Detection of BRAF V600E Mutations in Langerhans Cell Histiocytosis by Pyrosequencing
Ji Yuan, Gabriel C Caponetti, Debra Lytle, Timothy C Greiner. University of Nebraska Medical Center, Omaha, NE
Background: Langerhans cell histiocytosis (LCH) is a clonal proliferation of Langerhans-type cells without consistent cytogenetic abnormalities. A recent study has identified the BRAF V600E mutation (c.1799T>A) in 57% of LCH cases (Badalian-Very G. et al. Blood. 2010). We evaluated the frequency of the BRAF V600E mutation in our collection, including both pediatric and adult LCH patients, by using a lab-developed pyrosequencing assay.
Design: We have designed a sensitive BRAF pyrosequencing assay for the V600E mutation that is applicable to DNA from formalin-fixed paraffin-embedded (FFPE) tissue. The assay, which produces a 151bp product, has a sensitivity of 5% using the HT-29-cell line. DNA was extracted from FFPE tissue on 18 archived cases of LCH (7 pediatric and 11 adult), including 7 bone lesions, 4 lymph nodes, 3 cutaneous lesions, 3 pulmonary lesions, and 1 orbital mass. All cases contained at least 20% tumor cells by morphologic and immunophenotypic assessment of CD1a and S100.
Results: BRAF V600E mutations were identified in 7 of 18 (39%) LCH cases, including 3 of 7 (43%) pediatric and 4 of 11 (36%) adult specimens. The mean age of patients who carried the mutations was similar to that of patients who did not (25 vs. 32, P = 0.54). The cases with V600E mutations contained an average of 39% tumor cells (range from 20% to 70%) and an average of 10% of mutant alleles (range from 6% to 16%). There was no specific site associated with BRAF mutations, as these 7 cases involved three different sites (4 bone, 2 lung, and 1 skin). Interestingly, BRAF mutations were not detected in any of the four nodal specimens.
Conclusions: Compared to Badalian-Very et al, we found no age difference associated with BRAF mutant status. The frequency results are concordant with published data indicating that a subset of LCH harbors a BRAF V600E mutation, which may offer a therapeutic opportunity for RAF pathway inhibitors.
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 179, Tuesday Morning