The emerging role of measurable residual disease detection in AML in morphologic remission.

Despite the increasing knowledge of the genomic landscape of acute myeloid leukemia (AML), prediction merely based on genetics fails to anticipate outcome, presumably due to the heterogeneous composition of the leukemic clone determining complex interactions between different genetic abnormalities. Therefore, the introduction of a post-treatment biomarker exploring the quality of response to therapy such as assessment of measurable (previously minimal) residual disease (MRD) may lead to refinements of the prognostic assessment in AML. In this view, the European LeukemiaNet has recently endorsed the achievement of a MRD negative morphologic complete remission as a purpose the treatment. Techniques like multiparametric flow cytometry and reverse transcriptase-quantitative polymerase chain reaction have reached a level of sensitivity and specificity that make them ready for introduction in clinical practice. In the present review, we will give an update on the efforts in harmonization and/or standardization of MRD assessment in AML, focusing on the newest acquisitions in the clinical applications of MRD, and considering issues like relationship of MRD with leukemic stem cells or MRD assessment in peripheral blood.

A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia.

As relapses are common in acute myeloid leukemia (AML), early relapse prediction is of high importance. Although conventional minimal residual disease (MRD) measurement is carried out in bone marrow (BM), peripheral blood (PB) would be an advantageous alternative source. This study aims to investigate the specificity of leukemia-associated immunophenotypes used for MRD detection in blood samples. Consistency of PB MRD as compared with BM MRD was determined in flow cytometric data of 205 paired BM and PB samples of 114 AML patients. A significant correlation was found between PB and BM MRD (r=0.67, P<0.001), while median PB MRD percentage was factor 4-5 lower compared with BM MRD. Primitive blast (CD34+/CD117+/CD133+) frequency was significantly lower in PB (median factor 23.7), indicating that PB MRD detection is more specific than BM. Cumulative incidence of relapse 1 year after induction therapy was 29% for PB MRD-negative and 89% for PB MRD-positive patients (P<0.001). Three-year OS was 52% for MRD-negative and 15% for MRD-positive patients (P=0.034). Similar differences were found after consolidation therapy. As PB MRD appeared to be an independent predictor for response duration, the highly specific PB MRD assay may have a prominent role in future MRD assessment in AML.

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival.

Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.

Tumor heterogeneity makes AML a "moving target" for detection of residual disease.

Detection of minimal residual disease is recognized as an important post-therapy risk factor in acute myeloid leukemia patients. Two most commonly used methods for residual disease monitoring are real-time quantitative polymerase chain reaction and multiparameter flow cytometry. The results so far are very promising, whereby it is likely that minimal residual disease results will enable to guide future post-remission treatment strategies. However, the leukemic clone may change between diagnosis and relapse due to the instability of the tumor cells. This instability may already be evident at diagnosis if different subpopulations of tumor cells coexist. Such tumor heterogeneity, which may be reflected by immunophenotypic, molecular, and/or cytogenetic changes, can have important consequences for minimal residual disease detection, since false-negative results can be expected to be the result of losses of aberrancies used as minimal residual disease markers. In this review the role of such changes in minimal residual disease monitoring is explored. Furthermore, possible causes of tumor instability are discussed, whereby the concept of clonal selection and expansion of a chemotherapy-resistant subpopulation is highlighted. Accordingly, detailed knowledge of the process of clonal evolution is required to improve both minimal residual disease risk stratification and patient outcome.

Tumor heterogeneity makes AML a "moving target" for detection of residual disease.

Detection of minimal residual disease is recognized as an important post-therapy risk factor in acute myeloid leukemia patients. Two most commonly used methods for residual disease monitoring are real time quantitative polymerase chain reaction and multiparameter flow cytometry. Results so far are very promising, whereby it is likely that minimal residual disease results will enable to guide future post-remission treatment strategies. However, the leukemic clone may change between diagnosis and relapse due to instability of the tumor cells. This instability may already be evident at diagnosis if different subpopulations of tumor cells coexist. Such tumor heterogeneity, which may be reflected by immunophenotypic, molecular and/or cytogenetic changes, can have important consequences for minimal residual disease detection, since false-negative results can be expected to be the result of losses of aberrancies used as minimal residual disease markers. In this review the role of such changes in minimal residual disease monitoring is explored. Furthermore, possible causes of tumor instability are discussed, whereby the concept of clonal selection and expansion of a chemotherapy resistant subpopulation is highlighted. Accordingly, detailed knowledge of the process of clonal evolution is required to improve both minimal residual disease risk stratification and patient outcome. © 2013 Clinical Cytometry Society.

Defining consensus leukemia-associated immunophenotypes for detection of minimal residual disease in acute myeloid leukemia in a multicenter setting.

Flow-cytometric detection of minimal residual disease (MRD) has proven in several single-institute studies to have an independent prognostic impact. We studied whether this relatively complex approach could be performed in a multicenter clinical setting. Five centers developed common protocols to accurately define leukemia-associated (immuno)phenotypes (LAPs) at diagnosis required to establish MRD during/after treatment. List mode data files were exchanged, and LAPs were designed by each center. One center, with extensive MRD experience, served as the reference center and coordinator. In quarterly meetings, consensus LAPs were defined, with the performance of centers compared with these. In a learning (29 patients) and a test phase (35 patients), a mean of 2.2 aberrancies/patient was detected, and only 1/63 patients (1.6%) had no consensus LAP(s). For the four centers without (extensive) MRD experience, clear improvement could be shown: in the learning phase, 39-63% of all consensus LAPs were missed, resulting in a median 30% of patients (range 21-33%) for whom no consensus LAP was reported; in the test phase, 27-40% missed consensus LAPs, resulting in a median 16% (range 7-18%) of 'missed' patients. The quality of LAPs was extensively described. Immunophenotypic MRD assessment in its current setting needs extensive experience and should be limited to experienced centers.

The multidrug resistance protein breast cancer resistance protein (BCRP) protects adipose-derived stem cells against ischemic damage.

Adipose tissue-derived stem cells (ASCs) are promising candidates for regenerative therapy, like after myocardial infarction. However, when transplanted into the infarcted heart, ASCs are jeopardized by the ischemic environment. Interestingly, it has been shown that multidrug resistance (MDR) proteins like the breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) have a protective effect in haematopoietic stem cells. In ASC, however, only expression of BCRP was shown until now. In this study, we therefore analysed the expression and functional activity of BCRP and P-gp and their putative function in ischemia in ASC. BCRP and P-gp protein expression was studied over time (passages 2-6) using western blot analysis and immunohistochemical staining. MDR activity was analysed using protein-specific substrate extrusion assays. Ischemia was induced using metabolic inhibition. All analyses demonstrated protein expression and activity of BCRP in ASCs. In contrast, only minor expression of P-gp was found, without functional activity. BCRP expression was most prominent in early passage ASCs (p2) and decreased during culture. Finally, ischemia induced expression of BCRP. In addition, when BCRP was blocked, a significant increase in dead ASCs was found already after 1 h of ischemia. In conclusion, ASCs expressed BCRP, especially in early passages. In addition, we now show for the first time that BCRP protects ASCs against ischemia-induced cell death. These data therefore indicate that for transplantation of ASCs in an ischemic environment, like myocardial infarction, the optimal stem cell protective effect of BCRP theoretically will be achieved with early culture passages ASCs.

Minimal residual disease detection defined as the malignant fraction of the total primitive stem cell compartment offers additional prognostic information in acute myeloid leukaemia.

INTRODUCTION: Immunophenotypic detection of minimal residual disease (MRD) in bone marrow (BM) of acute myeloid leukaemia (AML) patients is of high prognostic relevance. Standard MRD percentage is assessed as a percentage of total white blood cells (WBCs) and is therefore highly dependent on WBC count. Peripheral blood (PB) contains more than five times lower MRD percentages. Therefore, PB in BM aspirates cause dilution of the MRD cells, possibly leading to false-negative results for BM MRD. The latter is avoided when relating the fraction of malignant primitive cells, identified by aberrant marker expression [aberrant primitive cells (aPC)], to the total population of primitive cells. Such a fraction may in addition reflect an important biological parameter. METHODS: As this approach is thus independent of WBC count and the total size of the primitive compartment, we investigated the role of aPC fractions on overall and relapse-free survival (RFS) in 98 patients with AML under the age of 60. RESULTS: We show that this approach identifies MRD-negative (as defined by % of WBC) but aPC-positive (as defined by % of primitive cells) patients with poor outcome after both first and second induction cycle of chemotherapy. CONCLUSION: As a result, in cases with a primitive marker present, RFS is best predicted when combining standard MRD percentage with aPC fractions.

The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse.

The majority of pediatric and younger adult (<60 years) AML patients achieve complete remission. However, 30-40% of patients relapse and display a dismal outcome. Recently we described a frequent instability of type I/II mutations between diagnosis and relapse. Here, we explored the hypothesis that these mutational shifts originate from clonal selection during treatment/disease progression. Subfractions of blasts from initial diagnosis samples were cell sorted and their mutational profiles were compared with those of the corresponding relapse samples of 7 CD34(+) AML patients. At diagnosis, subfractions of the CD45(dim)CD34(+)CD38(dim/-) compartment were heterogeneous in the distribution of mutations, when compared to the whole CD45(dim)CD34(+) blast compartment in 6 out of 7 patients. Moreover, within CD45(dim)CD34(+)CD38(dim/-) fraction of initial samples of 5 of these 6 AML patients, we found evidence for the presence of a minor, initially undetected subpopulation with a specific mutational profile that dominated the bulk of leukemic blasts at relapse. In conclusion, our findings lend support to the AML oligoclonality concept and provide molecular evidence for selection and expansion of a chemo-resistant subpopulation towards development of relapse. These results imply that early detection of pre-existing drug-resistant leukemic subpopulations is crucial for relapse prevention by proper timing of targeted treatment.

Residual normal stem cells can be detected in newly diagnosed chronic myeloid leukemia patients by a new flow cytometric approach and predict for optimal response to imatinib.

Insensitivity of chronic myeloid leukemia (CML) hematopoietic stem cells to tyrosine kinase inhibitors (TKIs) prevents eradication of the disease and may be involved in clinical resistance. For improved treatment results more knowledge about CML stem cells is needed. We here present a new flow cytometric approach enabling prospective discrimination of CML stem cells from their normal counterparts within single-patient samples. In 24 of 40 newly diagnosed CML patients residual normal CD34(+)CD38(-) stem cells could be identified by lower CD34 and CD45 expression, lower forward/sideward light scatter and by differences of lineage marker expression (CD7, CD11b and CD56) and of CD90. fluorescent in situ hybridization (FISH) analysis on Fluorescence-activated cell sorting sorted cells proved that populations were BCR-ABL positive or negative and long-term liquid culture assays with subsequent colony forming unit assays and FISH analysis proved their stem cell character. Patients with residual non-leukemic stem cells had lower clinical risk scores (Sokal, Euro), lower hematological toxicity of imatinib (IM) and better molecular responses to IM than patients without. This new approach will expand our possibilities to separate CML and normal stem cells, present in a single bone marrow or peripheral blood sample, thereby offering opportunities to better identify new CML stem-cell-specific targets. Moreover, it may guide optimal clinical CML management.

High TRAIL-R3 expression on leukemic blasts is associated with poor outcome and induces apoptosis-resistance which can be overcome by targeting TRAIL-R2.

Activation of the TNF-related apoptosis-inducing ligand (TRAIL) pathway can induce apoptosis in a broad range of human cancer cells. Four membrane-bound receptors have been identified. TRAIL-R1 and TRAIL-R2 contain a functional death domain; TRAIL-R3 and TRAIL-R4 lack a functional death domain and function as decoy receptors. Flow-cytometric analysis revealed that acute myeloid leukemic (AML) blasts expressed significantly more pro-apoptotic receptors compared to normal blasts. However, about 20% of AML patients highly expressed decoy receptor TRAIL-R3, which was strongly correlated to a shortened overall survival. TRAIL-R3 expression was also high on CD34+/CD38- cells, the compartment that harbors the leukemia initiating stem cell. Expression levels of pro-apoptotic TRAIL receptors were not correlated to the susceptibility for soluble TRAIL, which was generally low (mean level of cell death induction 14%). Cell death could be enhanced by down-modulation of TRAIL-R3, confirming its decoy function on AML blasts. Bypassing of TRAIL-R3 by treatment with antibodies directly targeting TRAIL-R2 resulted in higher rates of induced cell death (max. 80%). In conclusion, AML blasts do express pro-apoptotic TRAIL receptors. However, co-expression of decoy receptor TRAIL-R3 results in significant shortened overall survival. AML blasts could be targeted by anti-TRAIL-R2 antibodies, yielding a new therapeutic option for AML patients.

Clinical significance of flowcytometric minimal residual disease detection in pediatric acute myeloid leukemia patients treated according to the DCOG ANLL97/MRC AML12 protocol.

Analysis of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) may predict for clinical outcome. MRD levels were assessed by flowcytometric immunophenotyping in 94 children with AML enrolled into a single trial (United Kingdom Medical Research Council AML12 and similar Dutch Childhood Oncology Group ANLL97). An aberrant immunophenotype could be detected in 94% of patients. MRD levels after the first course of chemotherapy predicted for clinical outcome: 3-year relapse-free survival was 85%+/-8% (s.e.) for MRD-negative patients (MRD<0.1%), 64%+/-10% for MRD-low-positive patients (0.1%or=0.5%; P<0.001), whereas overall survival was 95%+/-5%, 70%+/-10% and 40%+/-13%, respectively, (P<0.001). Multivariate analysis allowing for age, karyotype, FLT3-internal tandem duplications and white blood cell count at diagnosis showed that MRD after the first course of chemotherapy was an independent prognostic factor. Although comparison of paired diagnosis-relapse samples (n=23) showed immunophenotypic shifts in 91% of cases, this did not hamper MRD analysis. In conclusion, flowcytometric MRD detection is possible in children with AML. The level of MRD after the first course of chemotherapy provides prognostic information that may be used to guide therapy.

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.

Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Aberrant marker expression patterns on the CD34+CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission.

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38- population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38- cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38- population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38- cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38- cells in complete remission, indicating the presence of malignant CD34+CD38- cells. Thus, by identifying residual malignant CD34+CD38- cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.

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.