Feasibility of the AML profiler (Skyline™ Array) for patient risk stratification in a multicentre trial: a preliminary comparison with the conventional approach.

Deoxyribonucleic acid microarrays allow researchers to measure mRNA levels of thousands of genes in a single experiment and could be useful for diagnostic purposes in patients with acute myeloid leukaemia (AML). We assessed the feasibility of the AML profiler (Skyline™ Array) in genetic stratification of patients with de novo AML and compared the results with those obtained using the standard cytogenetic and molecular approach. Diagnostic bone marrow from 31 consecutive de novo AML cases was used to test MLL-PTD, FLT3-ITD and TKD, NPM1 and CEBPAdm mutations. Purified RNA was used to assess RUNX1-RUNX1T1, PML-RARα and CBFß-MYH11 rearrangements. RNA remnants underwent gene expression profiling analysis using the AML profiler, which detects chromosomal aberrations: t(8;21), t(15;17), inv(16), mutations (CEBPAdm, ABD-NPM1) and BAALC and EVI1 expression. Thirty cases were successfully analysed with both methods. Five cases had FLT3-ITD. In one case, a t(8;21) was correctly detected by both methods. Four cases had inv(16); in one, the RNA quality was unsatisfactory and it was not hybridized, and in the other three, the AML profiler detected the genetic lesion - this being a rare type I translocation in one case. Two cases with acute promyelocytic leukaemia were diagnosed by both methods. Results for NPM1 mutations were concordant in all but two cases (2/11, non-ABD mutations). Analysis of costs and turnaround times showed that the AML profiler was no more expensive than the conventional molecular approach. These results suggest that the AML profiler could be useful in multicentre trials to rapidly identify patients with AML with a good prognosis. Copyright © 2016 John Wiley & Sons, Ltd.

A high proportion of cells carrying trisomy 12 is associated with a worse outcome in patients with chronic lymphocytic leukemia.

The prognosis of chronic lymphocytic leukemia (CLL) patients displaying trisomy 12 (+12) remains unclear. In this study, we analyzed the influence of the proportion of cells with +12, and other clinical and biologic factors, in time to first therapy (TTFT) and overall survival (OS), in 289 patients diagnosed with CLL carrying +12. Median OS was 129 months. One hundred seventy-four patients (60.2%) presented +12 in <60% of cells. TTFT and OS for this subgroup were longer than for the subgroup with +12 in ≥60% of cells, with a median TTFT of 49 months (CI95%, 39-58) vs 30 months (CI95%, 22-38) (P = 0.001); and a median OS of 159 months (CI95%, 119-182), vs 96 months (CI95%, 58-134) (P = 0.015). Other factors associated with a shorter TTFT were: Binet stage, B symptoms, lymphadenopathy, splenomegaly, high lymphocyte count, 11q-, high ß2 microglobulin, and high LDH. In the multivariate analysis, clinical stage, +12 in ≥60% of cells, high lymphocyte count, B symptoms, and 11q- in addition, resulted of significance in predicting shorter TTFT. Significant variables for OS were: Binet stage, lymphadenopathy, splenomegaly, high LDH, high ß2 microglobulin, 11q-, and CD38. In the multivariate analysis, only Binet stage, 11q-, and high ß2microglobulin significantly predicted shorter OS. CLL with +12 entails a heterogeneous group with intermediate prognosis. However, a high proportion of cells carrying +12 separates a subgroup of patients with poor outcome. Copyright © 2015 John Wiley & Sons, Ltd.

Multilineage dysplasia is associated with a poorer prognosis in patients with de novo acute myeloid leukemia with intermediate-risk cytogenetics and wild-type NPM1.

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

Biallelic losses of 13q do not confer a poorer outcome in chronic lymphocytic leukaemia: analysis of 627 patients with isolated 13q deletion.

Losses in 13q as a sole abnormality confer a good prognosis in chronic lymphocytic leukaemia (CLL). Nevertheless, its heterogeneity has been demonstrated and the clinical significance of biallelic 13q deletions remains controversial. We compared the clinico-biological characteristics of a series of 627 patients harbouring isolated 13q deletions by fluorescence in situ hybridization (FISH), either monoallelic (13q × 1), biallelic (13q × 2), or the coexistence of both clones (13qM). The most frequent 13q deletion was 13q × 1 (82·1%), while 13q × 2 and 13qM represented 8·6% and 9·3% of patients respectively. The median percentage of altered nuclei significantly differed across groups: 55%, 72·5% and 80% in 13q × 1, 13q × 2 and 13qM (P < 0·001). However, no significant differences in the clinical outcome among 13q groups were found. From 84 patients with sequential FISH studies, eight patients lost the remaining allele of 13q whereas none of them changed from 13q × 2 to the 13q × 1 group. The percentage of abnormal cells detected by FISH had a significant impact on the five-year cumulative incidence of treatment and the overall survival, 90% being the highest predictive power cut-off. In conclusion, loss of the remaining 13q allele is not enough to entail a worse prognosis in CLL. The presence of isolated 13q deletion can be risk-stratified according to the percentage of altered cells.

Structural alterations in chronic lymphocytic leukaemia. Cytogenetic and FISH analysis.

In this study, we described cytogenetics and fluorescence in situ hybridization (FISH) analysis performed in chronic lymphocytic leukaemia (CLL) patients with structural alterations. Results were correlated with clinical characteristics. A total of 38 CLL patients: 16 cases with complex and 22 with simple karyotypes were studied. For comparison of clinical parameters, a control group of 78 CLL patients with normal karyotype and without FISH genomic alterations were also evaluated. We found 38 structural abnormalities not previously described in the literature, 28 (74%) of them were translocations. In cases with complex karyotypes, chromosomes 6, 8 and 13 were the most frequently involved in new alterations (nine each), followed by chromosomes 12, 14 and 15 (six each). Chromosome 8p was particularly involved in losses, being 8p21-pter the commonest region of overlap. Cases with simple karyotypes, showed del(6q) as the most frequent alteration (39%). Del(9)(q11) was recurrent in our series. Analysis of clinical parameters showed significant differences in white blood count (p = 0.005) and platelet count (p = 0.015) between patients with structural alterations and the control group. In addition, patients with structural alterations had a significantly shorter time to first treatment (TFT) (29 months) than the control group (69 months) (p = 0.037). Cases with complex karyotypes had a lower proportion of patients in Rai 0 clinical stage (15.4% vs 75%) (p = 0.005) and higher ß2 microglobulin levels (3.3 vs 2.5 µg/mL) (p = 0.037) than those with simple karyotypes. Furthermore, a shorter TFT (13 months) and overall survival (56 months) in the complex karyotypes group compared with controls (69 and 144 months, respectively) (p = 0.015 and p = 0.005, respectively) were also found. Our results support the importance of cytogenetic analysis for clinical outcome in CLL and suggest that the diversity of genomic alterations is much greater than previously appreciated.

Core binding factor acute myeloid leukemia: the impact of age, leukocyte count, molecular findings, and minimal residual disease.

PURPOSE: Most patients with acute myeloid leukemia (AML) and genetic rearrangements involving the core binding factor (CBF) have favorable prognosis. In contrast, a minority of them still have a high risk of leukemia recurrence. This study investigated the adverse features of CBF AML that could justify investigational therapeutic approaches. PATIENTS AND METHODS: One hundred and fifty patients (median age 42 yr, range 16-69) with CBF AML (RUNX1-RUNX1T1 n = 74; CBFB-MYH11 n = 76) were prospectively enrolled into two consecutive CETLAM protocols at 19 Spanish institutions. Main clinic and biologic parameters were analyzed in the whole series. In non-selected cases with available DNA samples, the impact of molecular characterization and minimal residual disease (MRD) was also studied. RESULTS: Overall, complete remission (CR) rate was 89% (94% in ≤50 yr old and 72% in >50 yr, P = 0.002). At 5 yr, cumulative incidence of relapse (CIR) was 26 ± 1%, disease-free survival (DFS) 62 ± 6%, and overall survival (OS) 66 ± 4%. In multivariate analyses, leukocyte count above 20 × 10(9) /L, BAALC over-expression, and high copy numbers of RUNX1-RUNXT1 or CBFB-MYH11 after induction chemotherapy (CT) led to increased relapse rate. Regarding OS, age >50 yr, leukocyte count above 20 × 10(9) /L, and increased MN1 expression were adverse features. CONCLUSION: Age, leukocyte counts, BAALC, and MN1 gene expressions as well as high copy numbers of RUNX1-RUNXT1 or CBFB-MYH11 after induction chemotherapy are useful tools to predict the outcome and should be considered for risk-adapted therapy.

Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results.

Patients with chronic lymphocytic leukaemia (CLL) whose tumour cells harbour a 17p deletion (17p-) are universally considered to have a poor prognosis. The deletion can be detected at diagnosis or during the evolution of the disease, particularly in patients who have received chemotherapy. We sought to evaluate the natural history of patients with 17p- CLL, identify predictive factors within this prognostic subgroup, and evaluate the results of different therapeutic approaches. Data from 294 patients with 17p- CLL followed up at 20 different institutions was retrospectively collected and analysed. Median age was 68 (range 27-98) years at the time of fluorescence in situ hybridization analysis. After 17p- documentation, 52% received treatment, achieving an overall response rate of 50%. Median overall survival was 41 months, and was significantly shorter in patients with elevated beta(2)-microglobulin concentration (P < 0·001), B symptoms (P = 0·016), higher percentage of cells with deletion (P < 0·001), and acquired deletions (P = 0·012). These findings suggest that patients with 17p- CLL have a variable prognosis that can be refined using simple clinical and laboratory features, including 17p- clone size, beta2-microglobulin concentration, presence of B symptoms and type of deletion (de novo versus acquired).

Immunophenotype distinction between acute promyelocytic leukaemia and CD15- CD34- HLA-DR- acute myeloid leukaemia with nucleophosmin mutations.

Acute promyelocytic leukaemia (APL) is a unique clinicobiologic entity that can be successfully treated with All-trans Retinoic Acid ATRA-based regimens. Some cases of acute myeloid leukaemia (AML) with nucleophosmin (NPM) mutations have an immunophenotype that is similar to APL. The objective of the study is to compare antigenic expression in a group of APL patients with that in AML patients with NPM mutations and an APL-like immunophenotype (CD15- CD34- HLA-DR-). A consecutive series of 40 APL and 12 NPM patients with an APL-like phenotype were included in the study. Immunophenotypic patterns were investigated by multiparametric flow cytometry. Promyelocytic leukaemia-retinoic acid receptor-α transcript type, NPM and FLT3 mutations were investigated using conventional methods. Statistically significant differences were found between APL and NPM-mutated AML in CD33, CD13, CD2 and CD110 reactivity. CD2 expression was absent in every patient with NPM-mutated AML. In addition, mean fluorescence intensity and the coefficient of variation (cv) of CD33 and CD13 showed statistical differences between the two groups for CD33 (p = 0.007) and a trend to significance for CD13 (p = 0.05). Furthermore, among 45 evaluable patients, CD110 expression statistically differentiates between the two groups: [2/33 (6%) in the APL group and 8/12 (66.6%) in the NPM-mutated AML (p = 0.014)]. However, these traits were subtle, raising the possibility of practical diagnostic challenges. In conclusion, CD110 and CD33 reactivity may be useful to distinguish APL from NPM-mutated AML with CD15, CD34 and HLA-DR negativity. Nevertheless, cytogenetic and molecular characterization is necessary to establish the accurate diagnosis of AML.

The use of tetradecanoylphorbol acetate-stimulated peripheral blood cells enhances the prognostic value of interphase fluorescence in situ hybridization in patients with chronic lymphocytic leukemia.

Interphase fluorescence in situ hybridization (I-FISH) studies have a remarkable prognostic value in patients with chronic lymphocytic leukemia (CLL). I-FISH studies can be performed either on tetradecanoylphorbol acetate stimulated peripheral blood cells (I-FISH-TPA) or unstimulated peripheral blood mononuclear cells (I-FISH-PBMC). The aim of the study was to evaluate whether this finding was clinically relevant in a group of 235 patients with CLL. Fifty-six patients had both I-FISH-TPA and I-FISH-PBMC results. Compared with uncultured cells, the cytogenetic detection rate rose from 57 to 80% with the use of TPA-stimulated cells (P = 0.014). I-FISH-TPA provided a better prediction of treatment-free survival compared with I-FISH-PBMC (P = 0.031 vs. 0.166). Then, I-FISH-PBMC results from 93 historical patients were compared with 86 recent patients with I-FISH-TPA results. Genomic aberrations were detected in 46 and 67% of patients from the I-FISH-PBMC and I-FISH-TPA cohorts, respectively. The detection rate of 13q deletion as the only aberration increased from 10% with I-FISH-PBMC to 37% with I-FISH-TPA (P = 0.006). In conclusion, I-FISH-TPA increased the detection rate of 13q deletion and had an improved prognostic value compared with I-FISH-PBMC.

Additional chromosome abnormalities in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy.

BACKGROUND: Acute promyelocytic leukemia is a subtype of acute myeloid leukemia characterized by the t(15;17). The incidence and prognostic significance of additional chromosomal abnormalities in acute promyelocytic leukemia is still a controversial matter. DESIGN AND METHODS: Based on cytogenetic data available for 495 patients with acute promyelocytic leukemia enrolled in two consecutive PETHEMA trials (LPA96 and LPA99), we analyzed the incidence, characteristics, and outcome of patients with acute promyelocytic leukemia with and without additional chromosomal abnormalities who had been treated with all-trans retinoic acid plus anthracycline monochemotherapy for induction and consolidation. RESULTS: Additional chromosomal abnormalities were observed in 140 patients (28%). Trisomy 8 was the most frequent abnormality (36%), followed by abn(7q) (5%). Patients with additional chromosomal abnormalities more frequently had coagulopathy (P=0.03), lower platelet counts (P=0.02), and higher relapse-risk scores (P=0.02) than their counterparts without additional abnormalities. No significant association with FLT3/ITD or other clinicopathological characteristics was demonstrated. Patients with and without additional chromosomal abnormalities had similar complete remission rates (90% and 91%, respectively). Univariate analysis showed that additional chromosomal abnormalities were associated with a lower relapse-free survival in the LPA99 trial (P=0.04), but not in the LPA96 trial. However, neither additional chromosomal abnormalities overall nor any specific abnormality was identified as an independent risk factor for relapse in multivariate analysis. CONCLUSIONS: The lack of independent prognostic value of additional chromosomal abnormalities in acute promyelocytic leukemia does not support the use of alternative therapeutic strategies when such abnormalities are found.

Beta2-microglobulin is a better predictor of treatment-free survival in patients with chronic lymphocytic leukaemia if adjusted according to glomerular filtration rate.

Even in the era of newer and sophisticated prognostic markers, beta(2)-microglobulin (B2M) remains a simple but very powerful predictor of treatment-free survival (TFS) and overall survival (OS) in patients with chronic lymphocytic leukaemia (CLL). However, B2M levels are heavily influenced by the patient's glomerular filtration rate (GFR) and this study aimed to evaluate whether GFR-adjusted B2M (GFR-B2M) had improved prognostic value compared to unadjusted B2M in a cohort of over 450 consecutive CLL patients from two separate institutions. Multivariate analysis identified a significantly shorter TFS in patients who were ZAP-70 + (P < 0.001), with increased GFR-B2M (P < 0.001), and del(11q) or del(17p) as detected by fluorescence in situ hybridization (FISH; P < 0.001). When OS was evaluated by multivariate analysis, age 65 years or older (P < 0.001) and poor risk FISH abnormalities (P < 0.001) had a confirmed adverse prognostic impact, but the predictive value of GFR-B2M was lost in the validation analysis. In all survival models, B2M did not attain independent significance unless GFR-B2M was eliminated from the analysis. In conclusion, GFR-B2M is a better predictor of TFS than unadjusted B2M in CLL patients.

Epigenetic-based treatments emphasize the biologic differences of core-binding factor acute myeloid leukemias.

Acute myeloid leukemia (AML) is a heterogeneous group of disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. The most common chromosomal lesions in AML are the t(8;21) and inv(16). To better understand the leukemogenic mechanism of these fusion proteins, we performed gene expression studies in samples from (8;21), AML1 mutated and inv(16) patients, as well as from the Kasumi-1 cell line and a U937 cell line expressing the AML1-ETO fusion gene. To assess the influence of associated epigenetic lesions, we performed gene expression studies in Kasumi-1 cells and cells extracted from an Inv(16) patient, both treated with demethylating and HDAC inhibitor agents. Shared deregulated genes in the different types of core-binding factor leukemias were identified. We found a tight link between Inv(16) and mutant AML1 samples. Furthermore, some of the genes deregulated by the leukemogenic process reverted to their normal expression with demethylating and HDAC inhibitor treatment, highlighting the role of chromatin remodeling processes in AML.

Detection of chromosomal abnormalities in chronic lymphocytic leukemia increased by interphase fluorescence in situ hybridization in tetradecanoylphorbol acetate-stimulated peripheral blood cells.

Interphase fluorescent in situ hybridization on unstimulated peripheral blood mononuclear cells (I-FISH-PBMC) is used to detect chromosomal abnormalities such as 11q-, 13q-, 17p-, and trisomy 12 in chronic lymphocytic leukemia (CLL). A total of 56 samples from 49 patients with CLL were studied using commercially available probes for chromosome regions 11q22.3 (ATM), 13q14 (13S272), 17p13 (p53) and 12 centromere (D12Z3). We compared the results obtained with I-FISH-PBMC and those with I-FISH on TPA (tetradecanoylphorbol acetate; phorbol-12-myristate-13-acetate) stimulated peripheral blood cells (I-FISH-TPA) used for conventional cytogenetics, to evaluate the usefulness of I-FISH-TPA. The proportion of abnormal nuclei obtained with the I-FISH-TPA was higher than that found with I-FISH-PBMC (P < 0.001). Consequently, 15 cases with a negative or borderline result with I-FISH-PBMC became positive with I-FISH-TPA for 11q- (n = 2), 13q- (n = 9), and trisomy 12 (n = 4). In all but one of these, chromosomal abnormalities were confirmed by either metaphase FISH or conventional G-banding. Detection of cytogenetic aberrations thus increased from 51% with I-FISH-PBMC to 78% with I-FISH-TPA. Notably, all 15 of the cases that reached the diagnostic thresholds for 11q-, 13q-, and trisomy 12 had a slight lymphocytosis. An absolute lymphocyte count <8.7 x 10(9)/L was found to be the critical threshold (P = 0.037) below which I-FISH-TPA rather than I-FISH-PBMC should be performed. Not only could I-FISH-TPA detect a higher proportion of abnormal interphase nuclei, but it could also identify abnormal CLL cases that might be overlooked with use of I-FISH-PBMC, especially those with low absolute lymphocyte counts. I-FISH-TPA is thus a reliable technique for clinical diagnostics in CLL.

Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis.

The JAK2/V617F mutation has been noted in essential thrombocytemia. We investigated 19 cases with refractory anemia with ringed sideroblasts (RARS), including three RARS with thrombocytosis (RARS-T). Only the RARS-T patients showed this mutation. More cases need to be analyzed to determine the prevalence of the JAK2/V617F mutation in RARS-T.

A new D816 c-KIT gene mutation in refractory AML1-ETO leukemia.

One of the most common genetic events in acute myeloid leukemia (AML) is the t(8;21) (q22;q22) translocation, which contributes to leukemic transformation. However, different lines of evidence suggest that the AML1-ETO rearrangement is not sufficient to cause the full leukemic phenotype. Secondary genetic alterations such as mutations in receptor tyrosine kinases are thus required to induce overt AML. The incidence of c-KIT mutations in exon 17 was evaluated in 37 Spanish patients with AML1-ETO+ leukemias. c-KIT mutations were present in only two cases (6.6%) and were shown to be associated with an adverse outcome. The frequency of c-KIT mutations described here is much lower than in other reports.

Dual-color split signal fluorescence in situ hybridization assays for the detection of CALM/AF10 in t(10;11)(p13;q14-q21)-positive acute leukemia.

We developed dual-color split fluorescence in situ hybridization (FISH) assays to detect AF10 and/or CALM rearrangements. Among nine cases of acute leukemia with translocation breakpoints at 10p13 and 11q14-21, a CALM/AF10 rearrangement was found in seven and was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) in all. In 2/7 cases, FISH detected CALM/AF10 in extramedullary leukemic infiltrations in the mediastinum and breast. As expected, FISH was less sensitive than RT-PCR for disease monitoring of CALM-AF10 positive cases. This new FISH assay reliably discriminates between MLL/AF10 and CALM/AF10 genomic rearrangements, identifies variant and complex CALM/AF10 translocations and detects the CALM/AF10 rearrangement in extramedullary leukemic infiltrations.

The hypereosinophilic syndrome: fluorescence in situ hybridization detects the del(4)(q12)-FIP1L1/PDGFRA but not genomic rearrangements of other tyrosine kinases.

BACKGROUND AND OBJECTIVES: According to WHO criteria, the idiopathic hypereosinophilic syndrome (HES) is defined as persistent eosinophilia (>1.5x10(9)/L) without underlying causes, which is associated with signs or symptoms of organ involvement. Increased bone marrow blasts (>5%) or cytogenetic/genetic markers indicate chronic eosinophilic leukemia (CEL). A cryptic deletion of 4q12, i.e. del(4)(q12), producing the FIP1L1/PDGFRA fusion gene, identifies a distinct CEL subgroup (4q-/CEL). Our aims were: a) to use interphase-fluorescent in situ hybridization (FISH) to detect the cryptic 4q12 deletion; b) to compare the clinico-hematologic features of 4q-/CEL with other HES; c) to investigate whether PDGFRB, FGFR1, ABL1, and ETV6-activated tyrosine kinases are rearranged in CEL/HES. DESIGN AND METHODS: This multicenter study included 20 patients fulfilling the WHO criteria for HES and 6 patients without signs/symptoms of end-organ involvement. Double-color FISH was applied in all cases to investigate del(4)(q12). Further interphase-FISH assessed whether PDGFRB/5q33, FGFR1/8p11, ABL1/9q34, and ETV6/12p13, undergo rearrangements in HES. RESULTS: Ten of the 26 patients (9 males and 1 female) had a cryptic del(4)(q12)-FIP1L1/PDGFRA which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis in four. Hepatomegaly and splenomegaly were significantly more frequent in these 10 than in the other 16 patients. Seven of these 10 patients received imatinib mesylate therapy and all achieved hematologic remission. In 3 of the patients interphase-FISH and RT-PCR demonstrated cytogenetic and molecular remission. Improvements were observed in signs and symptoms of cardiac and central nervous system involvement in 2 and 1 patient, respectively. Rearrangements of PDGFRB, FGFR1, ABL1, or ETV6 were not detected in this study. INTERPRETATION AND CONCLUSIONS: FISH is a reliable diagnostic test for differentiating 4q-/CEL from other forms of HES, allowing an early diagnosis of good responders to imatinib mesylate therapy. For the first time we show that PDGFRB, FGFR1, ABL1 and ETV6 are not rearranged in HES and 4q-/CEL cases we studied.

Complex measurements may be required to establish the prognostic impact of immunophenotypic markers in AML.

OBJECTIVES: The prognostic impact of immunophenotypic markers in acute myeloid leukemia (AML) is controversial. METHODS: We retrospectively analyzed the value of CD34, CD117, CD7, and CD123 expression in a consecutive series of 592 adult patients with de novo AML. RESULTS: CD34+ measured as a percentage (≥2.88%) and CD34 mean fluorescence intensity (MFI) (≥146.79, arbitrary units [AU]) expression had a prognostic impact in terms of overall survival (OS; P = .005, P = .003), leukemia-free survival (LFS; P = .011, P < .001), and cumulative incidence of relapse (CIR; P = .014, P =. 001). The percentage of CD117+ cells (61.29%) was associated with shorter LFS (P =. 043), and CD117 MFI (≥284.01 AU) was associated with a shorter OS (P =. 033) and LFS (P =. 028). In the multivariate analysis, high CD34 MFI retained the independent value as predictor of LFS and CIR (P =. 012; hazard ratio [HR], 1.59; 95% confidence interval [CI], 1.11-2.28 and P =. 045; HR, 1.58; 95% CI, 1.01-2.46). CONCLUSIONS: CD34 positivity threshold with prognostic relevance is low (3% positive cells). Immunophenotypic findings in AML probably could only be fully exploited after a complex analysis that takes into account unconventional thresholds and the MFI.

FLT3 mutations are associated with other molecular lesions in AML.

The basic molecular defects underlying acute myeloid leukemias (AML) seem to be caused by inactivating mutations in transcription factors which control normal myeloid differentiation (Class II mutations) and genetic lesions in tyrosine kinases resulting in constitutive activation (Class I mutations). We sought to determine the frequency of associated mutations (Class I + Class II) in a consecutive series of adult de novo AML (353 patients) in order to stress the validity of this model. Mutations and rearrangements at the FLT3, AML1/ETO, CBFbeta/MYH11, AML1, CEBPalpha and MLL genes were investigated using standard molecular methods. Despite the limitations of the study (DNA availability hampered c-kit and ras mutational analysis), 3.4% of patients showed Class I + Class II mutations. Our findings could be consistent with the cooperative model. The search for new tyrosine kinases which can be the target of molecular lesions in AML warrants further investigation.

Id4 is deregulated by a t(6;14)(p22;q32) chromosomal translocation in a B-cell lineage acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: Chromosome translocations resulting in gene overexpression are commonly associated with lymphoid neoplasia. Enhancer elements of the immunoglobulin or T-cell receptor (TCR) loci are abnormally located in the vicinity of the entire coding sequences of genes which exert an influence on the normal maturation and differentiation program of lymphoid cells. DESIGN AND METHODS: A patient who presented with a B-cell lineage acute lymphoblastic leukemia had a t(6;14)(p22;q32). Cytogenetic and molecular findings confirmed the involvement of IgH. Molecular cloning of the breakpoint revealed that this was located near the coding sequence of the Id4 gene, a helix-loop-helix (HLH) inhibitor protein. Alu-repeated sequences at the 6p22 end flanked a short stretch of 10 bases shared by the 6p22 and 14q32 ends, suggesting that a deletion or a looping-Alu mediated mispairing mechanism may lead to this chromosome translocation. RESULTS: Northern blot and real-time polymerase chain reaction analyses showed that the Id4 mRNA was abnormally overexpressed in this case. Only the two smaller Id4 mRNA products were detected (1.6 and 1.1 kb). Immunohistochemical analysis of Id4 protein was also assayed in a series of hematologic malignancies. Marked overexpression was found in two cases of T-cell prolymphocytic leukemias and in four B-cell lineage acute lymphoblastic leukemia including one case with the t(8;14) and another case with a p53 mutation. INTERPRETATION AND CONCLUSIONS: The Id4 gene may behave as an oncogene in some human leukemias, perhaps through its capacity to sequester specific B-cell transcription factors. A genetic recombination between Alu-repeated sequences may not be the exclusive mechanism of generating pathogenic chromosomal translocations.