7.1 ANTIBODY REACTS WITH LEUKEMIC CELLS OF ACUTE MYELOID LEUKEMIA BEARING INV(16)
Authors
F Mannelli, S Bencini, I Cutini, G Gianfaldoni, B Scappini, A Bosi
Abstract
Background. Several antigenic patterns have been associated with genotypic aberrancies in acute leukemia. The expression of the human counterpart of mouse NG2 molecule, targeted by 7.1 monoclonal antibody (MoAb), has been demonstrated in association with rearrangement of the mixed lineage leukemia (MLL) gene in acute leukemia. Aims. The aim of the study was to investigate the expression of 7.1 MoAb in an unselected cohort of patients with acute myeloid leukemia (AML) in order to establish its correlation with specific genotypic groups. Methods. An extensive panel of quadruple combinations of MoAb was applied on a suspension of bone marrow or/and peripheral blood of AML samples at diagnosis. CD45 PerCP was applied in every tube to allow gating of cell populations. The expression of NG2 was assessed using the following staining: CD65-FITC/7.1-PE/CD45-PerCP/CD34-APC. MoAb 7.1 PE was purchased from Beckman-Coulter. 50,000 events per tube were acquired through a FACSCalibur flow cytometer. Analysis of antigen expression was performed through Infinicyt software. NG2 expression was explored on leukemic cells, gated on the basis of CD45 and side scatter, through: i) percentage of 7.1+ cells; ii) mean fluorescence intensity corrected for background fluorescence (MFI) determined by an isotype control; iii) coefficient of variation (CV) of fluorescence intensity. Cytogenetic aberrancies were determined by conventional karyotyping and by FISH for inv(16), t(8;21), t(15;17) and MLL; mutations of NPM1 gene were revealed by immunohistochemical detection of aberrant cytoplasmatic protein localization on trephine biopsy. Results. Cytogenetics. 64 AML patients were studied. According to genotype, they were subdivided as follows: a) t(15;17) # 6; b) t(8;21) # 4; c) inv(16) # 5; d) normal karyotype # 28, of whom 16 NPM1-wild type and 12 NPM1-mutated. No cases with MLL rearrangements were seen. According to SWOG stratification, 10 and 7 patients had high- and intermediate-risk (other than normal) karyotype respectively. Conventional karyotyping was not possible in 4 cases due to lack of growth. Flow cytometry. Leukemic cells were identified by dim expression of CD45 and low scatter signal; overall, blasts represented a median of 50.78% of global cellularity. No expression of 7.1 was detectable in patients with t(15;17), t(8;21) or other cytogenetic aberrancies. Neither within normal karyotype group, regardless of NPM1 status, 7.1 resulted positive in any case. Of note, we detected a partial expression of 7.1 on blasts from AML with inv(16). Specifically, all 5 patients with such genotype showed 7.1+ cells, with a median percentage of 23% (range 10-28) within blasts. Median MFI was 4.91 (range 3.47-9.86); median CV 275.41 (257.62-291.89). Apart from inv16, expression of 7.1 was seen in an AML case diagnosed as blastic plasmacytic dendritic cell neoplasm according to WHO classification; this finding was not unexpected since Orfao et al. (Haematologica 2004) have reported it in this entity. Summary/Conclusions. Our study describes the correlation between the expression of 7.1, so far associated with MLL abnormalities, and AML with inv(16). If confirmed within a larger cohort, this phenotypic aberrancy should be deepened for potential prognostic role and minimal residual disease assessment.
Figure. Expression in a case of AML with inv(16).