Value of Gene Expression Profiling by Taqman® Low Density Arrays in the Diagnosis and Prognosis of Acute Myeloid Leukemia.

So far, none of the high-density microarray studies in acute myeloid leukemia (AML) have provided a useful approach with relevant diagnostic and prognostic value for the routine clinical practice. We aimed to assess the clinical utility of a small combination of genes and fusion genes included in a microfluidic card by means of real-time quantitative PCR (RQ-PCR) in AML patients.

Ninety-six clinically relevant markers were analyzed simultaneously using TaqMan® Low Density Arrays (TLDAs) (Applied Biosystems, Foster City, CA). This plataform includes: 3 control genes (ABL1, GADPH and GUS), 36 AML specific fusion transcripts described, NPM1 mutations and 55 genes which RNA expression levels have been associated with prognosis or pathogenic process in AML. To assess the feasibility of the designed TLDA we evaluated the gene expression profile of 191 leukemia patients at diagnosis: 167 AML (inv(16): n=11, t(8;21): n=9, t(15;17): n=7, with 11q23 abnormalities: n=27, with other cytogenetic abnormalities: n=20 and normal karyotype n=93), 16 pre-B-ALL, 5 T-ALL and 3 CML with rare BCR-ABL fusions (b2a3, b3a3 and e19a2).

We observed a high correlation between TLDA results for fusion transcripts and those obtained by gold standard techniques (RQ-PCR based on Europe against Cancer protocols). Indeed, we identified 89 patients with different fusion transcripts, including 18 cases previously reported as negative by cytogenetics or FISH approach. Patients with a favorable karyotype showed a distinctive gene expression profile. Thus, inv(16) AML overexpressed: MYH11, CLIP3, CTNNB1, SPARC, SNAI1 and MN1; t(8;21) AML expressed high levels of ETO, POU4F1, CAV1, CD34, FOXO3A, PRAME and BAALC, and t(15;17) AML showed upregulation of HGF, FGF13, WT1 and PRAME. Further, the 51 NPM1 mutated patients (38 A, 5 B, 6 D and 2 DD1, confirmed by sequencing) also showed a specific gene profile, showing high NPM1, CD34, ABCB1, ABCG2, BAALC, PROM1 or BCL2 RNA levels and low HOXA7, HOXA9 and MEIS1 RNA levels. In contrast, AML with 11q23 abnormalities or with FLT3-ITD mutations did not exhibit a characteristic pattern. Several genes (EVI1, BAALC, ERG, PRAME or PIM1) showed prognostic significance in AML with normal karyotype and AML with NPM1 mutations, thus confirming TLDA as a useful approach for risk assessment in AML. Interestingly, PIM1 overexpression was an independent predictor for shorter relapse-free (RFS) and overall survival (OS) in patients with normal karyotype (p=0.012 and p<0.001, respectively) and patients harboring NPM1 mutations (p=0.004 and p=0.002). Moreover, patients with FLT3-ITD mutations and high levels of PIM1 showed an extremely poor prognosis (2-year RFS 26% and OS 30%). These results were confirmed in multivariate analyses.

We showed the diagnostic and prognostic value of the designed TLDA platform for evaluation of AML patients. Furthermore, in our series PIM1 has been identified as the most important prognostic marker and thus this oncogene could be used for a more accurate risk classification of AML patients.

Gutiérrez:Janssen Cilag: Honoraria; Celgene: Honoraria. Díaz-Mediavilla:Janssen-Cilag: Honoraria; Celgene: Honoraria.