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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

The effect of TspRI restriction enzyme treatment on the BRAF sequence analysis of patient samples (a) Patient no. 21, representative sample with no V600E mutation, was judged mutation (-):GTG after primary PCR, and this conclusion was not changed by treatment with TspRI followed by secondary PCR; (b) patient no. 23 was scored as mutation (-):GTG after primary PCR, but mutation (+):GAG after treatment with TspRI; (c) the mutation status of patient no. 6 was difficult to judge from the primary PCR because of overlap between mutation (-):GTG and mutation (+):GAG sequences, however, after treatment with TspRI, the mutation (+):GAG sequence was dominant, indicating a clear mutation (+):GAG status; (d) patient no. 3 was initially judged mutation (-):GTG after primary PCR, but mutation (+):GAT status was apparent after treatment with TspRI
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Fig3: The effect of TspRI restriction enzyme treatment on the BRAF sequence analysis of patient samples (a) Patient no. 21, representative sample with no V600E mutation, was judged mutation (-):GTG after primary PCR, and this conclusion was not changed by treatment with TspRI followed by secondary PCR; (b) patient no. 23 was scored as mutation (-):GTG after primary PCR, but mutation (+):GAG after treatment with TspRI; (c) the mutation status of patient no. 6 was difficult to judge from the primary PCR because of overlap between mutation (-):GTG and mutation (+):GAG sequences, however, after treatment with TspRI, the mutation (+):GAG sequence was dominant, indicating a clear mutation (+):GAG status; (d) patient no. 3 was initially judged mutation (-):GTG after primary PCR, but mutation (+):GAT status was apparent after treatment with TspRI

Mentions: From the sequence analysis of the primary PCR products, it was possible to confirm the presence of the BRAF V600E mutation in five cases. Twenty-three cases were determined to be mutation (-), and for four cases it was not possible to judge whether or not the mutation was present from the results (Table 1, (A) PCR). In the TspRI (-) group (B), the BRAF V600E mutation was detected in four cases, with 27 mutation-negative cases, and one indeterminate result. By contrast, in the TspRI (+) group (C), mutations were detected in 19 cases, including five where the mutation had been detected, four with indeterminate results, and ten cases judged to be mutation (-) by sequencing after only primary PCR. Our results also demonstrate that two of these 19 mutation (+) cases carried a mutation to GAT (D), rather than the more common GAG(E). Four representative examples of the sequence analyses with and without TspRI treatment are presented in Fig. 3.Fig. 3


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)

The effect of TspRI restriction enzyme treatment on the BRAF sequence analysis of patient samples (a) Patient no. 21, representative sample with no V600E mutation, was judged mutation (-):GTG after primary PCR, and this conclusion was not changed by treatment with TspRI followed by secondary PCR; (b) patient no. 23 was scored as mutation (-):GTG after primary PCR, but mutation (+):GAG after treatment with TspRI; (c) the mutation status of patient no. 6 was difficult to judge from the primary PCR because of overlap between mutation (-):GTG and mutation (+):GAG sequences, however, after treatment with TspRI, the mutation (+):GAG sequence was dominant, indicating a clear mutation (+):GAG status; (d) patient no. 3 was initially judged mutation (-):GTG after primary PCR, but mutation (+):GAT status was apparent after treatment with TspRI
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: The effect of TspRI restriction enzyme treatment on the BRAF sequence analysis of patient samples (a) Patient no. 21, representative sample with no V600E mutation, was judged mutation (-):GTG after primary PCR, and this conclusion was not changed by treatment with TspRI followed by secondary PCR; (b) patient no. 23 was scored as mutation (-):GTG after primary PCR, but mutation (+):GAG after treatment with TspRI; (c) the mutation status of patient no. 6 was difficult to judge from the primary PCR because of overlap between mutation (-):GTG and mutation (+):GAG sequences, however, after treatment with TspRI, the mutation (+):GAG sequence was dominant, indicating a clear mutation (+):GAG status; (d) patient no. 3 was initially judged mutation (-):GTG after primary PCR, but mutation (+):GAT status was apparent after treatment with TspRI
Mentions: From the sequence analysis of the primary PCR products, it was possible to confirm the presence of the BRAF V600E mutation in five cases. Twenty-three cases were determined to be mutation (-), and for four cases it was not possible to judge whether or not the mutation was present from the results (Table 1, (A) PCR). In the TspRI (-) group (B), the BRAF V600E mutation was detected in four cases, with 27 mutation-negative cases, and one indeterminate result. By contrast, in the TspRI (+) group (C), mutations were detected in 19 cases, including five where the mutation had been detected, four with indeterminate results, and ten cases judged to be mutation (-) by sequencing after only primary PCR. Our results also demonstrate that two of these 19 mutation (+) cases carried a mutation to GAT (D), rather than the more common GAG(E). Four representative examples of the sequence analyses with and without TspRI treatment are presented in Fig. 3.Fig. 3

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