BRAF V600E Mutations in Low Grade B-Cell Lymphomas
Eric Duncavage, Lauren Henke, Friederike Kreisel. Washington University, St. Louis, MO; Washington University School of Medicine, St. Louis, MO
Background: Activating mutations in the Serine/threonine-protein kinase b-raf (BRAF) including the common Valine to Glutamine substitution at coding position 600 (V600E) have been described in a variety of solid tumor types. The V600E mutation was recently reported to be present in all tested cases of Hairy Cell Leukemia (HCL), but not other low-grade B-cell lymphomas, suggesting it is both a sensitive and specific marker for HCL. We sought to validate these findings in low grade B-cell lymphomas including histologically similar lymphomas such as Splenic Marginal Zone Lymphoma (SMZL).
Design: We identified a set of low grade B-cell lymphomas including 5 HCLs, 5 SMZLs, 23 low-grade Follicular Lymphomas (FL), and 19 nodal small lymphocytic lymphomas (SLL). DNA was extracted from formalin-fixed tissue blocks and PCR amplified using primers targeting BRAF exon 15. DNA was then digested using the tspRI restriction enzyme that cuts DNA harboring the c1799 T->A sequence corresponding to BRAF V600E mutations. Select cases were then sequenced by both Sanger sequencing, BRAF exon 15, and Next Generation Sequencing (NGS), all BRAF exons, for confirmation.
Results: 5/5 HCL cases, 2/5 SMZL cases, 0/23 FL cases, and 0/19 SLL cases were positive for the BRAF V600E mutation by PCR and enzyme digest. However, when exon 15 was sequenced in the SMZL cases there was no evidence of a V600E mutation. We further sequenced all BRAF coding exons in the 2 PCR-positive SMLZ cases and saw no evidence of BRAF V600E or additional BRAF mutations. A histologic review of the PCR-positive SMZL cases revealed the spleen to show lymphomatous infiltration of the white and red pulp with no reactivity for Annexin A1 or CD103, CD25 and CD11c. These ancillary studies confirmed the diagnosis of SMZL.
Conclusions: We found the BRAF V600E mutation in all cases of HCL, but not in cases of SLL or FL, confirming what has been previously reported. In addition we found 2/5 cases of SMZL that were PCR-positive for BRAF V600E mutations, however we could not verify these findings by sequencing. Given the increased sensitivity of PCR-based detection methods over Sanger sequencing and NGS (20% and 10% respectively) it is unclear if the two BRAF V600E PCR-positive cases represent low level mutation frequency, tumor cell dilution, or false positive results. However, these finding suggest caution when using PCR-based BRAF V600E testing to classify cases as HCL if SMZL is included in the differential diagnosis.
Monday, March 19, 2012 1:00 PM
Poster Session II # 222, Monday Afternoon