Development and Validation of a Real Time Quantification Assay to Detect and Monitor BRAFV600E-Mutations in Hairy Cell Leukemia,

Abstract 3674 Introduction: So far, the diagnosis of hairy cell leukemia (HCL) was based on cytomorphology, immunophenotyping and histology. Monitoring of the disease during therapy is most sensitively performed by immunophenotyping. The genetics underlying HCL was unclear until very recently, when a BRAF V600E mutation was described to occur in all patients (48/48) with HCL (Tiacci et al., NEJM: 364, 2305–15, 2011). Aim: The aim of this study was to establish and evaluate a reliable genetic test to detect and monitor BRAFV 600E mutations with high specificity and sensitivity. Methods: After testing different variants of DNA or RNA based direct sequencing and melting curve assays we decided for an mRNA-based reverse transcription real time quantification (RQ-PCR) assay that yielded the best sensitivity and specificity. This assay includes a BRAF V600E mutation-specific primer and an additional mismatch nucleotide that further enhances the specific detection of mutated transcripts. The mutated transcripts were normalized against BRAF wildtype (BRAF wt) transcripts. Values are given in %BRAF V600E/BRAF wt. In total, 259 samples were analysed with this assay. First, the specificity of the assay was analysed in 96 samples with a “non-HCL” diagnosis (AML n=9, MDS n=7, MPN n=13, CML n=4, CML in MMR n=10, CMML n=4, CEL n=2, T-ALL n=1, B-ALL n=2, B-NHL n=35, CLL n=2, non-malignant diseases n=7). These samples were used to establish the non-specific background of the assay that can be caused by the normal wild type allele. Subsequently, 124 patients with a proven diagnosis of HCL were analysed (31 females/93 males median age: 59.6 yrs, range 24.3–88.4 yrs). In addition, 16 patients with HCL-variant were analyzed. In 19 of the HCL cases additional follow-up samples were investigated (range, 1–4 samples per patient, total number n=25). Results: Limited dilution series of DNA of 2 cases with highly infiltrated HCL in DNA of a normal control revealed a sensitivity of this assay of 1/10e4-1/10e5. Unspecific %BRAF V600E/BRAF wt values of “non-HCL” controls were very low (median: 0.003, range: 0.000–0.030, SD: 0.005). In the 16 cases with HCL-variant the %BRAF V600E/BRAF wt values were in the same range as in the “non-HCL” controls (median: 0.006, range: 0.000–0.012, SD: 0.004). Based on these results the cut-off level was defined as 0.018 %BRAF V600E/BRAF wt (3xSD over the median). 117/124 cases with proven HCL (94.4%) had a %BRAF V600E/BRAF wt expression that was above the cut-off as defined by normal controls (median: 16.9, range 0.077–280.3). There was a highly significant correlation between the BRAF expression and the percentage of HCL cells as determined by immunophenotyping (r=0.741, p=0.001). The remaining 7/124 (5.6%) HCL patients had %BRAF V600E/BRAF wt values in the range of normal controls and thus are regarded as not carrying the typical BRAF V600E. Deep sequencing using the 454 technology did not reveal any rare BRAF mutations even not at a low levels in any of these 7 cases (median coverage: 427 reads, range: 199–823 reads). In the 19 BRAF V600E-positive patients with follow-up samples an up to 4 log reduction of the %BRAF V600E/BRAF wt was observed during therapy. Comparison of this log reduction with the log reduction as determined by immunophenotyping again revealed a close correlation (r=0.892, p<0.001). In 5 samples values in remission were in the range of negative controls. In one case a relapse was predictable one month before cytomorphologic relapse appeared by an increasing %BRAF V600E/BRAF wt level, paralleling with an increasing percentage of HCL cell as determined by immunophenotyping. Conclusion: 1) BRAF V600E is a new marker that is useful for diagnosis of HCL. 2) This new assay can detect HCL in >94% of cases and BRAF V600E mutations with a specificity of 100%. 3) Given the high sensitivity of 1/10e4-1/10e5 it serves a valuable tool for minimal residual disease detection in HCL. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Wendland:MLL Munich Leukemia Laboratory: Employment. Ulke:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.