Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Abstract

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.

Figure 1.

(A) Immunoelectron microscopy images showing nucleolar NPM (Clone 376) in a lymphocyte (arrow), also shown at higher magnification, then a range of leukocytes from normal volunteer donors stained with NPM (Clone 376) with no pre-treatment and after pre-treatment (positive nucleoli staining shown in red). (B) A case of AML with NPM1 mutation showing NPM immunostaining (red, Clone 376) (arrows indicate blast cells with cytoplasmic staining) and at higher magnification. (C–D) Two cases of AML with NPM1 mutated showing nucleolar NPM immunostaining (red, Clone 376) with both positive and negative cytoplasmic labeling. (E–F) Two cases of AML with NPM1 wild-type stained for NPM (red, Clone 376), both with positive nucleoli. (E) Also shows positive cytoplasmic labeling. Then OCI-AML3 cell line (mixed with normal blood) shows positive NPM immunostaining (red, Clone 376) in the nucleoli and cytoplasm of the OCI-AML3 cell line (the arrow indicates a stained normal white blood cell).

Figure 2.

The case numbers correspond to Table 1. (A–B) AML with NPM1 mutation (case 1): (A) is the cell smear (NA24) and (B) the corresponding bone marrow trephine with cytoplasmic and nuclear labeling (clone 376). (C–D) AML NPM1 wild-type (case 20). (C) The cell smear with positive nucleoli and one cell with cytoplasmic labeling. (D) The corresponding bone marrow trephine with nuclear labeling (Clone 376).