Corine J. Hess

Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples

In acute myeloid leukemia (AML), activating mutations in the fms-like tyrosine kinase 3 (FLT3) gene predict poor prognosis. We determined FLT3 internal tandem duplications (FLT3/ITD) and D835 point mutations in paired initial and relapse samples from 80 pediatric and adult AML patients. One D835 point mutation was found in an initial pediatric AML sample. Fms-like tyrosine kinase 3/ITDs were present in 21 initial and 22 relapse samples (26.3 and 27.5%, respectively). Interestingly, FLT3/ITD positivity was related to a significantly shorter time to relapse, most pronounced when the ITD-positive status was found at relapse (P<0.001). However, FLT3/ITD status changed between diagnosis and relapse in 14 cases. In four patients, the FLT3/ITD became undetectable at relapse in five patients FLT3/ITDs were only detected at relapse, and in five patients the length or number of FLT3/ITDs changed. Gain of FLT3/ITDs may suggest oligoclonality with selective outgrowth of the FLT3/ITD-positive clone, whereas losses may reflect ITDs in the more mature leukemic cells rather than in the leukemic stem cell, or, alternatively, that other genetic aberrations provided a greater selective advantage. Studying FLT3/ITD kinetics in minimal residual disease setting may provide some answers for the changes we observed. Fms-like tyrosine kinase 3/ITD is a relevant marker for prognosis, and remains an important target for therapeutic inhibition.

High INDO (indoleamine 2,3-dioxygenase) mRNA level in blasts of acute myeloid leukemic patients predicts poor clinical outcome

In this study on patients with acute myeloid leukemia, a high INDO expression in leukemic blasts was associated with a poor outcome. Indoleamine 2,3-dioxygenase degrades the amino acid tryptophan which is essential for T cells. Tryptophan depletion causes T-cell cycle arrest and solid tumors that express high levels of indoleamine 2,3-dioxygenase can create immune suppression. Recently, blasts of patients with acute myeloid leukemia were shown to express indoleamine 2,3-dioxygenase. We determined INDO (encoding gene for indoleamine 2,3-dioxygenase) mRNA expression in leukemic blasts of 286 patients with acute myeloid leukemia by gene-expression profiling. Results were validated by quantitative polymerase chain reaction analysis in blasts of an independent cohort of 71 patients. High INDO expression was correlated to significantly shortened overall and relapse-free survival. Correlation of INDO expression to relevant known prognostic factors and survival identified high INDO expression as a strong negative independent predicting variable for overall and relapse-free survival. Inhibition of indoleamine 2,3-dioxygenase expressed by myeloid leukemic blasts may result in breaking immune tolerance and offers new therapeutic options for patients with acute myeloid leukemia.

Activated Intrinsic Apoptosis Pathway Is a Key Related Prognostic Parameter in Acute Myeloid Leukemia

PURPOSE By parallel assessment of multiple apoptosis-related transcripts, we aimed to refine the current concept of apoptosis resistance in acute myeloid leukemia (AML) and identify the combination of genes best predicting overall survival (OS). PATIENTS AND METHODS The reverse transcriptase multiplex ligation-dependent probe amplification technique was used for simultaneous quantification of 31 apoptosis-related transcripts in viable (7AAD-/AnnexinV-) blasts (CD45dim) from bone marrow aspirates of 120 newly diagnosed AML patients. By forward selection, a prognosis-predicting gene expression profile was constructed. The predictive validity of this profile was assessed by cross validation. RESULTS High transcript levels were associated with poor OS for seven of 31 genes, three of which were proapoptotic. The average expression of all 12 antiapoptotic genes was associated with poor OS (P = .029). A similar association with poor OS was found for the average expression of all 19 proapoptotic genes (P = .009). Forward selection and cross validation revealed the antiapoptotic gene BIRC3 and the proapoptotic genes BAX-(l) and BMF to optimally predict OS. Three equally sized patient groups, constructed by ranking the cross-validated prognoses of the patients, were clearly distinct (median OS times were 8.2, 16.7, and 85.6 months). CONCLUSION High expression of both pro- and antiapoptotic genes predicted poor OS, which postulates a mechanism of activation of the apoptosis pathway as a whole. This mechanism, which culminates in a three-gene expression signature, allows accurate clinical outcome prediction in AML and puts efforts to target single antiapoptosis genes in a new perspective.

Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia.

By assessment of the methylation status of 25 candidate tumor suppressor genes (TSGs) in 119 acute myeloid leukemia (AML) patients and 5 controls, we aimed to determine whether simultaneous methylation of multiple TSGs exerts prognostic impact. Methylation-specific multiplex ligation probe amplification (MS-MLPA) revealed methylation of at least one TSG in 59/119 patients, while no methylation was found in controls. Methylation of different TSGs within patients was substantially correlated (intra-class correlation; 0.38). ESR1 methylation (34/119) strongly predicted concurrent methylation of other genes, OR 7.33 (95%CI 4.13–12.99). A Cox regression model that included the three most frequently methylated TSGs ESR1, CDKN2B/p15 and IGSF4, showed ESR1 to have opposite effects on overall survival (OS) compared with the other two, HR 0.22 (95% CI 0.09–0.53) and HR 1.66 (95% CI 0.73–3.79), HR 1.61 (95%CI 0.66–3.93). By assessment of CDKN2B/p15 and IGSF4 methylation, patients with methylation at multiple loci can be identified. Accumulation of methylation aberrancies is much more pronounced in ESR1 methylated patients. When combined, the methylation status of ESR1, CDKN2B/p15 and IGSF4 enable identification of patient subgroups with large differences in OS (p <0.0001). This study shows that methylation profiling allows risk stratification in AML. In addition, ESR1 methylation may reflect a biological pathway that leads to hypermethylation of multiple genes, which is reflected by methylation of IGSF4 and/or CDKN2B/p15.

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100–1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01–0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.

Hypermethylation of the FANCC and FANCL promoter regions in sporadic acute leukaemia

Objective: Inactivation of the FA-BRCA pathway results in chromosomal instability. Fanconi anaemia (FA) patients have an inherited defect in this pathway and are strongly predisposed to the development of acute myeloid leukaemia (AML). Studies in sporadic cancers have shown promoter methylation of the FANCF gene in a significant proportion of various solid tumours. However, only a single leukaemic case with methylation of one of the FA-BRCA genes has been described to date, i.e. methylation of FANCF in cell line CHRF-288. We investigated the presence of aberrant methylation in 11 FA-BRCA genes in sporadic cases of leukaemia. Methods: We analyzed promoter methylation in 143 AML bone marrow samples and 97 acute lymphoblastic leukaemia (ALL) samples using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Samples with aberrant methylation were further analyzed by bisulphite sequencing and tested for mitomycin C sensitivity using Colony Forming Units assays. Results: MS-MLPA showed promoter methylation of FANCC in one AML and three ALL samples, while FANCL was found methylated in one ALL sample. Bisulphite sequencing of promoter regions confirmed hypermethylation in all cases. In addition, samples with hypermethylation of either FANCC or FANCL appeared more sensitive towards mitomycin C in Colony Forming Units assays, compared to controls. Conclusion: Hypermethylation of promoter regions from FA-BRCA genes does occur in sporadic leukaemia, albeit infrequently. Hypermethylation was found to result in hypersensitivity towards DNA cross-linking agents, a hallmark of the FA cellular phenotype, suggesting that these samples displayed chromosomal instability. This instability may have contributed to the occurrence of the leukaemia. In addition, this is the first report to describe hypermethylation of FANCC and FANCL. This warrants the investigation of multiple FA-BRCA genes in other malignancies.

Inhibition of the Intrinsic Apoptosis Pathway Downstream of Caspase-9 Activation Causes Chemotherapy Resistance in Diffuse Large B-Cell Lymphoma

Purpose: Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. Experimental Design: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9–mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry. Results: Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist. Conclusions: We conclude that the intrinsic, caspase-9–mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.

Flt-3 internal tandem duplication hampers differentiation of AML blasts towards leukemic dendritic cells

Flt-3 internal tandem duplication hampers differentiation of AML blasts towards leukemic dendritic cells