WESTERN BLOT IDENTIFICATION OF NPM1 LEUKEMIC MUTANTS IN CYTOLOGICAL AML SAMPLES: A POWERFUL DIAGNOSTIC TECHNIQUE

Nucleophosmin (NPM1) mutations occur in 50-60% of adult acute myeloid leukemia (AML) patients with normal karyotype and are predictors of favourable prognosis. For these reasons, analysis of NPM1 mutations has recently become a major new step in the diagnosis and prognostic stratification of AML patients. About 40 different molecular NPM1 mutation variants have been so far identified, with about 90% represented by mutation A (about 80%) and B (about 10%), other NPM1 mutations being extremely rare. Mutations of the NPM1 gene can be reliably identified by molecular biology techniques or by immunohistochemistry through detection of aberrant cytoplasmic NPM positivity (NPMc+). Mutational analysis is carried out only in specialized laboratories; immunohistochemistry overcome this problem but it is only applicable to paraffin sections from bone marrow biopsies. Hereby, we describe a highly sensitive and specific Western blot method that allows the easy identification of NPM1 leukemic mutants in cytological samples from AML patients. Rabbit polyclonal antibodies were generated against the altered C-terminal portion of the most common NPM mutant protein (type A). Western blot analysis of a selected number of AML samples proved that these antibodies reacted specifically with the NPM mutant but not with the wild-type NPM protein. These findings prompted us to use this method to analyze systematically cytological leukemic samples and to compare blindly the results obtained by Western Blot with those derived from immunohistochemical studies and, when available, from NPM1 mutational analysis. A total of 114 AML patients classified by immunohistochemistry into NPMc+ (cytoplasmic-positive; n=57) and NPMc- (cytoplasmic-negative; n=57) were enclosed in the study. Western Blot analysis was performed retrospectively in 83 AML cases and prospectively in 32 cases. We investigated a total of 174 cytological preparations (82 from NPMc+ and 92 from NPMc- AMLs) of various types, including frozen dry cell pellets of Ficoll-isolated leukemic cells; 1 to 2 drops of fresh whole bone marrow or peripheral blood; or even cells obtained scraping the surface of leukemic cytospins or smears. Western Blot analysis was performed according to standard procedures. Seventy-two out of 82 (88%) NPMc+ AML samples resulted positive at Western Blot analysis. Results obtained in cytological material of different types were comparable, indicating the high flexibility of the method. However, retrospective Western Blot analysis of badly preserved leukemic smears sometimes gave negative Results. In addition to NPM mutant A, the specific anti-NPM mutant antibodies recognized NPM mutant proteins of type B, D, E, and L. For patients studied prospectively, Western Blot analysis predicted NPM1 mutation in all (16 out of 16) NPMc+ AML patients investigated. Importantly, no false-positive results were registered. In conclusion, Western Blot analysis represents a new highly sensitive, specific and low cost assay for detecting the most common NPM1 mutations in AML cytological samples processed in different ways. This also represents the first example of employment of Western Blot for identification of a specific genetic lesion in AML, that should turn out to be very useful not only for leukemia diagnosis but also for research purposes.