BRAF V600 mutations in Langerhans cell histiocytosis with a simple and unique assay.
BACKGROUND
BRAF (V-raf murine sarcoma viral oncogene homolog B1) is a serine-threonine protein kinase involved in cell survival, proliferation, and differentiation. The most common missense mutation of BRAF (mainly V600E) contributes to the incidence of various cancers, including Langerhans cell histiocytosis (LCH). BRAF inhibitors molecularly targeting the V600E mutation have been developed to counteract the effect of the mutation. To ensure the administration of effective pharmacotherapy, it is therefore imperative to develop an effective assay to screen LCH patients for the V600E mutation. However, tumor tissues of LCH typically contain many inflammatory cells which make a correct judgement of the mutation status difficult in the DNA sequence analysis.


RESULTS
In this study, we present a new, highly sensitive analyzing method combining PCR, restriction enzyme digestion, and a sequencing assay using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens. TspRI is a restriction enzyme that cleaves the sequence encompassing the wild-type BRAF codon 600 into two fragments, which cannot be used as a template for subsequent BRAF PCR amplification. We therefore evaluated the sensitivity of BRAF V600 mutation detection by amplifying the primary PCR product digested with TspRI and sequencing the secondary PCR products. The V600E mutation was detected in FFPE tissue samples from 32 LCH patients; our assay was able to identify mutations in four samples that gave inconclusive results, and ten that were negative, according to standard PCR and sequencing.


CONCLUSIONS
We presented a new and highly sensitive method to detect BRAF V600 mutations. This screening method is expected to play an important role to select the most effective therapies.
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan. tatsuno-m@ncchd.go.jp.
Michiko
M
Tatsuno
Department of Children's Cancer Center, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Yoko
Y
Shioda
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Hideto
H
Iwafuchi
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.|Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Shigeki
S
Yamazaki
Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Kenta
K
Iijima
Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Chiaki
C
Takahashi
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.|Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Hiromi
H
Ono
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.|Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Kiyono
K
Uchida
Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Osamu
O
Okamura
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.|Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Mamoru
M
Matubayashi
Department of Clinical Laboratory Medicine, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Torayuki
T
Okuyama
Department of Children's Cancer Center, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Kimikazu
K
Matsumoto
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.
Takako
T
Yoshioka
Department of Pathology, National Center for Child Health and Development, PO Box 157-8535, 2-10-1 Okura, Setagaya-ku, Tokyo, Japan.|Department of Pathology, Tokai University School of Medicine, PO Box 259-1193, 143 Shimokasuya, Isehara-shi, Kanagawa, Japan.
Atsuko
A
Nakazawa
D000293:Adolescent; D018949:Antigens, CD1 / Q000032:analysis; D001483:Base Sequence; D016022:Case-Control Studies; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D005819:Genetic Markers; D020022:Genetic Predisposition to Disease; D006646:Histiocytosis, Langerhans-Cell / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D016612:Paraffin Embedding; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011379:Prognosis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D016707:Tissue Fixation
D018949:Antigens, CD1; C419393:CD1a antigen; D005404:Fixatives; D005819:Genetic Markers; D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
Formalin-fixed paraffin-embedded (FFPE); Langerhans cell histiiocytosis (LCH); TspRI; V-raf murine sarcoma viral oncogene homolog B1 (BRAF)