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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

Sequence analysis of samples from six mixtures containing 0–100 % BRAF V600Emutation(+) cells. (a) Sequences of undigested products, TspRI (-): Where material was derived from 100 % A2058 cells (top panel) the mutant sequence, GAG, contributed approximately 50 % of the total, allowing clear categorization of sample as mutation (+). In all other cases (0–50 %, A2058 cells), it was difficult to judge whether the samples were mutation (+) or (-). (b) Sequences of digested products, TspRI (+): All samples containing the mutated (GAG) sequences demonstrated superior amplification of this in comparison to the wild type (GTG) after treatment with TspRI. The negative control containing 0 % A2058 cells (bottom panel) did not show amplification of the mutant sequence. Hence, the BRAF mutation could detected in samples containing as little as 5 % of A2058 cells (equating to 2.5 % of total DNA)
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Fig2: Sequence analysis of samples from six mixtures containing 0–100 % BRAF V600Emutation(+) cells. (a) Sequences of undigested products, TspRI (-): Where material was derived from 100 % A2058 cells (top panel) the mutant sequence, GAG, contributed approximately 50 % of the total, allowing clear categorization of sample as mutation (+). In all other cases (0–50 %, A2058 cells), it was difficult to judge whether the samples were mutation (+) or (-). (b) Sequences of digested products, TspRI (+): All samples containing the mutated (GAG) sequences demonstrated superior amplification of this in comparison to the wild type (GTG) after treatment with TspRI. The negative control containing 0 % A2058 cells (bottom panel) did not show amplification of the mutant sequence. Hence, the BRAF mutation could detected in samples containing as little as 5 % of A2058 cells (equating to 2.5 % of total DNA)

Mentions: Six mixtures of cell lines with (A2058) and without (UE7T-13) the BRAF V600E mutation were prepared containing 0, 5, 10, 20, 50 and 100 % of A2058 cells. FFPE cell blocks were prepared from each mixture, and five 10 μm sections of each block were collected in 1.5 ml Eppendorf tubes. Three tubes were prepared for each FFPE blocks to examine in triplicate. DNA was extracted from the samples in each tube for subsequent PCR, TspRI treatment (+ or -), and sequence analysis. The A2058 cell line was heterozygous for the V600E mutation (Fig. 2, A2058 100 %, TspRI (-)), which was thus present in 50 % of BRAF sequences derived from this cell line. The sequence analysis of secondary PCR products derived from all proportions of A2058 cells, except 100 % (i.e., 0–50 %), demonstrated that when the primary PCR product template DNA was not treated with TspRI, the wild-type (mutation (-) sequence, GTG(V)), was preferentially amplified compared to the mutation(+) sequence, GAG(E). By contrast, treatment with TspRI prior to secondary PCR led to preferential amplification of the BRAF V600E mutation compared to the wild-type sequence, even in the presence of only 5 % of A2058 cells (Fig. 2, A2058 5 %, TspRI (+)). As expected, in the negative control containing UE7T-13 cells alone (A2058: 0 %), TspRI treatment prior to secondary PCR had no effect on the results of the sequencing analysis, indicating that these cells were mutation (-) (Fig. 2, A2058: 0 %, TspRI(+)).Fig. 2


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)

Sequence analysis of samples from six mixtures containing 0–100 % BRAF V600Emutation(+) cells. (a) Sequences of undigested products, TspRI (-): Where material was derived from 100 % A2058 cells (top panel) the mutant sequence, GAG, contributed approximately 50 % of the total, allowing clear categorization of sample as mutation (+). In all other cases (0–50 %, A2058 cells), it was difficult to judge whether the samples were mutation (+) or (-). (b) Sequences of digested products, TspRI (+): All samples containing the mutated (GAG) sequences demonstrated superior amplification of this in comparison to the wild type (GTG) after treatment with TspRI. The negative control containing 0 % A2058 cells (bottom panel) did not show amplification of the mutant sequence. Hence, the BRAF mutation could detected in samples containing as little as 5 % of A2058 cells (equating to 2.5 % of total DNA)
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Related In: Results  -  Collection

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Fig2: Sequence analysis of samples from six mixtures containing 0–100 % BRAF V600Emutation(+) cells. (a) Sequences of undigested products, TspRI (-): Where material was derived from 100 % A2058 cells (top panel) the mutant sequence, GAG, contributed approximately 50 % of the total, allowing clear categorization of sample as mutation (+). In all other cases (0–50 %, A2058 cells), it was difficult to judge whether the samples were mutation (+) or (-). (b) Sequences of digested products, TspRI (+): All samples containing the mutated (GAG) sequences demonstrated superior amplification of this in comparison to the wild type (GTG) after treatment with TspRI. The negative control containing 0 % A2058 cells (bottom panel) did not show amplification of the mutant sequence. Hence, the BRAF mutation could detected in samples containing as little as 5 % of A2058 cells (equating to 2.5 % of total DNA)
Mentions: Six mixtures of cell lines with (A2058) and without (UE7T-13) the BRAF V600E mutation were prepared containing 0, 5, 10, 20, 50 and 100 % of A2058 cells. FFPE cell blocks were prepared from each mixture, and five 10 μm sections of each block were collected in 1.5 ml Eppendorf tubes. Three tubes were prepared for each FFPE blocks to examine in triplicate. DNA was extracted from the samples in each tube for subsequent PCR, TspRI treatment (+ or -), and sequence analysis. The A2058 cell line was heterozygous for the V600E mutation (Fig. 2, A2058 100 %, TspRI (-)), which was thus present in 50 % of BRAF sequences derived from this cell line. The sequence analysis of secondary PCR products derived from all proportions of A2058 cells, except 100 % (i.e., 0–50 %), demonstrated that when the primary PCR product template DNA was not treated with TspRI, the wild-type (mutation (-) sequence, GTG(V)), was preferentially amplified compared to the mutation(+) sequence, GAG(E). By contrast, treatment with TspRI prior to secondary PCR led to preferential amplification of the BRAF V600E mutation compared to the wild-type sequence, even in the presence of only 5 % of A2058 cells (Fig. 2, A2058 5 %, TspRI (+)). As expected, in the negative control containing UE7T-13 cells alone (A2058: 0 %), TspRI treatment prior to secondary PCR had no effect on the results of the sequencing analysis, indicating that these cells were mutation (-) (Fig. 2, A2058: 0 %, TspRI(+)).Fig. 2

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