Distinguishing hairy cell leukemia variant from hairy cell leukemia: development and validation of diagnostic criteria

Abstract

Hairy cell leukemia (HCL) and hairy cell leukemia-variant (HCL-v) are rare diseases with overlapping clinico-pathological features. We performed flow cytometry analysis (FCM) of 213 cases (169 HCL, 35 HCL-v, 9 splenic marginal zone lymphoma (SMZL)), correlating results with available corresponding clinical and morphological data. FCM distinguished HCL-v from HCL and SMZL based solely upon expression of four antigens (CD11c, CD25, CD103, CD123) combined with B-cell markers (CD19, CD20, CD22). HCL-v expressed bright CD20, bright CD22, CD11c(100%), CD103(100%), dim(40%) or negative(60%) CD123, and uniformly lacked CD25(100%). HCL expressed bright CD20, bright CD22, bright CD11c, bright CD25, CD103, and bright homogeneous CD123(100%). Aberrant expression of CD5(2%/3%), CD10(12%/3%), CD23(21%/11%), CD38(14%/0%), CD2(2%/9%), CD4(0.5%/0%) and CD13(0.5%/3%), was observed in HCL/HCL-v, respectively. SMZL cases were CD103(-) and CD123(-) except for one case with dim CD123. HCL showed significantly greater marrow infiltration over HCL-v. Prominent nucleoli were observed in most HCL-v but rarely in HCL. A third of HCL and HCL-v marrows were hypocellular or aplastic-appearing. Detection of BRAFV600E mutation and annexin A1 were examined in a subset of cases to further validate FCM diagnostic criteria. HCL-v was negative for both annexin A1 (100%) and BRAFV600E mutation (100%), in contrast to HCL (74% positive for annexin A1; 76% positive for BRAFV600E mutation). HCL-v is resistant to traditional HCL therapy, making accurate diagnosis imperative. We have defined FCM criteria for differentiation of HCL-v from HCL and SMZL.

Figure 1

Hypercellular marrow with diffuse infiltrate of hairy cell leukemia cells (A, hematoxylin-eosin stain; B, CD20 immunostain, magnification ×400); Hypocellular marrow with interstitial infiltrate of hairy cell leukemia cells (C, hematoxylin-eosin stain, magnification ×1000; D, CD20 immunostain, magnification ×400); Hypocellular marrow with interstitial infiltrate of hairy cell leukemia variant cells (E, hematoxylin-eosin stain; F, CD20 immunostain, magnification ×400).

Figure 2

Difference in marrow involvement (% of marrow cellularity) between HCL and HCL-v marrow biopsies. Marrow involvement was 2.6 times as extensive in HCL and HCL-v (p=0.0066, Mann-Whitney).

Figure 3

Representative images of cytological features of hairy cell leukemia and hairy cell leukemia variant cells in the peripheral blood and bone marrow aspirates. (A, B and C) Hairy cell leukemia cells (Wright's Stain, magnification ×1000); (D and E) Hairy cell leukemia variant cells (Wright's Stain, magnification ×1000).

Figure 4

(A) Hairy cell leukemia cells with expression of CD5. (B) Hairy cell leukemia cells with expression of CD10. (C) Hairy cell leukemia variant cells with expression of CD5. (D) Hairy cell leukemia variant cells with expression of CD10. Analysis shows mononuclear cells, as determined by FSC and SSC characteristics, with HCL or HCL-v cells gated as red events.

Figure 5

(A) flow cytometry analysis demonstrating coexistent hairy cell leukemia and monoclonal B-cells consistent with chronic lymphocytic leukemia. Analysis shows mononuclear cells, as determined by FSC and SSC characteristics, with HCL cells gated as red events, and CLL cells gated as blue events. The chronic lymphocytic leukemia cells expressed dim CD20, dim CD22, dim CD11c, moderate CD25 and CD5, and showed dim kappa light chain restriction. (B) The hairy cell leukemia cells comprised approximately 0.55% of the cells, and were positive for CD20 (bright), CD11c (bright), CD25 and CD103. The hairy cell leukemia cells were lambda light chain restricted when gated on bright CD11c positive B-cells; (B–C) Flow cytometry analysis demonstrating a case of HCL-v with an apparent phenotypic change. Analysis shows mononuclear cells, as determined by FSC and SSC characteristics, with HCL-v cells gated as red events. (B) The hairy cell leukemia variant cells were negative for CD25. However, a small, minor subpopulation was positive for CD25. (C) Two years later, the neoplastic cells appeared to have acquired expression of CD25, but may actually represent an expansion of the minor CD25 positive population at diagnosis (B).