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Targeting the tyrosine kinase activity ofBCR-ABL represents a very promising therapeutic strategy in chronic myeloid leukemia (CML). Despite strong efficacy of the tyrosine kinase inhibitor STI571, resistance has been observed in a significant proportion of patients in advanced CML stage or in Ph-positive acute lymphoid leukemia (ALL). We investigated in this study the mechanism of resistance to STI571 through point mutations in the tyrosine kinase domain and/or BCR-ABL gene amplification in 24 patients (16 in chronic phase and 8 in accelerated phase of the disease) who obtained no cytogenetic response to STI571 treatment. Screening for the already-described Thr315Ile point mutation in the ABL domain using a reverse transcription polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) technique, 3 patients showed a proportion of mutated transcript at the time of resistance. The same technique failed to detect mutation at diagnosis, but a specific allele-specific oligonucleotide (ASO)–PCR on DNA for the Thr315Ile mutation and, after sequencing, for 2 newly described Phe311Leu and Met351Thr substitutions, showed the presence of rare mutated cells prior to STI571 therapy. Furthermore, the increased proportion of mutated cells during treatment detected by ASO-PCR strongly suggested clonal selection by the functional inhibiting effect of these mutations. Finally, no BCR-ABL gene amplification was detected by fluorescent in situ hybridization (FISH) in the 24 STI571-resistant patients. Our data support that in CML patients treated with STI571, ABL mutations are not restricted to the accelerated phase of the disease and that, at least in some cases, mutations seem to occur prior to STI571 therapy, probably as second mutational events during the course of CML.

Mutation of the nucleophosmin ( NPM ) gene has been reported as the most frequent mutation in acute myeloid leukemia (AML), especially in the presence of a normal karyotype. In this subgroup of intermediate-risk AML, the identification of other gene mutations (eg, FLT3 , CCAAT/enhancer-binding protein-α [ CEBPA ]) has helped to refine the prognosis. This study explored the prevalence and the prognostic impact of NPM mutations in a cohort of 106 patients with normal-karyotype AML. NPM exon 12 mutations were detected by polymerase chain reaction (PCR) and fragment analysis for the insertion/deletion globally resulting in a 4-bp insertion. NPM mutations were detected in 47% of patients and were associated with a high white blood cell count, involvement of the monocytic lineage (M4/M5), and a decreased prevalence of CEBPA mutations. Complete remission rate and long-term outcome did not differ between NPM-mutated and -nonmutated patients. Prospective studies are needed to confirm the definitive place of NPM mutation detection to predict AML response to therapy.

Mutations of the ten eleven translocation 2 gene (TET2) have recently been reported in myelodysplastic syndrome and myeloproliferative neoplasms. We analyzed the incidence and prognostic value of TET2 point mutations and other genomic alterations by direct sequencing and single nucleotide polymorphism microarray analysis in 111 de novo acute myeloid leukemia, who had all achieved complete remission (CR). Mutations were observed in 19 (17%) of the 111 patients compared with 10 (27%) of 36 patients who had failed to achieve CR (P = .2). In the 111 patients who had achieved CR, TET2 alterations were only significantly associated with NPM1 mutations but not with other pretreatment characteristics. TET2 gene status was not significantly correlated with disease-free survival and overall survival, both in the entire cohort and in patients with normal karyotype.

Familial platelet disorder (FPD), a rare autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities, is considered as a model of genetic predisposition to acute myeloid leukemia (AML). So far, monoallelic RUNX1 germline mutations have been found in 19 of 20 families with reported FPD, and the analysis of blast cells from only 5 patients at acute leukemia (AL) stage has shown no additional RUNX1 abnormality. Here, we performed RUNX1 analysis at constitutional and somatic levels in 8 persons with FPD who developed AL from 4 independent families. In addition to the germline RUNX1 mutation, we identified a second RUNX1 alteration in 6 AML cases (acquired point mutations in 4 cases and duplication of the altered RUNX1 allele associated with acquired trisomy 21 in 2 other cases). Although haploinsufficiency of RUNX1 causes FPD, our findings suggest that a second genetic event involving RUNX1 is often associated with progression to AML.

Acquired molecular abnormalities (mutations or chromosomal translocations) of the RUNX1 transcription factor gene are frequent in acute myeloblastic leukemias (AMLs) and in therapy-related myelodysplastic syndromes, but rarely in acute lymphoblastic leukemias (ALLs) and chronic myelogenous leukemias (CMLs). Among 18 BCR-ABL+ leukemias presenting acquired trisomy of chromosome 21, we report a high frequency (33%) of recurrent point mutations (4 in myeloid blast crisis [BC] CML and one in chronic phase CML) within the DNA-binding region of RUNX1. We did not found any mutation in de novo BCR-ABL+ ALLs or lymphoid BC CML. Emergence of the RUNX1 mutations was detected at diagnosis or before the acquisition of trisomy 21 during disease progression. In addition, we also report a high frequency of cryptic chromosomal RUNX1 translocation to a novel recently described gene partner, PRDM16 on chromosome 1p36, for 3 (21.4%) of 14 investigated patients: 2 myeloid BC CMLs and, for the first time, 1 therapy-related BCR-ABL+ ALL. Two patients presented both RUNX1 mutations and RUNX1-PRDM16 fusion. These events are associated with a short survival and support the concept of a cooperative effect of BCR-ABL with molecular RUNX1 abnormalities on the differentiation arrest phenotype observed during progression of CML and in BCR-ABL+ ALL.

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in approximately 50% of adult acute myeloid leukemia (AML) with normal karyotype. More than 40 mutant variants have been identified. Most of these mutations consist of a 4-bp insertion, which can be used as a target for minimal residual disease (MRD) monitoring. We previously checked the stability of NPM1 mutations at relapse in 21 NPM1-mutated patients at initial diagnosis. In this prospective study, we evaluated MRD by real-time quantitative PCR (RQ-PCR) in 36 NPM1-mutated AML patients corresponding to 33 adult and 3 pediatric cases, treated according to the French ALFA9801 or ALFA9802 and ELAM02 protocols, respectively. Out of these patients, 31/34 (91%) had normal karyotype, 13/33 (39%) had a high initial white blood cell count, and 10/36 (28%) were FLT3-Intern Tandem Duplication (FLT3-ITD) positive. 28 (78%) patients carry NPM mutation A, 3 (8%) mutation B and 5 (14%) other rare variants. RQ-PCR assays using a mutation-specific primer were performed on cDNA for mutation A and B and on genomic DNA for other NPM1 mutants. In our experiments, the maximal reproductible sensitivity of NPM1-based MRD detection is about 10−4 on genomic DNA and 10−5 to 10−6 on cDNA. The median follow-up was 260 days [40–791]. 2 to 9 follow-up samples from bone marrow and/or peripheral blood were analysed per patient. No correlation was found between leukocytosis at diagnosis and initial expression ratio of NPM1 mutation. The study of MRD log reduction after induction therapy shows that molecular responses are very heterogeneous (from 4.10−2 to more than 1.10−5), but 50% of patients reach at least a 4 log reduction in NPM1 levels. Patients with FLT3-ITD tend to have lower log reduction after induction than patients without FLT3-ITD, although not statistically significant (P=0.07). The analysis of NPM1-MRD in bone marrow and in peripheral blood at the same follow-up time-points shows that NPM1 levels can be until 1 log higher in bone marrow. This indicates that the evaluation of NPM1-MRD in bone marrow is more informative than in peripheral blood. We found all relapses had NPM1-MRD levels comparable to those observed at diagnosis. Among the 5 patients who relapsed so far, 2 were predictable by increasing MRD levels 1 to 4 months before hematological relapse. In 29 out of 36 cases, we could monitor MRD by both NPM1 mutation and WT1 gene expression. The comparison of the MRD profiles obtained by these two approaches reveals some discordant results, which can be, at least in part, explained by difference in the sensitivity of the RQ-PCR techniques, since the sensitivity of WT1 expression as MRD target is generally not higher than 10−3. In conclusion, NPM1 mutations are very specific and sensitive markers for MRD monitoring in AML. Further studies are required to determine if NPM1-MRD provides an independent prognostic factor and may be useful for therapeutic stratification in AML patients with intermediate risk cytogenetics.

Background: Besides the extensively studied point mutations in the BCR/ABL TK domain as the clinically most important cause of imatinib (IM) resistance either in CML and Philadelphia-positive (Ph+) ALL, additional acquired genetic events contributing to resistance/disease progression are not fully understood. Many evidences suggest that the enhanced survival and differentiation arrest of the CML blast crisis (BC) cells depends on the cooperation of BCR/ABL with other genes deregulated during disease progression. A recent study has identified RUNX1/AML1 transcription factor gene as a modulator of the cellular response towards IM in vitro and in vivo in mice, suggesting its possible involvement in disease persistence in IM-resistant CML patients. Purpose: We investigated RUNX1 molecular abnormalities (point mutations or cryptic chromosomal RUNX1 translocation to a novel recently described gene partner PRDM16 at 1p36) in 18 Ph+ leukemias. The observation that RUNX1 mutated allele is frequently duplicated by acquired trisomy of the altered chromosome 21 in acute myeloblastic leukemias (AML) prompt us to focus our analysis on a selected cohort (1 CP-CML, 3 AP-CML, 8 myeloid BC, 1 lymphoid BC, 2 de novo Ph+ ALL-B, 1 de novo AML and 1 therapy-related- Ph+ ALL) presenting acquired trisomy 21 along disease course. Methods: From peripheral blood leucocytes, recurrent mutations were investigated by sequencing DNA PCR fragments corresponding to exon 3 to 8 of RUNX1. RUNX1-PRDM16 fusion was investigated by RT-PCR (i.e. RUNX1 exon 5-PRDM16 exon 2 junction) and by FISH using the TEL/AML1 dual color/dual fusion translocation probes (Vysis, Downer’s grove, IL, USA). Results: We report a high frequency (33%) of recurrent point mutations (4 in myeloid BC CML and 1 in CP-CML) within the DNA-binding region of RUNX1. We did not find any mutation in de novo BCR/ABL+ ALLs or lymphoid BC CMLs. Onset of RUNX1 mutations was detected at diagnosis or before the acquisition of trisomy 21. We also report a high frequency of RUNX1-PRDM16 fusion for 3 out of 7 investigated patients: 2 CMLs and, for the first time reported to our knowledge, 1 therapy-related BCR/ABL+ ALL. RUNX1-PRDM16 is probably transcribed at low levels since only few bone marrow metaphases with trisomy 21 were t(1;21)(p36;q22) positive. Two patients presented both RUNX1 mutations and RUNX1-PRDM16 fusion. All these events are associated with a poor survival: 14 out of 18 patients died with a median survival of 3 months after the diagnosis of the acquired clonal trisomy 21. Furthermore, none of the patients in our study raised durable optimal responses to IM. Conclusion: Our data support the concept of a cooperative effect of BCR/ABL with molecular RUNX1 abnormalities on the differentiation arrest phenotype observed during progression of CML and in BCR/ABL+ ALL. Our findings also support that it may exist a heterogeneous genetic status at diagnosis of CML, underlying the need of further investigations able to define more precisely the molecular disease characteristics at diagnosis for better therapeutic decision adjustments.

A strong correlation between trisomy 13 and AML1 mutation has been recently reported in AML, particularly in M0 FAB subtype (Dicker et al, Silva et al). Whereas the exact mechanism of this coincidence has not been completely clarified, this correlation suggests a probable cooperation of these two events in leukemogenesis. FLT3, located on the 13q12 region, has been proposed to be implicated by over expression induced by gene dosage effect. However, trisomy 13 remains a rare event in AML and its association with AML1 mutations is not constant. To better document this association, we studied, a cohort of patients with trisomy 13 by array-CGH, which is a powerful tool to detect cryptic alterations as small size gene copy number variations (CNV). In addition we searched for AML1 mutations and performed the correlation with FLT3 alterations. We studied 12 AML with trisomy 13 (7 of which were M0 FAB subtype) and 6 additional patients affected by non-AML hematological malignancies (2 T-ALL, 1 NHL and 3 MPD). We performed direct sequencing of the exons 3 to 8 of AML1. Array-CGH was performed on 10/12 AML patients using Agilent CGH-Array 185k with pooled normal DNA as reference. GEP was performed using HG-U133 plus 2.0 Affymetrix Array on 5 patients characterized by trisomy 13 and AML1 mutations. AML1 mutations were observed in 8/18 patients including 6 of the 7 M0 AML, which represents a particularly high rate even in this subtype (86%). On the contrary, none of the non-AML patients harbored AML1 gene mutation. FLT3 locus analysis in the 12 AML patients showed the following results: None FLT3-ITD was observed whereas 5 FLT3-D835 mutations were detected (42%). FLT3 over expression was observed by GEP on 4/5 analyzed patients all without FLT3-D835 mutation. The remaining patient presented a FLT3-D835 mutation. CGH study confirmed trisomy 13 in 9/10 AML cases with weak amplitude for 4 patients, strongly suggesting that trisomy 13 was a sub-clonal anomaly, in accordance with caryotype results. Only one other recurrent abnormality was observed by CGH in 3 AML patients: 2 gains and one deletion affecting chromosome 21, all including AML1 locus. Moreover, CNV incidence was strong in these patients traducing a great genomic instability: there were 34 CNV gains and 23 deletions equally distributed all along the genome with a size extending from 300kb to complete chromosome. Among the interstitial CNV loci were included the following genes (CD68, CDH1, CDX2, CTNNA3, DOCK2, EIF3S9, EIF4A1, ERG, GNA12, IGF2, KLRC3, MAD1L1, MAFA, MKL2, VAV2) that could perhaps play a role in leukemogenesis. Here we confirmed the strong correlation between AML1 mutation and trisomy 13. Trisomy 13 is often sub-clonal indicating this alteration could be an additional event contributing to leukemia progression. In accordance with previous reported data, FLT3 over expression or FLT3-D835 mutation could cooperate with AML1 mutation. However, alteration of genes located on other CNV could not be excluded.

NPM mutation (NPMm) has been recently reported as the most frequent mutation in AML, especially in the presence of a normal karyotype. Association of NPMm with an increased complete remission (CR) rate has been suggested, but the long-term prognosis is not known. The abnormal mutated NPM protein shows an aberrant cytoplasmic localization that allows an immunohistochemical detection of NPM status. However, all mutations reported are insertion/deletion of exon 12 resulting in a global insertion of 4 nucleotides. We therefore developed a NPMm detection test based on DNA PCR amplification and fragment length analysis. As previously reported, we observed a higher frequency of NPMm in AML with normal karyotype (47%, 50/106) than in CBF AML (0%, 0/7) or in AML with poor risk cytogenetic (13%, 4/32). We further evaluated the clinical profile and the prognosis of NPM mutations in a retrospective cohort of 106 patients with normal karyotype treated according to the ALFA-9000 or ALFA-9802 protocols between 1990 and 2004. In these patients aged 17–65 years, NPMm was significantly associated with a high white blood cell count (69 vs 18 G/L, p

AML with normal karyotype constitutes a heterogeneous group considered of intermediate cytogenetic risk. Several gene mutations (CEBPAm, FLT3/ITD, MLL/PTD, NPMm) have recently helped to precise the prognostic in this AML subset. At the patient level, target gene expression profiles might be more accurate than gene mutations themselves to correctly assess prognosis. In this retrospective study including 120 AML patients with normal karyotype, we thus evaluated the global contribution of the expression of 7 genes, all reported to be individually associated with patient outcome: BAALC, EVI1, FLT3, HOXA9, TGIF1, TM4SF1, and TRIM16. Gene expression quantification was performed by real-time PCR using TBP as housekeeping gene. FLT3/ITD was present in 38/120 patients (32%), CEBPA mutation in 20/120 (17%) and MLL/PTD in 3/120 (3%). The recent NPM mutation (NPMm) was found in 50/106 screened patients (47%). We first performed an unsupervised hierarchical clustering analysis that delineated 4 subgroups of patients: 1) one group (n=13) with low HOXA9 expression and favorable prognosis, mostly composed of patients with CEBPAm (66%); 2) a second group (n=18) with high EVI1 expression, low rate of recurrent gene mutations, and poor outcome; 3–4) two groups (n=29 and 60) with high frequency of NPM mutations (70 and 56%) and intermediate outcome. Overall survival (OS) was significantly different in these 4 subgroups (p=.03). As no significant expression correlations were found between these 7 genes, we next considered each gene to contribute with an identical weight to patient outcome. Univariate analysis showed that all genes but BAALC were significantly associated with either OS and/or event-free survival (EFS). We built a molecular risk index based on best prediction cut-off analyses for the 6 remaining genes. This 3-class index (favorable risk, n=22; intermediate risk, n=57; high risk, n=41) was highly predictive of complete remission rate (100%, 91%, and 76%; p=.01), OS (6y-OS: 77%, 37%, and 25%; p=.0001) and EFS (6y-EFS: 62%, 29%, and 13%; p

Introduction: In core binding factor (CBF) acute myeloid leukemia (AML), the disruption of CBFa (AML1) or CBFb genes through t(8;21) or inv(16) impairs normal hematopoietic differentiation, but is considered to be insufficient to induce leukemogenesis. The need of an additionnal oncogenic event that promotes cellular proliferation has been suggested. This proliferative signal may be delivered by mutated receptor tyrosine kinases (RTKs) or RAS proto-oncogenes. In a retrospective study, we evaluate the incidence and the prognosis of RTKs (FLT3/D835, KIT/D816 and KIT/Ex8 insertion/deletion) and N/K-RAS mutations in patients with CBF AML. Patients: Genomic DNA were obtained from the bone marrow or blood of 81 patients under 60 years with CBF AML (t(8;21)/inv(16) : 48/33) : 21 treated in the pediatric LAME trial (27/6) and 60 in the adult ALFA trial (33/15) between 1990 and 2004. The median follow-up is 5 years. Results: RTKs mutations are found in 15/74 (20%) patients. KIT mutations are present in 11/74 (15%) patients, at a lower rate than previously described. KIT/D816 mutations are observed in 2/49 (4%) t(8;21) patients and 1/32 (3%) inv(16) patients. KIT/Ex8 mutations are slightly more present in inv(16) than in t(8;21) patients (5/32 vs 3/42, 16% vs 7%). One of the 5/81 patients (6%) with FLT3/D835 mutations also presents a KIT/Ex8 mutation. RAS mutations rates are significantly higher in inv(16) than in t(8;21) patients (11/33 vs 4/38, 33% vs 11%), with mostly N-RAS mutations (75%). No correlation between RTKs or RAS mutations and age or WBC is observed. Four patients did not achieve complete remission (CR rate : 95%), 2 with FLT3/D835 and 3 with RAS mutations. In univariate analysis, RTKs mutations are significantly associated with a shorter OS and EFS (p=.001 and p=.02, resp.) KIT mutations are associated with a shorter EFS and RFS (p=.002 and p=.003) in t(8;21) patients but do not affect the outcome of inv(16) patients in our study. Patients with FLT3/D835 mutations have significantly shorter OS, EFS and RFS (p Conclusion: Inv(16) and t(8;21) cytogenetics are classically associated with a low risk of relapse. Patients with CBF AML are no more considered by many teams for allogeneic stem cell transplantation, even with a related donor. However, screening for RTKs mutations may identify patients with a more adverse outcome and thus susceptible to benefit from intensified protocols or tyrosine kinase inhibitors.

Background: The relatively low incidence of Ras mutations in acute lymphoblastic leukemia (ALL) has suggested that Ras pathway plays only a minor role in the disease. However, the recent finding of activating mutations in PTPN11, which encodes SHP-2 phosphatase, FLT3, or suggest that Ras can also be activated through upstream mechanisms in childhood ALL. Assessing the frequency, spectrum, and relation with clinical features of these mutations should permit to better evaluate the implication of Ras pathway activation. Methods: 209 children (aged 1 to 17 yrs) with B-lineage ALL were studied at diagnosis. ALL with a t(12;21) were excluded from the study since no mutation of the Ras pathway has been found previously in this type of ALL. All patients were enrolled in the EORTC 58951 trial and the median follow up was 3 years. Relapse samples were also studied in 20/26 children who relapsed. Mutations of PTPN11, N-Ras, and H-Ras were screened by direct sequencing. FLT3 length mutations were screened by capillary electrophoresis, and FLT3 activating loop mutations (D835/I836) by EcoRV digestion of PCR products. FLT3 mutations were further caracterized by sequencing. Results: N-Ras or K-Ras was mutated in 28 patients (13%). PTPN11 mutations were found in 10 (5%) cases (9 in exon 3, 1 in exon 13) and were absent at remission, excluding a Noonan syndrome in these patients. FLT3 was mutated in 6 (3%) cases (one internal tandem duplication, and 5 ins/del). Other specific mutations were MLL rearrangement in 2 (1%) cases, E2A-PBX1 in 10 (5%) cases, and BCR-ABL in 8 (4%) cases. With exception of one patient having both E2A-PBX1 and a N1-Ras mutation, genetic deffects were never found together. No difference was observed at presentation between ALL with Ras, PTPN11 and FLT3 mutations and other ALL regarding age, gender, WBC, or risk group. A trend toward over-representation of hyperdiploid caryotype was observed in mutated ALL (58% vs 46% in non mutated ALL) but it was not significant. No difference was either observed in response to prephase, minimal residual disease or relapse rate. The Ras mutation found at presentation was lost in 3 out of 4 patients who relapsed. In one of these patients, a new Ras mutation appeared at relapse. In an additional patient Ras mutation was only found at relapse. Conclusion: We confirmed the presence of FLT3, and PTPN11 activating mutations in childhood ALL. However, mutation of either of these genes was only found in 21% of TEL-AML1 negative B-lineage childhood ALL, which is lower than previously described. The mutual exclusion of t(9;22), Ras, PTPN11, and FLT3 mutations can be explained by their partially redundant effect on Ras pathway and is in favor of their implication in leukemogenesis. The frequency of mutations activating directly or undirectly Ras pathway suggests that, contrary to what what was previously thought, Ras could represent a major pathway in ALL Surprisingly, while FLT3 and PTPN11mutations remained stable during the course of the disease, this was not the case Ras for mutations. This could explain the lack of prognostic relevance of these mutations and possibly reduce the interest of treatment strategies specifically targeting Ras in ALL.