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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

Optimization of TspRI digestion for the BRAF amplicons. 100 ng of BRAF PCR fragments amplified from normal DNA were digested with TspRI. Lane 1: No TspRI, 65 °C, 15 min. Lane 2: 5U TspRI, 65 °C, 5 min. Lane 3: 10U TspRI, 65 °C, 10 min. Lane 4: 20U TspRI, 65 °C, 15 min, leading to digestion of the majority of BRAF PCR fragments
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Fig1: Optimization of TspRI digestion for the BRAF amplicons. 100 ng of BRAF PCR fragments amplified from normal DNA were digested with TspRI. Lane 1: No TspRI, 65 °C, 15 min. Lane 2: 5U TspRI, 65 °C, 5 min. Lane 3: 10U TspRI, 65 °C, 10 min. Lane 4: 20U TspRI, 65 °C, 15 min, leading to digestion of the majority of BRAF PCR fragments

Mentions: A 209 bp PCR product including BRAF V600E wild-type was amplified from DNA extracted from FFPE blocks of human tonsil tissue (Fig. 1, lane 1). The PCR products were treated with TspRI under various conditions. After digestion at 65 °C for 15 min, the products were almost completely cleaved to give two fragments of 124 and 94 bp (Fig. 1, lane 4).Fig. 1


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)

Optimization of TspRI digestion for the BRAF amplicons. 100 ng of BRAF PCR fragments amplified from normal DNA were digested with TspRI. Lane 1: No TspRI, 65 °C, 15 min. Lane 2: 5U TspRI, 65 °C, 5 min. Lane 3: 10U TspRI, 65 °C, 10 min. Lane 4: 20U TspRI, 65 °C, 15 min, leading to digestion of the majority of BRAF PCR fragments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Optimization of TspRI digestion for the BRAF amplicons. 100 ng of BRAF PCR fragments amplified from normal DNA were digested with TspRI. Lane 1: No TspRI, 65 °C, 15 min. Lane 2: 5U TspRI, 65 °C, 5 min. Lane 3: 10U TspRI, 65 °C, 10 min. Lane 4: 20U TspRI, 65 °C, 15 min, leading to digestion of the majority of BRAF PCR fragments
Mentions: A 209 bp PCR product including BRAF V600E wild-type was amplified from DNA extracted from FFPE blocks of human tonsil tissue (Fig. 1, lane 1). The PCR products were treated with TspRI under various conditions. After digestion at 65 °C for 15 min, the products were almost completely cleaved to give two fragments of 124 and 94 bp (Fig. 1, lane 4).Fig. 1

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