Phospho-ERKThr202/Tyr204 Is Overexpressed in Hairy Cell Leukemia and Is a Useful Diagnostic Marker in Bone Marrow Trephine Sections
Douglas W Warden, Sarah Ondrejka, Jeffrey Lin, Lisa Durkin, Juraj Bodo, Eric D Hsi. Cleveland Clinic, Cleveland, OH
Background: BRAF V600E mutations are present in virtually all cases of hairy cell leukemia (HCL) and absent in other small B-cell leukemias and lymphomas. This activating mutation results in constitutive BRAF signaling, which is manifested by phosphorylation of ERK. We hypothesized that detection of phospho-ERK (pERK) in tissue sections may be a specific and useful marker for diagnosis of HCL.
Design: We performed a pERK/CD20 double-stain in 89 bone marrow samples involved by B-cell lymphoproliferative disorders of small lymphocytes, including 27 cases of HCL confirmed by flow cytometry. Immunostaining was performed on decalcified formalin-fixed paraffin embedded bone marrow sections with an automated immunostainer (Ventana Medical Systems). The double staining protocol used a rabbit monoclonal antibody to pERK1/2Thr202/Tyr204 (Cell Signaling Technology, clone D13.14.4E) and mouse monoclonal antibody for CD20 (Dako, Carpinteria, CA, clone L26). Allele-specific PCR for the BRAF V600E mutation was performed on 11 cases of HCL with available DNA and in 1 non-HCL case where pERK staining was positive.
Results: pERK staining (cytoplasmic and nuclear localization) in at least 70% of B-cells was observed in all 27 cases of HCL tested. B-cells in all cases of CLL/SLL (n=12), follicular lymphoma (n=7), splenic marginal zone lymphoma (n=9), mantle cell lymphoma (n=9), CD5- B-cell leukemia/lymphoma not otherwise specified (NOS, n=10), lymphoplasmacytic lymphoma (n=11), and hairy cell leukemia variant (n=2) were negative for pERK. One of two cases of atypical CLL, likely representing mixed-cell CLL with 17p deletion, was pERK-positive. Allele-specific PCR on 11 cases of HCL contained the BRAF V600E variant in all 11 cases. The one non-HCL case that was positive for p-ERK also contained the BRAF V600E. Overall, while 100% of HCL cases expressed pERK, only 1 of 64 (1.6%) of other small B-cell lymphoma/leukemias in bone marrow expressed pERK. Thus, the sensitivity and specificity of pERK for diagnosis of HCL in our hands is 100 and 98%.
Conclusions: BRAF V600E mutations are present in all HCL cases. Immunohistochemistry for pERK can be reliably applied on routinely processed bone marrow trephine sections and is highly sensitive and specific for HCL. It appears to be a useful tool in the differential diagnosis of small B-cell leukemias/lymphomas, and pERK is a surrogate marker for BRAF V600E in the rare non-HCL leukemias with this mutation.
Monday, March 19, 2012 11:30 AM
Platform Session: Section C, Monday Morning