The Role of Novel Dual Color Immunohistochemistry in Detection of Minimal Hairy Cell Leukemia in Bone Marrow: A Study of 148 Cases
Authors: Gupta Gaurav K. , Sun Xiaoping , Yuan Constance M. , Stetler-Stevenson Maryalice , Kreitman Robert J. , Maric Irina 
Published: October 2019

Abstract
Background:
Hairy-cell leukemia (HCL) is a B-cell lymphoproliferative disorder characterized by distinct immunophenotype (positive for CD19, CD20, PAX5, CD22, CD11c, CD25, CD103, CD123 and CD200). Both flow cytometry (FC) and immunohistochemistry (IHC) can be used to determine these markers, while the expression of another marker (TRAP) is IHC specific. Both trephine bone marrow biopsy and aspirate are vital for assessment of extent of bone marrow infiltration. However, in some cases a cellular aspirate cannot be obtained due to extensive fibrosis, ie "dry tap". In such cases, IHC stains are crucial for assessment of bone marrow leukemic involvement. So far, IHC detection of HCL in the bone marrow sections has been limited to overt disease and could not be reliably used in cases with minimal HCL involvement. Novel automated dual-antibody immunohistochemistry techniques can identify aberrant antigen co-expression in neoplastic cells with high sensitivity. Consistent detection of minimal disease involvement is crucial given that treatment decisions are sometimes based on these data (Grever et. al. Blood 2017). In this study, we evaluated the efficacy of two novel dual IHC assays in assessing minimal HCL involvement in the core biopsies and compared the results with concurrently collected flow cytometric data.
Design:
We analyzed data on 148 cases of HCL (123 male, 25 female; mean age 59.8, range 25-81). All cases had multiparameter flow cytometry performed using CD19, CD20, CD22, CD11c, CD25, CD103, CD123, surface light chains, CD5 and CD23. In parallel, bone marrow IHC was done using PAX5/CD103 and PAX5/TRAP dual stains and the automated stainer (Ventana Ultra).
Results:
Cases were divided into three groups based on the combined results of PAX5/CD103 and PAX5/TRAP stains: negative (82; 55.4%); rare dual positive cells (less than 5% of total cells) (21; 14.1%) and positive (45; 30.4%). Flow cytometry data concurred with IHC results in all IHC positive cases (median 7.05% HCL cells out of all lymphoid cells by FC; range 0.05%-91.7%) and all rare dual-cell IHC positive cases (median 0.98% HCL cells; range 0.02%-19.04%). Overall sensitivity of dual IHC was 81.4%, positive predictive value 100% and negative predictive value 81.7%. In dual IHC negative group, 15/82 cases (18.3%) were low level positive by FC analysis (median 0.13% HCL cells; range 0.01%-9.21%). When dual IHC results were analyzed separately, PAX5/CD103 results were similar to the combined results. PAX5/TRAP staining alone was slightly less sensitive in IHC negative cases; 22/82 (26.8%) of PAX5/TRAP negative or non-evaluable cases were positive by FC analysis (median 0.27% HCL cells, range 0.01%-29.5%).
Conclusion:
Dual color IHC is a sensitive tool in detecting HCL, even in cases with minimal disease involvement. All IHC positive cases concurred with flow cytometry data, even when HCL burden was extremely low (as low as 0.02% of all lymphoid cells by flow cytometric analysis). Only 18.3% of dual IHC negative cases were positive for low level involvement by FC analysis. Therefore, dual IHC is a sensitive new tool for evaluation of minimal marrow involvement by HCL.
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Disclosures
Kreitman: NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox.