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

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.

Keywords: Formalin-fixed paraffin-embedded (FFPE); Langerhans cell histiiocytosis (LCH); TspRI; V-raf murine sarcoma viral oncogene homolog B1 (BRAF).

Fig. 1

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

Fig. 2

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)

Fig. 3

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

Fig. 4

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