Evaluation of gene expression signatures predictive of cytogenetic and molecular subtypes of pediatric acute myeloid leukemia

Background

Pediatric acute myeloid leukemia is a heterogeneous disease characterized by non-random genetic aberrations related to outcome. The genetic subtype is currently detected by different diagnostic procedures which differ in success rate and/or specificity.

Design and Methods

We examined the potential of gene expression profiles to classify pediatric acute myeloid leukemia. Gene expression microarray data of 237 children with acute myeloid leukemia were collected and a double-loop cross validation approach was used to generate a subtype-predictive gene expression profile in the discovery cohort (n=157) which was then tested for its true predictive value in the independent validation cohort (n=80). The classifier consisted of 75 probe sets, representing the top 15 discriminating probe sets for MLL-rearranged, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q21;q22) and t(7;12)(q36;p13)-positive acute myeloid leukemia.

Results

These cytogenetic subtypes represent approximately 40% of cases of pediatric acute myeloid leukemia and were predicted with 92% and 99% accuracy in the discovery and independent validation cohort, respectively. However, for NPM1, CEBPA, MLL(-PTD), FLT3(-ITD), KIT, PTPN11 and N/K-RAS gene expression signatures had limited predictive value. This may be caused by a limited frequency of these mutations and by underlying cytogenetics. This latter is exemplified by the fact that different gene expression signatures were discovered for FLT3-ITD in patients with normal cytogenetics and in those with t(15;17)(q21;q22)-positive acute myeloid leukemia, which pointed to HOXB-upregulation being specific for FLT3-ITD⁺ cytogenetically normal acute myeloid leukemia.

Conclusions

In conclusion, gene expression profiling correctly predicted the most prevalent cytogenetic subtypes of pediatric acute myeloid leukemia with high accuracy. In clinical practice, this gene expression signature may replace multiple diagnostic tests for approximately 40% of pediatric acute myeloid leukemia cases whereas only for the remaining cases (predicted as ‘acute myeloid leukemia-other’) are additional tests indicated. Moreover, the discriminative genes reveal new insights into the biology of acute myeloid leukemia subtypes that warrants follow-up as potential targets for new therapies.     

Prognostic impact of white blood cell count in intermediate risk acute myeloid leukemia: relevance of mutated NPM1 and FLT3-ITD

Background

High white blood cell count at presentation is an unfavorable prognostic factor for treatment outcome in intermediate cytogenetic risk acute myeloid leukemia. Since the impact of white blood cell count on outcome of subgroups defined by the molecular markers NPMc⁺ and FLT3-internal tandem duplication (ITD) is unknown, we addressed this issue.

Design and Methods

We studied the effect of white blood cell count on outcome in a clinically and molecularly well-defined cohort of 525 patients with acute myeloid leukemia using these molecular markers. In addition, since an increased white blood cell count has been associated with an increased FLT3-ITD/FLT3 (wild-type) ratio, we investigated whether the effect of white blood cell count on outcome could be explained by the FLT3-ITD/FLT3 ratio.

Results

This analysis revealed that white blood cell count had no impact on outcome in patients with the genotypic combinations ‘NPMc⁺ without FLT3-ITD’ and ‘NPM1 wild-type with or without FLT3-ITD’. In contrast, white blood cell count had a significant impact on complete remission rate (P=0.034), event-free survival (P=0.009) and overall survival (P<0.001) in patients with the genotypic combination ‘NPMc⁺ with FLT3-ITD’. A FLT3-ITD/FLT3 ratio greater than 1 was also associated with a reduced complete remission rate (P=0.066) and significantly reduced event-free survival (P= 0.001) and overall survival (P=0.001) in patients with the genotypic combination ‘NPMc⁺ with FLT3-ITD’. Multivariable analysis revealed that white blood cell count and FLT3-ITD/FLT3 ratio were independent prognostic indicators for outcome in the subgroup with the genotypic combination ‘NPMc⁺ with FLT3-ITD’.

Conclusions

Our results demonstrate that both high white blood cell count and FLT3-ITD/FLT3 ratio are prognostic factors in patients with acute myeloid leukemia with the genotypic combination ‘NPMc⁺ with FLT3-ITD''.     

Integrative analysis of type-I and type-II aberrations underscores the genetic heterogeneity of pediatric acute myeloid leukemia

Background

Several studies of pediatric acute myeloid leukemia have described the various type-I or type-II aberrations and their relationship with clinical outcome. However, there has been no recent comprehensive overview of these genetic aberrations in one large pediatric acute myeloid leukemia cohort.

Design and Methods

We studied the different genetic aberrations, their associations and their impact on prognosis in a large pediatric acute myeloid leukemia series (n=506). Karyotypes were studied, and hotspot regions of NPM1, CEPBA, MLL, WT1, FLT3, N-RAS, K-RAS, PTPN11 and KIT were screened for mutations of available samples. The mutational status of all type-I and type-II aberrations was available in 330 and 263 cases, respectively. Survival analysis was performed in a subset (n=385) treated on consecutive acute myeloid leukemia Berlin-Frankfurt-Munster Study Group and Dutch Childhood Oncology Group treatment protocols.

Results

Genetic aberrations were associated with specific clinical characteristics, e.g. significantly higher diagnostic white blood cell counts in MLL-rearranged, WT1-mutated and FLT3-ITD-positive acute myeloid leukemia. Furthermore, there was a significant difference in the distribution of these aberrations between children below and above the age of two years. Non-random associations, e.g. KIT mutations with core-binding factor acute myeloid leukemia, and FLT3-ITD with t(15;17)(q22;q21), NPM1- and WT1-mutated acute myeloid leukemia, respectively, were observed. Multivariate analysis revealed a ‘favorable karyotype’, i.e. t(15;17)(q22;q21), t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22). NPM1 and CEBPA double mutations were independent factors for favorable event-free survival. WT1 mutations combined with FLT3-ITD showed the worst outcome for 5-year overall survival (22±14%) and 5-year event-free survival (20±13%), although it was not an independent factor in multivariate analysis.

Conclusions

Integrative analysis of type-I and type-II aberrations provides an insight into the frequencies, non-random associations and prognostic impact of the various aberrations, reflecting the heterogeneity of pediatric acute myeloid leukemia. These aberrations are likely to guide the stratification of pediatric acute myeloid leukemia and may direct the development of targeted therapies.     

Incorporating FLT3 inhibitors in the frontline treatment of FLT3 mutant acute myeloid leukemia

FLT3 mutations occur in up to a third of newly diagnosed patients with acute myeloid leukemia (AML) and confer poor prognosis. Clinical development of FLT3 tyrosine kinase inhibitors for AML initially involved broad-spectrum inhibitors (midostaurin, sorafenib) targeting multiple kinases. Addition of midostaurin to upfront intensive chemotherapy for younger patients with FLT3 mutant AML significantly improved overall survival and validated FLT3 as a therapeutic target. Other regimens such as sorafenib and hypomethylating agents (azacitidine, decitabine) have expanded the use of FLT3 inhibitors to other populations with FLT3 mutant AML. However, emerging data on new highly potent and specific FLT3 inhibitors such as quizartinib, gilteritinib, and crenolanib suggest that these agents may soon supplant midostaurin and sorafenib in the upfront setting. Using case presentations, this review provides guidelines and practical management strategies for frontline therapy of patients with newly diagnosed FLT3 mutant AML in the current era.     

Correlation of minimal residual disease cell frequency with molecular genotype in patients with acute myeloid leukemia

Background

About 70–80 percent of patients with acute myeloid leukemia enter complete remission, but at least half of these patients who achieve remission go on to relapse. Improved treatment is likely to come from increasing the time to relapse, especially for younger patients. With the vastly increasing number of targeted therapies there is a strong need for short-term end-points to efficiently test such therapies for further pursuance. Minimal residual disease assessment may offer such an end-point since it is a strong independent prognostic factor. As proof of principle we examined this concept for FLT3-ITD status at diagnosis.

Design and Methods

We determined FLT3-ITD status in bone marrow samples from 196 patients with newly diagnosed acute myeloid leukemia. The frequencies of residual leukemic cells of these 196 patients were assessed in 267 follow-up bone marrow samples using immunophenotypic assessment of minimal residual disease.

Results

The median frequency of residual leukemic cells after the first cycle of chemotherapy was 8.5-fold higher in patients with FLT3-ITD than in those with wild type FLT3. Such a difference translates into differences in survival, even if other potentially outcome-modulating mutations, such as NPM1, KIT, NRAS, KRAS, FLT3-exon 20 and PTPN11 are included in the analysis.

Conclusions

This study shows that it could be possible to study the efficacy of FLT3 inhibitors using the level of minimal residual disease as a short-term end-point.     

Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia

Background

C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models.

Design and Methods

C-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated.

Results

C-type lectin-like molecule-1 was expressed in 86.5% (45/52) of cases of acute myeloid leukemia, in 54.5% (12/22) of acute myeloid leukemia CD34⁺/CD38⁻ stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemia-derived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to C-type lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells.

Conclusions

Our results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.     

Co-administration of the mTORC1/TORC2 inhibitor INK128 and the Bcl-2/Bcl-xL antagonist ABT-737 kills human myeloid leukemia cells through Mcl-1 down-regulation and AKT inactivation

Effects of concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL in human acute myeloid leukemia cells were examined. Tetracycline-inducible Bcl-2/Bcl-xL dual knockdown markedly sensitized acute myeloid leukemia cells to the dual TORC1/2 inhibitor INK128 in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/xL antagonist ABT-737 sharply induced cell death in multiple acute myeloid leukemia cell lines, including TKI-resistant FLT3-ITD mutants and primary acute myeloid leukemia blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras, while exerting minimal toxicity toward normal hematopoietic CD34⁺ cells. Combined treatment was particularly active against CD34⁺/CD38⁻/CD123⁺ primitive leukemic progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal microenvironment. Notably, INK128 was more potent than the TORC1 inhibitor rapamycin in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737 activity. Mcl-1 ectopic expression dramatically attenuated INK128/ABT-737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 actions. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak knockdown reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic acute myeloid leukemia xenograft model. Analysis of subcutaneous acute myeloid leukemia-derived tumors revealed significant decrease in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with results obtained in vitro. These findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in acute myeloid leukemia.     

The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine

Background

Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double-stranded breaks and have been front-line drugs for the treatment of leukemia for many years. Voreloxin is a first-in-class anti-cancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. The efficacy and mechanisms of action of voreloxin in acute myeloid leukaemia were addressed in this study.

Design and Methods

Primary acute myeloid leukemia blasts (n = 88) and myeloid cell lines were used in vitro to study voreloxin through viability assays to assess cell killing and synergy with other drugs. Apoptosis and cell cycling were assessed by flow cytometry. DNA relaxation assays were utilized to determine that voreloxin was active on topoisomerase II.

Results

The mean lethal dose 50% (LD₅₀) (± standard deviation) of voreloxin for primary acute myeloid leukemia blasts was 2.30 μM (± 1.87). Synergy experiments between voreloxin and cytarabine identified synergism in 22 of 25 primary acute myeloid leukemia samples tested, with a mean combination index of 0.79. Apoptosis was shown to increase in a dose-dependent manner. Furthermore, voreloxin was active in the p53-null K562 cell line suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay.

Conclusions

Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of acute myeloid leukemia, a disease with a poor long-term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and cytarabine recommends further investigation of this topoisomerase II inhibitor.     

Clinical impact of FLT3 mutation load in acute promyelocytic leukemia with t(15;17)/PML-RARA

Background

Combined treatment with all-trans-retinoic acid and chemotherapy is extremely efficient in patients with acute promyelocytic leukemia with t(15;17)/PML-RARA, but up to 15% of patients relapse.

Design and Methods

To further clarify the prognostic impact of parameters such as FLT3 mutations, we comprehensively characterized the relation between genetic features and outcome in 147 patients (aged 19.7–86.3 years) with acute promyelocytic leukemia.

Results

Internal tandem duplications of the FLT3 gene (FLT3-ITD) were detected in 47/147 (32.0%) and tyrosine kinase domain mutations (FLT3-TKD) in 19/147 (12.9%) patients. FLT3-ITD or FLT3-TKD mutation status did not have a significant prognostic impact, whereas FLT3-ITD mutation load, as defined by a mutation/wild-type ratio of less than 0.5 was associated with trends to a better 2-year overall survival rate (86.7% versus 72.7%; P=0.075) and 2-year event-free survival rate (84.5% versus 62.1%, P=0.023) compared to the survival rates of patients with a ratio of 0.5 or more. Besides the t(15;17), an additional chromosomal abnormality was detected in 57 of 147 cases and did not show a significant impact on survival. White blood cell counts of 10×10⁹/L or less versus more than 10×10⁹/L were associated with a better 2-year overall survival rate (88.3% versus 69.4%, respectively; P=0.015), as was male sex (P=0.040). In multivariate analysis, only higher age had a significant adverse impact.

Conclusions

Prospective trials should further investigate the clinical impact of the FLT3-ITD/wild-type mutation load aiming to evaluate whether this parameter might be included in risk stratification in patients with acute promyelocytic leukemia.     

Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations

OBJECTIVE: Activating mutations in FLT3 are known to be a frequent transforming event in acute myeloid leukemia. Small molecule-inhibitor therapy targeting the FLT3 kinase is, therefore, an attractive strategy. FLT3 kinase inhibitors, such as PKC412, have already entered clinical trials. Even though results are encouraging, emergence of primary and secondary resistance does occur in the majority of patients. Thus, it will be crucial to carefully characterize the activity of every single compound against different activating and resistance FLT3-internal tandem duplication (ITD) mutations. Here we tested the efficacy of sunitinib and sorafenib to inhibit primary FLT3 activating mutations (ITD and D835Y) and of secondary resistance mutations. METHODS: Ba/F3 cell lines stably expressing oncogenic FLT3 mutations were used to calculate cellular IC(50) values for sunitinib and sorafenib using cell proliferation assays. Differential IC(50) values for sorafenib toward FLT3-ITD and FLT3-D835Y were confirmed by Western blotting. Cell death was measured by propidium-iodide staining and flow cytometry. RESULTS: Sorafenib inhibits FLT3-ITD more potent than FLT3-D835Y, while sunitinib is equally effective against both mutant forms of FLT3. Importantly, sensitivity toward sorafenib and sunitinib varies between the different secondary FLT3-ITD resistance mutations. CONCLUSIONS: These results establish sensitivity profiles for the FLT3 inhibitors sunitinib and sorafenib. This may help to develop rational treatment strategies for acute myeloid leukemia with these compounds.     

Hypocellular acute myeloid leukemia in adults: analysis of the clinical outcome of 123 patients

Background

The hypocellular variant of acute myeloid leukemia accounts for less than 10% of all cases of adult acute myeloid leukemia. It is defined by having less than 20 percent of cellular bone marrow in a biopsy at presentation. It is unclear in the literature whether the outcome of hypocellular acute myeloid leukemia differs from that of non-hypocellular acute myeloid leukemia.

Design and Methods

We retrospectively analyzed all the cases reported to be hypocellular acute myeloid leukemia between 2000 and 2009. A second pathology review was conducted and the diagnosis was confirmed in all cases.

Results

One hundred twenty-three (9%) patients were identified: patients with hypocellular acute myeloid leukemia were older than those with non-hypocellular acute myeloid leukemia (P=0.009) and more frequently presented with cytopenias (P<0.001). Forty-one patients with hypocellular acute myeloid leukemia had an antecedent hematologic disorder and 11 patients had received prior chemo-radiotherapy for non-hematopoietic neoplasms. On multivariate analysis, overall survival, remission duration and event-free survival were comparable to those of other patients with acute myeloid leukemia.

Conclusions

The outcome of hypocellular acute myeloid leukemia does not differ from that of non-hypocellular acute myeloid leukemia.     

FLT3-internal tandem duplication and age are the major prognostic factors in patients with relapsed acute myeloid leukemia with normal karyotype

Background

Several genetic aberrations with prognostic impact in first-line therapy have been described in patients with acute myeloid leukemia and normal karyotype. However, little is known about the influence of these aberrations on outcome after relapse. This study aimed to identify clinical and molecular risk factors for patients with relapsed acute myeloid leukemia with normal karyotype.

Design and Methods

We analyzed 94 patients with acute myeloid leukemia and normal karyotype after first relapse for clinical and molecular risk factors for survival. All patients had received first-line treatment and follow-up within two prospective, multicenter trials. Leukemic blasts were analyzed at diagnosis for genetic aberrations in the FLT3, NPM1, CEBPA, WT1, IDH1 and IDH2 genes by polymerase chain reaction and/or direct sequencing.

Results

A second complete remission was achieved in 52% of patients who received re-induction therapy. The presence of an FLT3-internal tandem duplication, duration of first complete remission less than 6 months and age above the median of 47 years were associated with a significantly lower rate of second complete remission. The median survival after relapse was 11 months and the 6-year survival rate was 28%. In multivariate analysis, FLT3-internal tandem duplication and age above the median were the only independent negative prognostic factors for survival. The 6-year survival rate of patients with none of these factors was 56%, whereas it was significantly inferior in patients with one or both of these factors (15% and 6%, respectively). This was also true for patients who underwent allogeneic stem cell transplantation after relapse.

Conclusions

FLT3-internal tandem duplication and age are the major prognostic factors in patients with relapsed acute myeloid leukemia with a normal karyotype. Patients with at least one of these risk factors have a dismal outcome and might be considered for investigational treatment approaches after relapse. (ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT00209833","term_id":"NCT00209833"}}NCT00209833)     

Sorafenib plus all‐trans retinoic acid for AML patients with FLT3‐ITD and NPM1 mutations

Knowledge of the molecular basis of acute myeloid leukaemia has increased considerably in the past few years, and therapies targeting specific molecular defects of this disease are intensively investigated. Patients with both NPM1 and FLT3‐ITD mutations encompass 20% of cytogenetically normal AML. The multikinase and FLT3 inhibitor, sorafenib, has shown some efficacy in patients with relapsed FLT3‐ITD⁺ AML. In addition, it is suggested that all‐trans retinoic acid (ATRA) used in combination with chemotherapy has shown to improve outcome of patients harbouring NPM1 mutations. We report here the clinical course of three patients with refractory or relapsed FLT3‐ITD⁺/NPM1⁺ AML who achieved significant response upon sorafenib and ATRA combination.     

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

Background

Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established.

Design and Methods

We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005.

Results

FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis.

Conclusions

FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.     

Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses

Background

Disease recurrence in patients with acute myeloid leukemia may be partially explained by the escape of leukemic blasts from CD4⁺ T-cell recognition. The current study investigates the role of aberrant HLA class II antigen presentation on leukemic blasts by determining both the clinical and functional impact of the class II-associated invariant chain peptide (CLIP).

Design and Methods

The levels of expression of CLIP and HLA-DR on blood and bone marrow samples from 207 patients with acute myeloid leukemia were correlated with clinical outcome. Irradiated CLIP⁻ and CLIP⁺ leukemic blasts were compared for their ability to induce CD4⁺ T cells during mixed leukocyte reactions. To discriminate between these blasts, we down-modulated CLIP expression on myeloid leukemic cell lines by RNA interference of the invariant chain, a chaperone protein critically involved in HLA-DR processing, and performed flow cytometric sorting for their isolation from primary acute myeloid leukemia samples.

Results

We found that patients with leukemic blasts characterized by a high amount of HLA-DR occupied by CLIP (relative amount of CLIP) had a significantly shortened disease-free survival. The clear reductions in amount of HLA-DR occupied by CLIP on blasts of the THP-1 and Kasumi-1 myeloid leukemic cell lines after treatment with invariant chain short interfering RNA resulted in enhanced rates of allogeneic CD4⁺ T-cell proliferation. Similar findings were obtained in an autologous setting, in which there were strong increases in proliferation of remission CD4⁺ T cells stimulated with CLIP⁻-sorted leukemic blasts from HLA-DR⁺ acute myeloid leukemia patients, in contrast to CLIP⁺-sorted leukemic blasts from the same patients.

Conclusions

These data highlight the relevance of CLIP expression on leukemic blasts and the potential of CLIP as a target for immunomodulatory strategies to enhance HLA class II antigen presentation and CD4⁺ T-cell reactivity in acute myeloid leukemia.     

Maintenance therapy for FLT3-ITD-mutated acute myeloid leukemia

FLT3-ITD is a constitutively activated variant of the FLT3 tyrosine kinase receptor. Its expression in acute myeloid leukemia (AML) is associated with a poor prognosis. Due to this, the development of tyrosine kinase inhibitors (TKI) blocking FLT3-ITD became a rational therapeutic concept. This review describes key milestones in the clinical development of different FLT3-specific TKI with a particular focus on FLT3-TKI maintenance therapy in remission after allogeneic hematopoietic stem cell transplantation (HCT). Recent evidence from randomized trials using sorafenib in FLT3-ITD mutated AML provided a proof of concept that targeted post-HCT maintenance therapy could become a new treatment paradigm in AML.     

RHAMM/HMMR (CD168) is not an ideal target antigen for immunotherapy of acute myeloid leukemia

Background

Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored.

Design and Methods

Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation.

Results

High levels of RHAMM were expressed by CD34⁺CD38⁺ and CD34^- acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34⁺CD38^- leukemic stem cells, and was not different from that in CD34⁺CD38^- hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34⁺ cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells.

Conclusions

RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy.Key words: leukemic stem cell, acute myeloid leukemia, cell therapy and immunotherapy, HMMR, RHAMM     

Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells

Background

The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia.

Design and Methods

Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels.

Results

We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4⁺CD25⁺ Foxp3⁺ T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4⁺CD25⁺ T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms’ tumor protein.

Conclusions

These data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.