Background
V-raf murine sarcoma viral oncogene homolog B1 (BRAF) is a serine-threonine protein kinase that functions through the RAS/MAPK (RAS-RAF-MEK-ERK) [
1] signaling cascade, regulating cell survival, proliferation, and differentiation. Missense mutations in the
BRAF gene contribute to the incidence of various types of cancer [
2,
3]. The V600 mutations account for the majority of
BRAF mutations and are observed in Langerhans cell histiocytosis (LCH) [
4], Erdheim-Chester disease (ECD) [
5], melanoma [
6], papillary thyroid carcinoma [
7,
8], colorectal cancer [
9], hairy cell leukemia (HCL) [
10], and chronic lymphocytic leukemia (CLL) [
11].
Recently,
BRAF mutations were shown to interfere with pharmacotherapies targeting the epithelial growth factor receptor (EGFR) [
12,
13]. Accordingly, BRAF inhibitors targeting the V600 mutations were developed to preserve EGFR responses in melanoma, and they are anticipated to be effective in various cancers associated with
BRAF V600 mutations. Therefore, it is imperative to develop a sensitive screening method for the detection of V600 mutations to determine which patients may require V600 inhibitors and to ensure the efficiency of EGFR-targeted therapy.
In this study, we present a highly sensitive assay using a combination of PCR, restriction enzyme cleavage, and a sequence analysis of DNA extracted from formalin-fixed paraffin-embedded (FFPE) sections. The sensitivity of the assay was determined by inspecting several samples derived from mixtures of two cell lines, one with the BRAF V600E mutation and another with wild-type BRAF.In addition, the results using this new method were compared with those from standard PCR and sequencing, and the two methods were evaluated using FFPE tissue section from LCH patients, the tumor content ratio determined by CD1a immunostaining.
Methods
Subjects and samples
This study was conducted using FFPE tissue from LCH patients diagnosed at the National Center for Child Health and Development. There were 32 patients in total, 16 males and 16 females. Twenty-three patients were aged ≤5 years and nine were aged ≥6 years. The FFPE samples were from the bone or tissue (n = 14), or subcutaneous tissues (n = 18). Normal control DNA was extracted from a human tonsil FFPE block. This study was approved by the Ethics Committee of the National Center of Child Health and Development (No. 559, 1035), and patients (or their guardians) provided their written informed consent for the use of their samples in this study.
Cell lines
The BRAF V600E mutant cell line, A2058, and the wild-type cell line, UE7T-13, were used to verify the efficiency of BRAF mutation detection. A2058 and UE7T-13 were acquired from the JCRB Cell Bank (National Institute of Biomedical Innovation). Cells were cultured in DMEM (GIBCO: catalogue number 12430-054) containing 10 % FCS (Sigma-Aldrich).
Six mixtures were prepared by combining various proportions (0, 5, 10, 20, 50 and 100 %) of the A2058 BRAF V600E mutation (+) cell line with the UE7T-13 BRAF mutation (-) cell line; FFPE cell blocks were prepared for each mixture using the Shandon Cytoblock kit (Thermo Scientific), and 5 × 10 μm sections were cut from each block. Three of these five sections were collected in 1.5 ml Eppendorf tubes for DNA extraction and molecular analyses in triplicate.
DNA was extracted from the FFPE sections using the ReliaPrep FFPE gDNA Miniprep System (Promega) or the NucleoSpin FFPE DNA Kit (Macherey-Nagel) according to the manufacturer’s protocols. The concentration of the extracted DNA was determined using a NanoDrop spectrophotometer (Thermo Scientific).
PCR and sequence analysis of BRAF
We designed one set of primers to PCR amplify BRAF exon 15, including the codon 600 sequence, and generate a product of 209 bp. The forward primer sequence was BRAF-F: 5′-TCATAATGCTTGCTTGCTCTGATAGGA-3′ and the reverse primer was BRAF-R: 5′-CAGTGGAAAAATAGCCTC-3′ (nucleotides 147–169 and 355–338 of GenBank: M95712.2, respectively). PCR was performed with 25 μl reaction mixtures, using the HotStarTaq Master Mix Kit (Qiagen), containing 12.5 μl of 2 × reaction master mix, each primer at a final concentration of 0.4 μM and 1 μl of template. The PCR conditions were as follows: an initial denaturation at 95 °C for 15 min, followed by 42 cycles of amplification (30 s at 95 °C, 40 s at 56 °C, and 40 s at 72 °C), and a final step of 72 °C for 10 min. These PCR conditions were used for first and second PCR. Molecular sizes and concentrations of the PCR products were determined using a Bioanalyzer (Agilent).
The PCR products were treated with ExoSAP-IT (Affymetrix) to remove unconsumed dNTPs and primers. A sequence analysis was subsequently performed according to the BigDye Terminator v3.1 Cycle Sequencing kit protocol (Applied Biosystems) using the forward or reverse primer. Free dye terminators were removed from the completed sequencing reactions using a DyeEx 2.0 Spin kit (Qiagen), followed by ethanol precipitation and resuspension in Hi-Di Formamide (Applied Biosystems). The sequence data were detected using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), and the mutation sequence was confirmed by both the forward and reverse sequence data.
Restriction enzyme analysis
TspRI (New England Biolabs) is a restriction enzyme that cuts at the sequence 5′-NNCASTGNN-3′ (3′-NNGTSACNN-5′); it can cut at the wild-type V600 BRAF codon in PCR amplicons across this region to give two fragments, but not sequences with V600E or V600D mutations. Digestion of PCR products with TspRI was performed at 65 °C in 1 × CutSmart Buffer (New England Biolabs). After the digestion, a second PCR was performed using the digested PCR products as templates.
Immunohistochemistry
CD1a immunostaining was performed using an automatic immunostaining machine HISTOSTAINER 48A (Nichirei Corporation, Japan). The primary antibody was CD1a mouse monoclonal antibody (Leica Biosystem: NCL-CD1a-220) using a 1:50 dilution, and the secondary antibody was Histofine Simple Stain MAX-PO (MULTI) (Nichirei Corporation, Japan). Images of immunostained samples were captured using a NanoZoomer-XR digital scanner (Hamamatsu Photonics). The tumor content was calculated as the ratio of the CD1a (+) area to the whole specimen, using the NDP.analyze U12356 software program.
Discussion
LCH is classified into two categories, single organ and multi-organ involvement. The prognosis is almost good in the case of single organ disease, however the course often leads to a poor prognosis in the case of multi-organ involvement, necessitating chemotherapy. Therefore, the selection of effective therapy is important, since relapse after remission is associated with high mortality [
14].
The
BRAF gene is located on chromosome 7q34, composed of 18 exons, and encodes a mRNA transcript of 2478 bp.
BRAF genetic missense mutations are associated with the incidence of various types of cancers.
BRAF mutations have been confirmed, not only in LCH, but also in ECD, melanoma, papillary thyroid cancer, colorectal cancer, HCL and CLL. The V600E mutation accounts for the majority of these mutations, especially in melanoma where approximately 60–70 % cases of melanoma patients have
BRAF mutations, and about 90 % of these are V600E. Melanoma tumor containing the
BRAF V600E mutation are resistant to EGFR inhibitors, and treatment with BRAF inhibitors demonstrates remarkable effects on the tumor. Vemurafenib, dabrafenib and trametinib have been used in the treatment of such patients as targeted therapy [
15‐
17]. However, these treatments are not effective in patients without
BRAF mutations. Immunotherapy with ipilimumab, which targets tumors without mutant
BRAF, is appropriate for these patients [
18]. Hence, sensitive methods for the detection of
BRAF genetic mutations are important for the selection of effective therapies and in the diagnosis. Generally, tumor tissue biopsies from LCH patients include some normal tissue and many inflammatory cells. When analyzing the DNA sequences from such samples, the proportion of tumor cells is an important issue that can affect the analysis and correct judgment of the mutation status.
To address this problem, we verified the accuracy of our new method for mutation detection by comparing sequences of the
BRAF V600E mutation amplified by the popular method of PCR alone, with those generated by our method combining PCR with TspRI restriction enzyme digestion. A total of 32 tissue samples from LCH patients were screened for the presence of
BRAF V600E mutation. Based on the findings of conventional PCR and a sequence analysis, we confirmed only five cases of
BRAF V600E mutation, with four cases giving ambiguous results. Treatment with TspRI reduced the amount of DNA template from wild-type tissue; consequently, we were able to detect 19 cases with clear mutations, with four cases where a clear result could not be obtained. In addition, we were able to detect not only the V600E mutation, but also V600D. The detection of 19
BRAF V600 (E or D) mutations from 32 patients, corresponds to 59 % of all cases in this series. This incidence is similar to that found by another study (57 %), which used iPLEX chemistry methodology [
19,
20]. In addition, our results from DNA samples derived from a
BRAF V600E heterozygous mutant cell line mixed with a wild-type cell line indicate that the mutation can be detected when only 5 % of cells are from the mutation (+) cell line, equal to 2.5 % of all cells.
For the
BRAF V600E mutation analysis, we assessed several methods for DNA extraction from the FFPE samples. However, some methods were limited to only samples with more than 50 % of tumor tissue, or the necessity for microdissection of the tumor tissue [
5,
6]. These requirements can preclude an analysis due to the tumor size or condition, or require specialists who are able to discriminate tumor tissue, and are therefore likely to be performed inconsistently in different laboratories. Furthermore, these methods require specialized and expensive equipment, which will limit analyses to those laboratories with access to such facilities. The method we herein propose only requires access to a regular thermal cycler and sequencer and does not require difficult pathology methods of tissue selection by microdissection. As it is now common for commercial companies to provide sequencing services, it will be possible to use this method for the diagnosis, not only in specialized facilities, but also in general clinical laboratories.
Acknowledgments
The authors thank Dr. Akihiro Umezawa, Deputy Director of Laboratory and Manager of the Department of Cell Engineering, Department of Reproductive Biology, and Dr. Kenichiro Kobayashi, Chief of the Department of Pediatric Hematology and Oncology Research, for proofreading and editing.
This work was supported in part by a grant from the National Center for Child Health and Development of Japan (24-4, 26-20).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MT participated in the design of the study, carried out molecular experiments, performed statistical analysis and drafted the manuscript. AN, TY, HI and SY participated in the design of the study, performed histological analysis of all samples and participated in drafting the manuscript. YS participated in the design of the study and drafted the manuscript. KI, CT, HO and KU performed collection and pre-treatment of all samples. KM, TO, MM and OO participated in the design of the study and in drafting the manuscript. All authors read and approved the final manuscript.