Daniela Diverio

Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin

Acute myeloid leukemia (AML) carrying NPM1 mutations and cytoplasmic nucleophosmin (NPMc+ AML) accounts for about one-third of adult AML and shows distinct features, including a unique gene expression profile. MicroRNAs (miRNAs) are small noncoding RNAs of 19–25 nucleotides in length that have been linked to the development of cancer. Here, we investigated the role of miRNAs in the biology of NPMc+ AML. The miRNA expression was evaluated in 85 adult de novo AML patients characterized for subcellular localization/mutation status of NPM1 and FLT3 mutations using a custom microarray platform. Data were analyzed by using univariate t test within BRB tools. We identified a strong miRNA signature that distinguishes NPMc+ mutated (n = 55) from the cytoplasmic-negative (NPM1 unmutated) cases (n = 30) and includes the up-regulation of miR-10a, miR-10b, several let-7 and miR-29 family members. Many of the down-regulated miRNAs including miR-204 and miR-128a are predicted to target several HOX genes. Indeed, we confirmed that miR-204 targets HOXA10 and MEIS1, suggesting that the HOX up-regulation observed in NPMc+ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD+ samples were characterized by up-regulation of miR-155. Further experiments demonstrated that the up-regulation of miR-155 was independent from FLT3 signaling. Our results identify a unique miRNA signature associated with NPMc+ AML and provide evidence that support a role for miRNAs in the regulation of HOX genes in this leukemia subtype. Moreover, we found that miR-155 was strongly but independently associated with FLT3-ITD mutations.

Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that encode aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia.

The acute promyelocytic leukaemia (APL) 15;17 translocation generates a PML/RAR alpha chimeric gene which is transcribed as a fusion PML/RAR alpha mRNA. Molecular studies on a large series of APLs revealed great heterogeneity of the PML/RAR alpha transcripts due to: (i) variable breaking of chromosome 15 within three PML breakpoint cluster regions (bcr1, bcr2 and bcr3), (ii) alternative splicings of the PML portion and (iii) alternative usage of two RAR alpha polyadenylation sites. Nucleotide sequence analysis predicted two types of proteins: multiple PML/RAR alpha and aberrant PML. The PML/RAR alpha proteins varied among bcr1, 2 and 3 APL cases and within single cases. The fusion proteins contained variable portions of the PML N terminus joined to the B‐F RAR alpha domains; the only PML region retained was the putative DNA binding domain. The aberrant PML proteins lacked the C terminus, which had been replaced by from two to ten amino acid residues from the RAR alpha sequence. Multiple PML/RAR alpha isoforms and aberrant PML proteins were found to coexist in all APLs. These findings indicate that two potential oncogenic proteins are generated by the t(15;17) and suggest that the PML activation pathway is altered in APLs.

Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is characterised by a unique fusion transcript, PML/RAR alpha. We tested for this transcript in 35 APL patients who were in apparent remission after various treatments. 11 of 13 patients who tested positive 4 months after achieving remission were in relapse 1-4 months later. All 22 patients who tested negative at 4 months were disease-free after a further 3 months to five years. The test may therefore prove useful in determining the need for additional treatment during clinical remission.

Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580–5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.

Immunophenotype of adult and childhood acute promyelocytic leukaemia: correlation with morphology, type of PML gene breakpoint and clinical outcome. A cooperative Italian study on 196 cases

Acute promyelocytic leukaemia (APL), characterized by a specific PML‐RARα fusion gene resulting from translocation t(15;17) and by a high response rate to differentiation therapy with all‐trans retinoic acid, presents clinical (varying WBC counts, age and treatment outcome), morphological (hypergranular M3 and hypogranular M3V) and molecular (three isoforms of PML breakpoint) heterogeneity.

Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol.

Alterations in the FLT3 gene, including internal tandem duplications (ITDs) and D835 mutations occur frequently in acute myelogenous leukemia. We investigated the prevalence and clinico-biological correlations of FLT3 ITDs and D835 mutations in 90 patients with acute promyelocytic leukemia (APL) receiving the AIDA protocol. Twenty patients in which both presentation and relapse material was available were analyzed sequentially. Thirty-three patients (37%) harbored the ITD, and seven (7.7%) the D835 mutation in blasts obtained at diagnosis. Presence of ITDs was strongly associated with high WBC count (P = 0.0001), M3 variant (P = 0.0004), and the short (BCR3) PML/RARα isoform (P = 0.003). There was no difference in response to induction in the two ITD+ve and ITD−ve groups, while a trend towards inferior outcome was observed for ITD+ve cases when analyzing disease-free survival (DFS) and relapse risk (RR). These differences, however, did not reach statistical significance. Sequential studies showed variable patterns in diagnostic and relapse material, ie ITD (−ve/−ve, +ve/+ve, +ve/−ve, −ve/+ve) and D835 (−ve/−ve, +ve/−ve, −ve/+ve). Our results indicate that FLT3 alterations are associated in APL with more aggressive clinical features and suggest that these lesions may not play a major role in leukemia progression.

AIDA 0493 protocol for newly diagnosed acute promyelocytic leukemia: very long-term results and role of maintenance

All-trans-retinoic acid (ATRA) has greatly modified the prognosis of acute promyelocytic leukemia; however, the role of maintenance in patients in molecular complete remission after consolidation treatment is still debated. From July 1993 to May 2000, 807 genetically proven newly diagnosed acute promyelocytic leukemia patients received ATRA plus idarubicin as induction, followed by 3 intensive consolidation courses. Thereafter, patients reverse-transcribed polymerase chain reaction-negative for the PML-RARA fusion gene were randomized into 4 arms: oral 6-mercaptopurine and intramuscular methotrexate (arm 1); ATRA alone (arm 2); 3 months of arm1 alternating to 15 days of arm 2 (arm 3); and no further therapy (arm 4). Starting from February 1997, randomization was limited to ATRA-containing arms only (arms 2 and 3). Complete remission was achieved in 761 of 807 (94.3%) patients, and 681 completed the consolidation program. Of these, 664 (97.5%) were evaluated for the PML-RARA fusion gene, and 586 of 646 (90.7%) who tested reverse-transcribed polymerase chain reaction-negative were randomized to maintenance. The event-free survival estimate at 12 years was 68.9% (95% confidence interval, 66.4%-71.4%), and no differences in disease-free survival at 12 years were observed among the maintenance arms.

CD7 positive acute myeloid leukaemia: a subtype associated with cell immaturity.

Seventeen patients with acute myeloid leukaemia (AML) whose blasts co‐expressed the T‐cell associated CD7 antibody were identified among 160 consecutive AML cases. Fourteen had FAB defined AML according to morphocyto‐chemical criteria, whereas three patients were classified as ‘MO’ on the basis of immunophenotype. The incidence of CD7 positivity was particularly significant in the less differentiated subtypes MO and M1 compared with other FAB groups (P < 0.001). In all cases the myeloid determinants CD13 and/or CD33 were associated with CD7 expression. Other B‐lymphoid (CD10, CD19) or T‐lymphoid (CD2, surface and cytoplasmic CD3) markers were analysed and found to be negative. Five out of 15 cases examined were TdT+. Clonal rearrangements of the immunoglobulin heavy chain (IgH) and/or T‐cell receptor (TcR) β chain genes were identified in only three out of 13 cases. Among these, one out of five coexpressing TdT showed IgH rearrangement when analysed at the DNA level.

PAX5 Expression in Acute Leukemias Higher B-Lineage Specificity Than CD79a and Selective Association with t(8;21)-Acute Myelogenous Leukemia

The transcription factor PAX5 plays a key role in the commitment of hematopoietic precursors to the B-cell lineage, but its expression in acute leukemias has not been thoroughly investigated. Hereby, we analyzed routine biopsies from 360 acute leukemias of lymphoid (ALLs) and myeloid (AMLs) origin with a specific anti-PAX5 monoclonal antibody. Blasts from 150 B-cell ALLs showed strong PAX5 nuclear expression, paralleling that of CD79a in the cytoplasm. Conversely, PAX5 was not detected in 50 T-cell ALLs, including 20 cases aberrantly coexpressing CD79a. Among 160 cytogenetically/molecularly characterized AMLs, PAX5 was selectively detected in 15 of 42 cases bearing the t(8;21)/AML1-ETO rearrangement. Real-time reverse transcription-PCR studies in t(8;21)-AML showed a similar up-regulation of PAX5 transcript in all of the 8 tested samples (including 4 cases that were negative at anti-PAX5 immunostaining), suggesting that PAX5 is expressed in t(8;21)-AML more widely than shown by immunohistochemistry. Interestingly, PAX5+ t(8;21)-AML also expressed CD79a and/or CD19 (major transcriptional targets of PAX5 in B-cells) in 10 of 12 evaluable cases. Our results indicate that PAX5 is a more specific marker than CD79a for B-cell ALL diagnosis. Moreover, among AMLs, PAX5 expression selectively clusters with t(8;21), allowing its immunohistochemical recognition in a proportion of cases, and likely explaining a peculiar biological feature of this subset of myeloid leukemias, i.e. the aberrant expression of B-cell genes.

Early prediction of treatment outcome in acute myeloid leukemia by measurement of WT1 transcript levels in peripheral blood samples collected after chemotherapy.

The Wilms’ tumor gene WT1 is a reliable marker for minimal residual disease assessment in acute leukemia patients. The study was designed to demonstrate the potential use of WT1 to establish quality of remission in acute leukemia patients for early identification of patients at high risk of relapse. A prospective study based on a quantitative Real–Time PCR (TaqMan) assay in 562 peripheral blood samples collected from 82 acute leukemia patients at diagnosis and during follow-up was established. The evaluation of WT1 in peripheral blood samples after induction chemotherapy can distinguish the continuous complete remission patients from those who obtain only an “apparent” complete remission and who could relapse within a few months. WT1 helps identify patients at high risk of relapse soon after induction chemotherapy allowing post-induction therapy in high risk patients to be intensified.