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BRAF V600 mutations in Langerhans cell histiocytosis with a simple and unique assay.

Tatsuno M, Shioda Y, Iwafuchi H, Yamazaki S, Iijima K, Takahashi C, Ono H, Uchida K, Okamura O, Matubayashi M, Okuyama T, Matsumoto K, Yoshioka T, Nakazawa A - Diagn Pathol (2016)

Bottom Line: BRAF inhibitors molecularly targeting the V600E mutation have been developed to counteract the effect of the mutation.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.This screening method is expected to play an important role to select the most effective therapies.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

CD1a immunohistochemistry FFPE tissue section from LCH patients stained with CD1a. (a) Two samples that were V600E mutation (+) after both primary PCR amplification and TspRI followed by additional PCR amplification. Four of five samples in this category demonstrated up to 70 % tumor content. These cases were easy to detect by primary PCR. (b) Examples of staining of samples that were BRAF mutation (-) after primary PCR, but mutation (+) after TspRI treatment. Seven of eight such samples contained <50 % tumor content. These samples were not dissected to isolate tumor tissue; nevertheless, a BRAF V600E mutation was detected after treatment with TspRI. (c) Two samples that were BRAF V600E mutation (-) after both primary PCR and after TspRI digestion followed by additional amplification. Tumor content was determined in 11 of 13 samples in this category and ranged from 4.9 to 100 %, hence mutation detection was independent of tumor content
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Fig4: CD1a immunohistochemistry FFPE tissue section from LCH patients stained with CD1a. (a) Two samples that were V600E mutation (+) after both primary PCR amplification and TspRI followed by additional PCR amplification. Four of five samples in this category demonstrated up to 70 % tumor content. These cases were easy to detect by primary PCR. (b) Examples of staining of samples that were BRAF mutation (-) after primary PCR, but mutation (+) after TspRI treatment. Seven of eight such samples contained <50 % tumor content. These samples were not dissected to isolate tumor tissue; nevertheless, a BRAF V600E mutation was detected after treatment with TspRI. (c) Two samples that were BRAF V600E mutation (-) after both primary PCR and after TspRI digestion followed by additional amplification. Tumor content was determined in 11 of 13 samples in this category and ranged from 4.9 to 100 %, hence mutation detection was independent of tumor content

Mentions: From our experiments using mixtures of cell lines, it was possible to detect the V600E mutation when 5 % of the cells in a mixture were mutation (+). We next examined the relationship between mutation detection by the sequence analysis after TspRI treatment and the ratio of tumor content in patient specimens by immunohistochemical staining. The area of the tumor tissue was calculated as the area of anti-CD1a staining in an FFPE section. Specimens for both immunohistochemical staining and DNA extraction were obtained from the same blocks (Table 1 and Fig. 4).Fig. 4


BRAF V600 mutations in Langerhans cell histiocytosis with a simple and unique assay.

Tatsuno M, Shioda Y, Iwafuchi H, Yamazaki S, Iijima K, Takahashi C, Ono H, Uchida K, Okamura O, Matubayashi M, Okuyama T, Matsumoto K, Yoshioka T, Nakazawa A - Diagn Pathol (2016)

CD1a immunohistochemistry FFPE tissue section from LCH patients stained with CD1a. (a) Two samples that were V600E mutation (+) after both primary PCR amplification and TspRI followed by additional PCR amplification. Four of five samples in this category demonstrated up to 70 % tumor content. These cases were easy to detect by primary PCR. (b) Examples of staining of samples that were BRAF mutation (-) after primary PCR, but mutation (+) after TspRI treatment. Seven of eight such samples contained <50 % tumor content. These samples were not dissected to isolate tumor tissue; nevertheless, a BRAF V600E mutation was detected after treatment with TspRI. (c) Two samples that were BRAF V600E mutation (-) after both primary PCR and after TspRI digestion followed by additional amplification. Tumor content was determined in 11 of 13 samples in this category and ranged from 4.9 to 100 %, hence mutation detection was independent of tumor content
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837618&req=5

Fig4: CD1a immunohistochemistry FFPE tissue section from LCH patients stained with CD1a. (a) Two samples that were V600E mutation (+) after both primary PCR amplification and TspRI followed by additional PCR amplification. Four of five samples in this category demonstrated up to 70 % tumor content. These cases were easy to detect by primary PCR. (b) Examples of staining of samples that were BRAF mutation (-) after primary PCR, but mutation (+) after TspRI treatment. Seven of eight such samples contained <50 % tumor content. These samples were not dissected to isolate tumor tissue; nevertheless, a BRAF V600E mutation was detected after treatment with TspRI. (c) Two samples that were BRAF V600E mutation (-) after both primary PCR and after TspRI digestion followed by additional amplification. Tumor content was determined in 11 of 13 samples in this category and ranged from 4.9 to 100 %, hence mutation detection was independent of tumor content
Mentions: From our experiments using mixtures of cell lines, it was possible to detect the V600E mutation when 5 % of the cells in a mixture were mutation (+). We next examined the relationship between mutation detection by the sequence analysis after TspRI treatment and the ratio of tumor content in patient specimens by immunohistochemical staining. The area of the tumor tissue was calculated as the area of anti-CD1a staining in an FFPE section. Specimens for both immunohistochemical staining and DNA extraction were obtained from the same blocks (Table 1 and Fig. 4).Fig. 4

Bottom Line: BRAF inhibitors molecularly targeting the V600E mutation have been developed to counteract the effect of the mutation.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.This screening method is expected to play an important role to select the most effective therapies.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus