8]. So far, although BRAF mutations are detectable in about 55% of metastatic melanomas [9–11], no significant correlation with disease outcome was demonstrated [12]. In addition, BRAF mutations are also detectable in benign melanocytic nevi, suggesting a limited role in tumorigenesis, with additional mutations required for melanoma
⁎ Corresponding author. Tel.: + 39 055 4271441; fax: + 39 055 4271371. E-mail address: [email protected]fi.it (P. Pinzani). 1 These authors contributed equally to this paper. 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.05.024
development [13]. In human melanoma, BRAF mutations are predominantly clustered in exon 15. The T1799A transversion (BRAFV600E) accounts for 92% of all BRAF variants in melanoma [14]. This mutation significantly increases the kinase activity [14–16] leading to continuous transcription-mediated proliferation and neoplastic growth [12]. Inhibition of the BRAF pathway is currently considered a promising target for melanoma treatment and selective kinase inhibitors targeting the BRAF–MEK–ERK pathway are being developed. Preclinical studies revealed that tumors bearing BRAFV600E are more sensitive than wild type to these inhibiting agents [17]. The genotyping of melanoma to select patients will be critical in the future development of agents targeting this pathway. Thus, analysis of BRAF mutations in tissues will be needed for patient stratification. Alternatively, blood can be used to detect BRAF variants, since free circulating DNA in serum and plasma has been shown to contain DNA carrying the same alterations of corresponding cancer [18,19]. In this study, we developed a novel real-time PCR assay based on the use of a locked nucleic acid (LNA) probe and an allele specific primer. This allele specific real-time PCR method was used to detect plasma-circulating BRAFV600E DNA in a series of cutaneous melanoma patients and controls. Results were evaluated both as percentages of total DNA and as absolute concentration (pg/ml). The purpose of the
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study was to determine whether this method allows the accurate and sensitive detection of BRAFV600E alleles in plasma DNA of melanoma patients and to evaluate the clinical sensitivity and specificity of the test.
used to obtain samples with known percentage of mutated alleles, to be used as standards.
2. Material and methods
Two 5 ml-aliquots of peripheral blood were collected in EDTA tubes, transported within one hour to the laboratory and centrifuged twice at 4 °C for 10 min (1600 rcf and 14000 rcf). Plasma aliquots were stored at − 80 °C before use. DNA was extracted from 500 μl of plasma, using the QIAamp DSP Virus Kit (Qiagen, Italy) and RNAse digestion to prevent RNA interference during assay reaction. DNA was extracted from cell lines with QIAamp DNA Blood Mini kit (Qiagen). DNA from formalin-fixed paraffin-embedded tissues was extracted using the FFPE Tissue kit (Qiagen).
2.1. Patients and cell lines Fifty-five consecutive patients treated at the Department of Dermatological Sciences of the University of Florence, were evaluated for BRAFV600E mutation in plasma DNA. The series included patients undergone surgery for in situ melanoma (3 females and 6 males; age range: 39–76 years, median 60 years; n = 9, location: 1 limb 1 acral and 7 chest) and invasive melanoma (21 females and 25 males; age range: 25–88 years, median 66 years; n = 46). For additional baseline and clinical characteristics of invasive melanoma see Table 1. In addition, subjects undergoing surgical excision of non-melanoma skin cancer (superficial basal cell carcinomas, n = 10; squamous cell carcinomas n = 3; location: 7 face and 6 chest; 8 females and 5 males; age range: 35–85 years, median 64 years) were studied. As the control population we enrolled 18 healthy subjects (9 females and 9 males; age range: 29–81 years, median 59.5 years) who voluntarily donated their blood to be submitted to BRAFV600E plasma measurement. Blood samples were collected during the dermatologic examination and before surgery. For 56/68 subjects (12 non-melanoma skin cancer, 7 in situ melanomas and 37 invasive melanomas), paired formalin-fixed paraffin-embedded (FFPE) tissues were also analyzed. Only FFPE tumor tissues containing at least 70% of tumor cells were included. The research protocol was approved by the review board of the Department of Physiopathology of the University of Florence and all the patients signed an informed consent. Control DNA from the melanoma cell line SKMEL28, homozygote for BRAFV600E mutation, and from the wild type cell line MCF7 was included in each run. Serial dilutions of SKMEL28 and MCF7 DNA were Table 1 Differences in BRAFV600E mutated DNA quantity (both as % of total DNA and as absolute concentration) in patients affected by invasive melanoma divided on the basis of location, Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity. BRAFV600E mutated DNA (%)
BRAFV600E mutated DNA (pg/ml plasma)
Mean
Mean
Parameter
Cases (n)
Total Location Head and neck Limbs Chest Acral Genital Thickness ≤1 mm 1.01–2.0 mm 2.01–4.0 mm N4 mm Clark level II III IV Ulceration Absent Present Sentinel lymph node Positive Negative Not done
46
4.2 ± 1.3
4 15 22 3 2
0.7 ± 0.4 5.9 ± 3.8 4.7 ± 1.6 1.6 ± 1.6 3.1 ± 0.7
0.88⁎
77 ± 49 481 ± 240 545 ± 216 202 ± 202 353 ± 33
0.87⁎
23 13 6 4
4.8 ± 2.6 5.8 ± 2.8 4.2 ± 2.8 1.8 ± 0.9
0.94⁎
388 ± 161 670 ± 362 688 ± 479 91 ± 77
0.68⁎
9 14 23
3.4 ± 1.4 8.8 ± 4.8 2.9 ± 0.9
0.24⁎
263 ± 75 815 ± 387 375 ± 159
0.29⁎
33 12
5.3 ± 2.0 3.3 ± 1.7
0.59⁎⁎
498 ± 163 433 ± 294
0.85⁎⁎
3 17 26
0.8 ± 0.8 7.2 ± 3.4 –
0.45⁎⁎
144 ± 144 662 ± 279 –
0.46⁎⁎
p value
p value
2.2. DNA extraction
2.3. Real-time PCR to quantify total free circulating DNA with APP single copy gene Absolute quantification of the single copy gene APP (amyloid precursor protein, chr.4q11–q13) was performed in plasma samples to accurately measure the total amount of free circulating DNA per ml plasma, using primers and probe as previously reported [20]. This assay provides results for the normalization of the DNA volume to be used in the real-time PCR method for BRAFV600E. Quantification of DNA concentration was obtained by interpolation on an external reference curve ranging from 10 to 105 pg/tube of genomic DNA extracted from a blood pool of healthy donors and measured spectrophotometrically (Nanodrop ND1000, Nanodrop, USA). Analysis of 7 different runs provided the following values (mean ± S.E.): slope = −3.45 ± 0.05 (mean efficiency = 0.95) and Y-intercept = 40.2 ± 0.93 with coefficient of correlation always higher than 0.99. Results were expressed as ng circulating DNA/ml of plasma. Real-time PCR was run in the 7900HT Fast (Applied Biosystems), by denaturation at 95 °C for 10 min and 45 cycles of PCR (95 °C for 15 s, 60 °C for 60 s). 2.4. Allele specific real-time PCR to detect BRAFV600E mutation For the real-time detection of BRAFV600E we designed a mutationspecific forward primer (5′-AAA ATA GGT GAT TTT GGT CTA GCT ACA GA-3′), while reverse primer (5′-GAC AAC TGT TCA AAC TGA TGG-3′) was common to wild type and mutated sequences. A dual-labelled LNA probe (5′-FAM-T[+C]GAGA[+T]TT[+C][+T][+C]TG[+T]AG[+C]TBHQ1–3′, Sigma, USA) was designed on the complementary strand to that of the BRAFV600E allele specific primer (Fig. 1). For the measurement
430 ± 124
⁎ p values have been calculated by ANOVA test. ⁎⁎ p values have been calculated by Student t-test.
Fig. 1. Upper panel. Allele specific Taqman-based real-time PCR assay design and sequences of primers and probe for the quantifications of BRAFV600E mutated DNA. Lower panel. Amplification plots obtained for wild type (WT) and samples containing known percentage (100, 50, 20, 10 and 1%) of BRAFV600E mutated alleles (MUT).
P. Pinzani et al. / Clinica Chimica Acta 411 (2010) 1319–1324
of BRAFV600E allele concentration, we used 0.5 ng/reaction DNA from plasma, tissues and cell lines (as determined by qPCR for APP), in a 20 μl total PCR volume. Each sample was assayed in duplicate. Real-time PCR was performed by denaturation at 95 °C for 10 min and 50 cycles of PCR (95 °C for 15 s, 64 °C for 60 s) using Quantitect® Probe PCR Master Mix (Qiagen). The standard curve for BRAFV600E consisted of six dilutions (100%, 50%, 20%, 10%, 1% and 0% mutated alleles) obtained by mixing DNA from mutant SKMEL28 and wild type MCF7 with a dynamic range of about 10 Cq (quantification cycle) [21] (Fig. 1). Linear regression analysis (n = 7) of the Cq versus % mutated DNA provided the mean equation y = −3.22 ± 0.10x − 36.80 ± 0.87 (mean efficiency= 1.04), with a coefficient of correlation constantly higher than 0.99. Sample results were expressed as % of mutated DNA. From this first result, we also derived the absolute concentration of BRAFV600E in terms of mutated DNA (ng/ml plasma) using the following formula:
BRAF
V600E
mutated DNAðpg = ml plasmaÞ
= ðð%BRAF
V600E
× total DNAÞ = 100Þ*1000:
In order to evaluate the sensitivity of the standard curve of our real-time assay, we performed 5 replicates of the wt sample in the same assay run. Mean Cq − 2 standard deviations was interpolated on the standard curve to calculate the theoric detection limit, resulting to 0.3% BRAFV600E mutated DNA. All available tissue samples were submitted to the newly developed method as well as direct sequencing as previously reported [22]. For FFPE tumors, BRAFV600E positivity was assessed by values Nmean + 2 SD of results obtained from normal skin samples (n = 5). 2.5. Statistical analysis Statistical analysis was carried out using the SPSS software package 15.0 (SPSS, Chicago, USA). Statistical differences between qualitative results were assessed by the Fisher's exact test, while quantitative data were evaluated by either ANOVA or Student t-test. p values lower than 0.05 were considered statistically significant. Clinical sensitivity and specificity were assessed by receiver-operating characteristics (ROC) curve analysis. 3. Results 3.1. Total cell-free DNA concentration in plasma When submitted to real-time PCR for the APP quantification in plasma DNA, all samples from patients and controls showed positive amplification plots. In control subjects plasma DNA level (mean± SE: 5.5 ± 0.7 ng/ml) was significantly lower than in in situ (16.0 ± 3.6 ng/ml) (p b 0.05) and in invasive melanomas (14.5± 1.4 ng/ml) (p b 0.001) without any differences between the two latter groups. Cell-free DNA concentrations in non-melanoma skin cancer group was (7.9 ± 1.5 ng/ml) significantly lower than that found in both the in situ and invasive melanomas (p b 0.05) (Fig. 2 upper panel left). In order to provide a better understanding of the variability of our results we reported the histograms of distribution of total DNA concentration per class of subjects (Fig. 2 lower panels A–D, left). We did not detect any difference in terms of plasma free DNA concentration when patients were classified on the basis of melanoma location, Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity (Table 1).
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3.2. BRAFV600E percentage and concentrations in plasma Plasma percentage of BRAFV600E DNA was 0.8 ± 0.2% in control subjects, 0.3 ± 0.2% in non-melanoma skin cancer, 2.6 ± 1.3% in patients with in situ melanoma and 4.2 ± 1.3% in invasive melanoma patients (Fig. 2 upper panel centre), with a significant difference between the control group and invasive melanomas (p b 0.05). The differences became even more evident when BRAFV600E results were expressed as pg/ml of plasma (p b 0.005); BRAFV600E DNA concentration was 44.6 ± 12.9 pg/ml in control subjects, 11.7 ± 7.9 pg/ml in non-melanoma skin cancer, 267.9 ± 110.6 pg/ml in patients with in situ melanoma and 430.3 ± 123.9 pg/ml in invasive melanoma patients. A significant difference was found between the nonmelanoma skin cancer group and both the in situ (p b 0.05) and invasive melanomas (p b 0.005) (Fig. 2 upper panel right). In the lower panels (Fig. 2, A–D centre and right), we report the histograms representing the distribution of plasma BRAFV600E DNA concentrations in our case study. Quantification of mutated BRAFV600E did not reveal differences on the basis of melanoma location, Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity (Table 1). 3.3. Clinical penetrance of the measurement of plasma BRAFV600E ROC curve analysis was performed to define the cut-off values of total DNA levels and BRAFV600E mutated DNA which better designed maximal clinical sensitivity and specificity (Fig. 3). The calculated cutoff values with the corresponding sensitivity and specificity values are reported in Table 2. The separated evaluation of the quantity of plasma free DNA and the percentage of mutated BRAFV600E showed comparable clinical sensitivity and specificity. However, from the combination of the two parameters and the consequent evaluation of the effective concentration of BRAFV600E we obtained the maximal sensitivity (83%) maintaining an elevated specificity (97%). 3.4. BRAFV600E in paired melanoma tissues and plasma samples For 56/68 subjects, FFPE tissues were analyzed for BRAFV600E mutation by the newly developed real-time method and confirmed by direct sequencing (as the reference method). This comparison was conducted on tissue specimens and a full concordance between the two methods was found for all the wild type samples corresponding to realtime quantitative values lower than the cut-off as well as samples with a percentage of BRAFV600E variant allele higher than 10%. Discordant values were found in the interval between the cut-off value and 10% due to the different sensitivity characteristics of the two techniques. Comparison of plasma and tissue results in the same subject was obtained by evaluating results expressed as percentage of total mutated alleles on the basis of the above reported cut-off values. A statistically significant correlation was found when analyzing the concordance between plasma and tissue samples by Fisher's exact test (p b 0.001) (Table 3). Overall, 45 subjects obtained concordant results on tissue as well as on plasma, 24 resulting wild type and 21 showing the mutation in both samples. Eight mutated tissues did not find correspondence in plasma DNA, while 3 subjects showed the BRAFV600E mutation only in plasma. 4. Discussion The current study describes the development of a minimally invasive method to quantify rare BRAFV600E DNA sequences into the circulation. In particular, a real-time PCR method was developed by using a mutation-specific forward primer and a reverse primer common to both wild type and mutated BRAF sequences. A dual-labelled LNA probe was designed on the complementary strand to that recognized and
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Fig. 2. Quantitative results of total cell-free DNA (ng/ml of plasma) (upper panel left), BRAFV600E mutated DNA expressed as percent of total DNA (%) (upper panel centre) as well as absolute quantity (pg/ml of plasma) (upper panel right). Columns represent mean ± standard error of control group (A) (n = 18), non-melanoma skin cancer (B) (n = 13), in situ melanomas (C) (n = 9) and invasive melanomas (D) (n = 46). Histograms of distributions of values of total cell-free DNA (lower panels A–D left), BRAFV600E mutated plasma DNA (% and pg/ml of plasma) per class of subjects ((lower panels A–D, centre and right respectively).
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Table 2 Cut-off values for total cell-free DNA, BRAFV600E mutated DNA (%) and BRAFV600E mutated DNA (pg/ml plasma) as resulting from ROC curves. Parameter Total cell-free DNA (ng/ml plasma) BRAFV600E mutated DNA (%) BRAFV600E mutated DNA (pg/ml plasma)
Cutt-off
Sensitivity (%)
Specificity (%)
7.0 1.9 100
79 76 97
75 75 83
Table 3 Comparison of BRAFV600E detection in plasma and corresponding formalin-fixed and paraffin-embedded (FFPE) tissue samples (p b 0.001 Fisher's exact test). FFPE tissue Negative Positive Total
Fig. 3. Receiver-operating characteristics (ROC) curves of total cell-free DNA (ng/ml of plasma), BRAFV600E mutated DNA expressed as percent of total DNA (%) as well as absolute quantity (pg/ml of plasma). The area under the curve for each parameter is reported in the table.
hybridized by the BRAFV600E allele specific primer. The innovative design of the assay achieves a dynamic range of about 10 cycles of difference between the 100% mutated and wild type samples, with an assay sensitivity up to 0.3% of mutated alleles. The method was used to detect BRAFV600E in DNA from cell-free plasma compartment of melanoma patients and healthy controls. Neither the enrichment of epithelial cells nor whole genome amplification procedures were used, as reported for example by Oldenburg et al. [23]. PCR amplification was possible in as little as 0.5 ng of plasma cell-free DNA. In the above cited report [23] a LNA probe was used as well, but with a two-step approach, blocking the wild type DNA to prevent its amplification, and using a specific primer for the mutant target in a second PCR reaction. On this basis, our method appears more direct and faster, consisting only in a single real-time PCR. Accurate quantitative measurement of plasma total DNA concentration was preliminarily performed by real-time PCR method since previous studies indicated a relationship between plasma free DNA concentration and cancer presence [24]. The measurement of total plasma DNA concentration actually evidenced a significant difference between control group and in situ (p b 0.001) or invasive melanomas (p b 0.001). In particular, we demonstrated that, as a mean, DNA concentrations are about two fold higher in melanoma patients. These results are concordant with many research on different cancer types (such as prostate adenocarcinoma, lung cancer, ovarian cancer, colorectal carcinoma, pancreatic adenocarcinoma, melanoma, breast cancer, head and neck squamous cell carcinoma), which have been extensively revised by Fleischaker and Smith [24]. The two authors report that several studies observed no correlation between plasma DNA concentration and tumor size, stage or location, similarly to what we observed in our work. Moreover a recent review [25], besides confirming higher DNA yields for cancer patients in comparison to control subjects, underlined the need for research on the origin, function and significance of these nucleic acid molecules as imperative in order to clarify the significance of changes in their amount and
Plasma
Total
Negative
Positive
24 8 32
3 21 24
27 29 56
properties before circulating DNA can be used with confidence as a biomarker. The difference between melanomas and controls increased when BRAFV600E percentage was calculated (p = 0.01) and even more when we combined the two previous indexes to obtain the effective concentration of BRAFV600E in terms of pg mutated alleles/ml of plasma (p = 0.002). These results were confirmed taking into account clinical sensitivity and specificity of the analyzed parameters: in fact the combination of BRAFV600E percentage and DNA concentration in plasma (resulting in BRAFV600E absolute concentration) turned out to be the most sensitive biomarker to discriminate between melanoma patients and healthy subjects. We did not detect any statistical difference in terms of plasma DNA concentration and BRAFV600E mutated DNA levels when melanoma patients were stratified on the basis of Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity. From the analytical point of view, the assay resulted highly sensitive and accurate in evidencing either heterogeneity of tumor tissue (as regards to BRAFV600E mutated cells) or the absolute quantity of mutated allele present in plasma. The allele specific Taqman-based real-time PCR assay shows the ability to overcome problems related to DNA quality often encountered when dealing with FFPE- or plasma-derived specimens. Result validation was obtained by direct sequencing with full concordance of the results in the ranges 0–2% and N10%. Interestingly, comparison of the results obtained in plasma with those found in corresponding FFPE tissues indicated an 80% overall concordance. Other authors reported similar discordance probably due to the possibility of sampling of non-mutation-bearing portions of the tumor or to factors affecting the release of tumor DNA in plasma (including tumor size, vascularisation, anatomic site, biological characteristics, rate of apoptosis and necrosis, and renal excretion) [18,26]. Previous works reporting data from plasma detection of BRAFV600E mutation were based on conventional PCR [27], mutant allele specific amplification (MASA) plus denaturing high performance liquid chromatography (DHPLC) [28], and allele specific PCR assays [18] but did not show quantitative results. Quantitative information in our hands better evidenced differences between the control group and melanoma patients, representing a useful biomarker for clinical diagnosis. In conclusion, the allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAFV600E mutated DNA in plasma.
Acknowledgements This work has received financial support from the Istituto Toscano Tumori to C.O. and MIUR-PRIN 2008 to D.M.
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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
Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients Pamela Pinzani a,⁎,1, Francesca Salvianti a,1, Roberta Cascella 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 Human Pathology, University of Florence, Viale G.B. Morgagni 85, 50139 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 13 January 2010 Received in revised form 13 May 2010 Accepted 13 May 2010 Available online 1 June 2010 Keywords: Cell-free DNA Allele specific real-time PCR BRAFV600E mutation Melanoma Molecular marker Noninvasive diagnosis
a b s t r a c t Background: BRAF is the most frequently mutated oncogene in melanoma with BRAFV600E mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods: We propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAFV600E concentration in patients affected by cutaneous melanoma (n = 55) and non-melanoma skin cancers (n = 13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma. Results: A significant difference between the control group and invasive melanomas (p b 0.01) was evidenced in BRAFV600E concentration, either as relative percentage or absolute values. ROC curve indicated that BRAFV600E absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance. Conclusions: The allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAFV600E mutated DNA in plasma. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Somatic mutations of BRAF oncogene are the most frequent molecular variants in cutaneous melanoma [1–3]. BRAF mutants lead to a constitutive activation of the RAF/MEK pathway, a membrane-to nucleus signaling system that controls proliferation, differentiation and apoptosis in mammalian cells [1,4]. The activation of this pathway has been associated with proliferation of melanocytes and melanoma cells [5,6]. BRAF mutations are likely to be an early event in melanoma. Previous studies suggested that BRAF-mutated melanomas are dependent upon BRAF for proliferation and survival [7,8]. So far, although BRAF mutations are detectable in about 55% of metastatic melanomas [9–11], no significant correlation with disease outcome was demonstrated [12]. In addition, BRAF mutations are also detectable in benign melanocytic nevi, suggesting a limited role in tumorigenesis, with additional mutations required for melanoma
⁎ Corresponding author. Tel.: + 39 055 4271441; fax: + 39 055 4271371. E-mail address: [email protected]fi.it (P. Pinzani). 1 These authors contributed equally to this paper. 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.05.024
development [13]. In human melanoma, BRAF mutations are predominantly clustered in exon 15. The T1799A transversion (BRAFV600E) accounts for 92% of all BRAF variants in melanoma [14]. This mutation significantly increases the kinase activity [14–16] leading to continuous transcription-mediated proliferation and neoplastic growth [12]. Inhibition of the BRAF pathway is currently considered a promising target for melanoma treatment and selective kinase inhibitors targeting the BRAF–MEK–ERK pathway are being developed. Preclinical studies revealed that tumors bearing BRAFV600E are more sensitive than wild type to these inhibiting agents [17]. The genotyping of melanoma to select patients will be critical in the future development of agents targeting this pathway. Thus, analysis of BRAF mutations in tissues will be needed for patient stratification. Alternatively, blood can be used to detect BRAF variants, since free circulating DNA in serum and plasma has been shown to contain DNA carrying the same alterations of corresponding cancer [18,19]. In this study, we developed a novel real-time PCR assay based on the use of a locked nucleic acid (LNA) probe and an allele specific primer. This allele specific real-time PCR method was used to detect plasma-circulating BRAFV600E DNA in a series of cutaneous melanoma patients and controls. Results were evaluated both as percentages of total DNA and as absolute concentration (pg/ml). The purpose of the
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study was to determine whether this method allows the accurate and sensitive detection of BRAFV600E alleles in plasma DNA of melanoma patients and to evaluate the clinical sensitivity and specificity of the test.
used to obtain samples with known percentage of mutated alleles, to be used as standards.
2. Material and methods
Two 5 ml-aliquots of peripheral blood were collected in EDTA tubes, transported within one hour to the laboratory and centrifuged twice at 4 °C for 10 min (1600 rcf and 14000 rcf). Plasma aliquots were stored at − 80 °C before use. DNA was extracted from 500 μl of plasma, using the QIAamp DSP Virus Kit (Qiagen, Italy) and RNAse digestion to prevent RNA interference during assay reaction. DNA was extracted from cell lines with QIAamp DNA Blood Mini kit (Qiagen). DNA from formalin-fixed paraffin-embedded tissues was extracted using the FFPE Tissue kit (Qiagen).
2.1. Patients and cell lines Fifty-five consecutive patients treated at the Department of Dermatological Sciences of the University of Florence, were evaluated for BRAFV600E mutation in plasma DNA. The series included patients undergone surgery for in situ melanoma (3 females and 6 males; age range: 39–76 years, median 60 years; n = 9, location: 1 limb 1 acral and 7 chest) and invasive melanoma (21 females and 25 males; age range: 25–88 years, median 66 years; n = 46). For additional baseline and clinical characteristics of invasive melanoma see Table 1. In addition, subjects undergoing surgical excision of non-melanoma skin cancer (superficial basal cell carcinomas, n = 10; squamous cell carcinomas n = 3; location: 7 face and 6 chest; 8 females and 5 males; age range: 35–85 years, median 64 years) were studied. As the control population we enrolled 18 healthy subjects (9 females and 9 males; age range: 29–81 years, median 59.5 years) who voluntarily donated their blood to be submitted to BRAFV600E plasma measurement. Blood samples were collected during the dermatologic examination and before surgery. For 56/68 subjects (12 non-melanoma skin cancer, 7 in situ melanomas and 37 invasive melanomas), paired formalin-fixed paraffin-embedded (FFPE) tissues were also analyzed. Only FFPE tumor tissues containing at least 70% of tumor cells were included. The research protocol was approved by the review board of the Department of Physiopathology of the University of Florence and all the patients signed an informed consent. Control DNA from the melanoma cell line SKMEL28, homozygote for BRAFV600E mutation, and from the wild type cell line MCF7 was included in each run. Serial dilutions of SKMEL28 and MCF7 DNA were Table 1 Differences in BRAFV600E mutated DNA quantity (both as % of total DNA and as absolute concentration) in patients affected by invasive melanoma divided on the basis of location, Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity. BRAFV600E mutated DNA (%)
BRAFV600E mutated DNA (pg/ml plasma)
Mean
Mean
Parameter
Cases (n)
Total Location Head and neck Limbs Chest Acral Genital Thickness ≤1 mm 1.01–2.0 mm 2.01–4.0 mm N4 mm Clark level II III IV Ulceration Absent Present Sentinel lymph node Positive Negative Not done
46
4.2 ± 1.3
4 15 22 3 2
0.7 ± 0.4 5.9 ± 3.8 4.7 ± 1.6 1.6 ± 1.6 3.1 ± 0.7
0.88⁎
77 ± 49 481 ± 240 545 ± 216 202 ± 202 353 ± 33
0.87⁎
23 13 6 4
4.8 ± 2.6 5.8 ± 2.8 4.2 ± 2.8 1.8 ± 0.9
0.94⁎
388 ± 161 670 ± 362 688 ± 479 91 ± 77
0.68⁎
9 14 23
3.4 ± 1.4 8.8 ± 4.8 2.9 ± 0.9
0.24⁎
263 ± 75 815 ± 387 375 ± 159
0.29⁎
33 12
5.3 ± 2.0 3.3 ± 1.7
0.59⁎⁎
498 ± 163 433 ± 294
0.85⁎⁎
3 17 26
0.8 ± 0.8 7.2 ± 3.4 –
0.45⁎⁎
144 ± 144 662 ± 279 –
0.46⁎⁎
p value
p value
2.2. DNA extraction
2.3. Real-time PCR to quantify total free circulating DNA with APP single copy gene Absolute quantification of the single copy gene APP (amyloid precursor protein, chr.4q11–q13) was performed in plasma samples to accurately measure the total amount of free circulating DNA per ml plasma, using primers and probe as previously reported [20]. This assay provides results for the normalization of the DNA volume to be used in the real-time PCR method for BRAFV600E. Quantification of DNA concentration was obtained by interpolation on an external reference curve ranging from 10 to 105 pg/tube of genomic DNA extracted from a blood pool of healthy donors and measured spectrophotometrically (Nanodrop ND1000, Nanodrop, USA). Analysis of 7 different runs provided the following values (mean ± S.E.): slope = −3.45 ± 0.05 (mean efficiency = 0.95) and Y-intercept = 40.2 ± 0.93 with coefficient of correlation always higher than 0.99. Results were expressed as ng circulating DNA/ml of plasma. Real-time PCR was run in the 7900HT Fast (Applied Biosystems), by denaturation at 95 °C for 10 min and 45 cycles of PCR (95 °C for 15 s, 60 °C for 60 s). 2.4. Allele specific real-time PCR to detect BRAFV600E mutation For the real-time detection of BRAFV600E we designed a mutationspecific forward primer (5′-AAA ATA GGT GAT TTT GGT CTA GCT ACA GA-3′), while reverse primer (5′-GAC AAC TGT TCA AAC TGA TGG-3′) was common to wild type and mutated sequences. A dual-labelled LNA probe (5′-FAM-T[+C]GAGA[+T]TT[+C][+T][+C]TG[+T]AG[+C]TBHQ1–3′, Sigma, USA) was designed on the complementary strand to that of the BRAFV600E allele specific primer (Fig. 1). For the measurement
430 ± 124
⁎ p values have been calculated by ANOVA test. ⁎⁎ p values have been calculated by Student t-test.
Fig. 1. Upper panel. Allele specific Taqman-based real-time PCR assay design and sequences of primers and probe for the quantifications of BRAFV600E mutated DNA. Lower panel. Amplification plots obtained for wild type (WT) and samples containing known percentage (100, 50, 20, 10 and 1%) of BRAFV600E mutated alleles (MUT).
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of BRAFV600E allele concentration, we used 0.5 ng/reaction DNA from plasma, tissues and cell lines (as determined by qPCR for APP), in a 20 μl total PCR volume. Each sample was assayed in duplicate. Real-time PCR was performed by denaturation at 95 °C for 10 min and 50 cycles of PCR (95 °C for 15 s, 64 °C for 60 s) using Quantitect® Probe PCR Master Mix (Qiagen). The standard curve for BRAFV600E consisted of six dilutions (100%, 50%, 20%, 10%, 1% and 0% mutated alleles) obtained by mixing DNA from mutant SKMEL28 and wild type MCF7 with a dynamic range of about 10 Cq (quantification cycle) [21] (Fig. 1). Linear regression analysis (n = 7) of the Cq versus % mutated DNA provided the mean equation y = −3.22 ± 0.10x − 36.80 ± 0.87 (mean efficiency= 1.04), with a coefficient of correlation constantly higher than 0.99. Sample results were expressed as % of mutated DNA. From this first result, we also derived the absolute concentration of BRAFV600E in terms of mutated DNA (ng/ml plasma) using the following formula:
BRAF
V600E
mutated DNAðpg = ml plasmaÞ
= ðð%BRAF
V600E
× total DNAÞ = 100Þ*1000:
In order to evaluate the sensitivity of the standard curve of our real-time assay, we performed 5 replicates of the wt sample in the same assay run. Mean Cq − 2 standard deviations was interpolated on the standard curve to calculate the theoric detection limit, resulting to 0.3% BRAFV600E mutated DNA. All available tissue samples were submitted to the newly developed method as well as direct sequencing as previously reported [22]. For FFPE tumors, BRAFV600E positivity was assessed by values Nmean + 2 SD of results obtained from normal skin samples (n = 5). 2.5. Statistical analysis Statistical analysis was carried out using the SPSS software package 15.0 (SPSS, Chicago, USA). Statistical differences between qualitative results were assessed by the Fisher's exact test, while quantitative data were evaluated by either ANOVA or Student t-test. p values lower than 0.05 were considered statistically significant. Clinical sensitivity and specificity were assessed by receiver-operating characteristics (ROC) curve analysis. 3. Results 3.1. Total cell-free DNA concentration in plasma When submitted to real-time PCR for the APP quantification in plasma DNA, all samples from patients and controls showed positive amplification plots. In control subjects plasma DNA level (mean± SE: 5.5 ± 0.7 ng/ml) was significantly lower than in in situ (16.0 ± 3.6 ng/ml) (p b 0.05) and in invasive melanomas (14.5± 1.4 ng/ml) (p b 0.001) without any differences between the two latter groups. Cell-free DNA concentrations in non-melanoma skin cancer group was (7.9 ± 1.5 ng/ml) significantly lower than that found in both the in situ and invasive melanomas (p b 0.05) (Fig. 2 upper panel left). In order to provide a better understanding of the variability of our results we reported the histograms of distribution of total DNA concentration per class of subjects (Fig. 2 lower panels A–D, left). We did not detect any difference in terms of plasma free DNA concentration when patients were classified on the basis of melanoma location, Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity (Table 1).
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3.2. BRAFV600E percentage and concentrations in plasma Plasma percentage of BRAFV600E DNA was 0.8 ± 0.2% in control subjects, 0.3 ± 0.2% in non-melanoma skin cancer, 2.6 ± 1.3% in patients with in situ melanoma and 4.2 ± 1.3% in invasive melanoma patients (Fig. 2 upper panel centre), with a significant difference between the control group and invasive melanomas (p b 0.05). The differences became even more evident when BRAFV600E results were expressed as pg/ml of plasma (p b 0.005); BRAFV600E DNA concentration was 44.6 ± 12.9 pg/ml in control subjects, 11.7 ± 7.9 pg/ml in non-melanoma skin cancer, 267.9 ± 110.6 pg/ml in patients with in situ melanoma and 430.3 ± 123.9 pg/ml in invasive melanoma patients. A significant difference was found between the nonmelanoma skin cancer group and both the in situ (p b 0.05) and invasive melanomas (p b 0.005) (Fig. 2 upper panel right). In the lower panels (Fig. 2, A–D centre and right), we report the histograms representing the distribution of plasma BRAFV600E DNA concentrations in our case study. Quantification of mutated BRAFV600E did not reveal differences on the basis of melanoma location, Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity (Table 1). 3.3. Clinical penetrance of the measurement of plasma BRAFV600E ROC curve analysis was performed to define the cut-off values of total DNA levels and BRAFV600E mutated DNA which better designed maximal clinical sensitivity and specificity (Fig. 3). The calculated cutoff values with the corresponding sensitivity and specificity values are reported in Table 2. The separated evaluation of the quantity of plasma free DNA and the percentage of mutated BRAFV600E showed comparable clinical sensitivity and specificity. However, from the combination of the two parameters and the consequent evaluation of the effective concentration of BRAFV600E we obtained the maximal sensitivity (83%) maintaining an elevated specificity (97%). 3.4. BRAFV600E in paired melanoma tissues and plasma samples For 56/68 subjects, FFPE tissues were analyzed for BRAFV600E mutation by the newly developed real-time method and confirmed by direct sequencing (as the reference method). This comparison was conducted on tissue specimens and a full concordance between the two methods was found for all the wild type samples corresponding to realtime quantitative values lower than the cut-off as well as samples with a percentage of BRAFV600E variant allele higher than 10%. Discordant values were found in the interval between the cut-off value and 10% due to the different sensitivity characteristics of the two techniques. Comparison of plasma and tissue results in the same subject was obtained by evaluating results expressed as percentage of total mutated alleles on the basis of the above reported cut-off values. A statistically significant correlation was found when analyzing the concordance between plasma and tissue samples by Fisher's exact test (p b 0.001) (Table 3). Overall, 45 subjects obtained concordant results on tissue as well as on plasma, 24 resulting wild type and 21 showing the mutation in both samples. Eight mutated tissues did not find correspondence in plasma DNA, while 3 subjects showed the BRAFV600E mutation only in plasma. 4. Discussion The current study describes the development of a minimally invasive method to quantify rare BRAFV600E DNA sequences into the circulation. In particular, a real-time PCR method was developed by using a mutation-specific forward primer and a reverse primer common to both wild type and mutated BRAF sequences. A dual-labelled LNA probe was designed on the complementary strand to that recognized and
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Fig. 2. Quantitative results of total cell-free DNA (ng/ml of plasma) (upper panel left), BRAFV600E mutated DNA expressed as percent of total DNA (%) (upper panel centre) as well as absolute quantity (pg/ml of plasma) (upper panel right). Columns represent mean ± standard error of control group (A) (n = 18), non-melanoma skin cancer (B) (n = 13), in situ melanomas (C) (n = 9) and invasive melanomas (D) (n = 46). Histograms of distributions of values of total cell-free DNA (lower panels A–D left), BRAFV600E mutated plasma DNA (% and pg/ml of plasma) per class of subjects ((lower panels A–D, centre and right respectively).
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Table 2 Cut-off values for total cell-free DNA, BRAFV600E mutated DNA (%) and BRAFV600E mutated DNA (pg/ml plasma) as resulting from ROC curves. Parameter Total cell-free DNA (ng/ml plasma) BRAFV600E mutated DNA (%) BRAFV600E mutated DNA (pg/ml plasma)
Cutt-off
Sensitivity (%)
Specificity (%)
7.0 1.9 100
79 76 97
75 75 83
Table 3 Comparison of BRAFV600E detection in plasma and corresponding formalin-fixed and paraffin-embedded (FFPE) tissue samples (p b 0.001 Fisher's exact test). FFPE tissue Negative Positive Total
Fig. 3. Receiver-operating characteristics (ROC) curves of total cell-free DNA (ng/ml of plasma), BRAFV600E mutated DNA expressed as percent of total DNA (%) as well as absolute quantity (pg/ml of plasma). The area under the curve for each parameter is reported in the table.
hybridized by the BRAFV600E allele specific primer. The innovative design of the assay achieves a dynamic range of about 10 cycles of difference between the 100% mutated and wild type samples, with an assay sensitivity up to 0.3% of mutated alleles. The method was used to detect BRAFV600E in DNA from cell-free plasma compartment of melanoma patients and healthy controls. Neither the enrichment of epithelial cells nor whole genome amplification procedures were used, as reported for example by Oldenburg et al. [23]. PCR amplification was possible in as little as 0.5 ng of plasma cell-free DNA. In the above cited report [23] a LNA probe was used as well, but with a two-step approach, blocking the wild type DNA to prevent its amplification, and using a specific primer for the mutant target in a second PCR reaction. On this basis, our method appears more direct and faster, consisting only in a single real-time PCR. Accurate quantitative measurement of plasma total DNA concentration was preliminarily performed by real-time PCR method since previous studies indicated a relationship between plasma free DNA concentration and cancer presence [24]. The measurement of total plasma DNA concentration actually evidenced a significant difference between control group and in situ (p b 0.001) or invasive melanomas (p b 0.001). In particular, we demonstrated that, as a mean, DNA concentrations are about two fold higher in melanoma patients. These results are concordant with many research on different cancer types (such as prostate adenocarcinoma, lung cancer, ovarian cancer, colorectal carcinoma, pancreatic adenocarcinoma, melanoma, breast cancer, head and neck squamous cell carcinoma), which have been extensively revised by Fleischaker and Smith [24]. The two authors report that several studies observed no correlation between plasma DNA concentration and tumor size, stage or location, similarly to what we observed in our work. Moreover a recent review [25], besides confirming higher DNA yields for cancer patients in comparison to control subjects, underlined the need for research on the origin, function and significance of these nucleic acid molecules as imperative in order to clarify the significance of changes in their amount and
Plasma
Total
Negative
Positive
24 8 32
3 21 24
27 29 56
properties before circulating DNA can be used with confidence as a biomarker. The difference between melanomas and controls increased when BRAFV600E percentage was calculated (p = 0.01) and even more when we combined the two previous indexes to obtain the effective concentration of BRAFV600E in terms of pg mutated alleles/ml of plasma (p = 0.002). These results were confirmed taking into account clinical sensitivity and specificity of the analyzed parameters: in fact the combination of BRAFV600E percentage and DNA concentration in plasma (resulting in BRAFV600E absolute concentration) turned out to be the most sensitive biomarker to discriminate between melanoma patients and healthy subjects. We did not detect any statistical difference in terms of plasma DNA concentration and BRAFV600E mutated DNA levels when melanoma patients were stratified on the basis of Breslow thickness, Clark level, presence of ulceration and sentinel lymph node positivity. From the analytical point of view, the assay resulted highly sensitive and accurate in evidencing either heterogeneity of tumor tissue (as regards to BRAFV600E mutated cells) or the absolute quantity of mutated allele present in plasma. The allele specific Taqman-based real-time PCR assay shows the ability to overcome problems related to DNA quality often encountered when dealing with FFPE- or plasma-derived specimens. Result validation was obtained by direct sequencing with full concordance of the results in the ranges 0–2% and N10%. Interestingly, comparison of the results obtained in plasma with those found in corresponding FFPE tissues indicated an 80% overall concordance. Other authors reported similar discordance probably due to the possibility of sampling of non-mutation-bearing portions of the tumor or to factors affecting the release of tumor DNA in plasma (including tumor size, vascularisation, anatomic site, biological characteristics, rate of apoptosis and necrosis, and renal excretion) [18,26]. Previous works reporting data from plasma detection of BRAFV600E mutation were based on conventional PCR [27], mutant allele specific amplification (MASA) plus denaturing high performance liquid chromatography (DHPLC) [28], and allele specific PCR assays [18] but did not show quantitative results. Quantitative information in our hands better evidenced differences between the control group and melanoma patients, representing a useful biomarker for clinical diagnosis. In conclusion, the allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAFV600E mutated DNA in plasma.
Acknowledgements This work has received financial support from the Istituto Toscano Tumori to C.O. and MIUR-PRIN 2008 to D.M.
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