Precision and prognostic value of clone-specific minimal residual disease in acute myeloid leukemia.

The genetic landscape of adult acute myeloid leukemias (AML) has been recently unraveled. However, due to their genetic heterogeneity, only a handful of markers are currently used for the evaluation of minimal residual disease (MRD). Recent studies using multi-target strategies indicate that detection of residual mutations in less than 5% of cells in complete remission is associated with a better survival. Here, in a series of 69 AMLs with known clonal architecture, we design a clone-specific strategy based on fluorescent in situ hybridization and high-sensitivity next generation sequencing to detect chromosomal aberrations and mutations, respectively, in follow-up samples. The combination of these techniques allows tracking chromosomal and genomic lesions down to 0.5-0.4% of the cell population in remission samples. By testing all lesions in follow-up samples from 65 of 69 evaluable patients, we find that initiating events often persist and appear to be, on their own, inappropriate markers to predict short-term relapse. In contrast, the persistence of two or more lesions in more than 0.4% of the cells from remission samples is strongly associated with lower leukemia-free and overall survivals in univariate and multivariate analyses. Although larger prospective studies are needed to extend these results, our data show that a personalized, clone-specific, MRD follow up strategy is feasible in the vast majority of AML cases.

Prognostic impact of high ABC transporter activity in 111 adult acute myeloid leukemia patients with normal cytogenetics when compared to FLT3, NPM1, CEBPA and BAALC.

ATP-binding cassette transporter (and specially P-glycoprotein) activity is a well known prognostic factor in acute myeloid leukemia, but when compared to other molecular markers its prognostic value has not been well studied. Here we study relationships between this activity, fms-like tyro-sine kinase 3(FLT3/ITD), nucleophosmin(NPM1), CAAT-enhancer binding protein alpha(CEBPα), and brain and acute leukemia cytoplasmic protein (BAALC), in 111 patients with normal cytogenetics who underwent the same treatment, and evaluate its prognostic impact. Independent factors for survival were age (P=0.0126), ATP-binding cassette transporter activity (P=0.018) and duplications in the fms-like tyrosine kinase 3 (P=0.0273). In the 66 patients without fms-like tyrosine kinase 3 duplication and without nucleophosmin mutation, independent prognostic factors for complete remission achievement and survival were age and ATP-binding cassette transporter activity. In conclusion, ATP-binding cassette transporter activity remains an independent prognostic factor, and could assist treatment decisions in patients with no nucleophosmin mutation and no fms-like tyrosine kinase 3 duplication.

High Id1 expression is associated with poor prognosis in 237 patients with acute myeloid leukemia.

Inhibitors of differentiation (Id) are a group of dominant inhibitors of basic helix-loop-helix transcriptional factors, which promote excessive proliferation, and also protect cells against drug-induced apoptosis in mammalians. Recently, Id1 has been identified as a common downstream target of several constitutively activated oncogenic tyrosine kinase, such as FLT3 internal tandem duplication, in leukemia cells. We analyzed Id1 expression as possible prognostic factor in 237 acute myeloid leukemia (AML) patients. High Id1 expression was associated with older age (P = .009) and with FLT3 internal tandem duplication (P = .003). However, 61% of the patients in the group of FLT3(-) AML were Id1(+), suggesting that other tyrosine kinases are involved. In whole population, high Id1 expression independently predicted shorter disease-free survival (P = .05) and overall survival (P = .003). In young patients (age

ATP Binding Cassette transporters associated with chemoresistance: transcriptional profiling in extreme cohorts and their prognostic impact in a cohort of 281 acute myeloid leukemia patients.

BACKGROUND: A major issue in the treatment of acute myeloid leukemia is resistance to chemotherapeutic drugs. An increasing number of ATP-Binding-Cassette transporters have been demonstrated to cause resistance to cancer drugs. The aim of this study was to highlight the putative role of other ATP-Binding-Cassette transporters in primary chemoresistant acute myeloid leukemia. DESIGN AND METHODS: In the first part of this study, using taqman custom arrays, we analyzed the relative expression levels of 49 ATP-Binding-Cassette genes in 51 patients divided into two extreme cohorts, one very sensitive and one very resistant to chemotherapy. In the second part of this study, we evaluated the prognostic impact, in a cohort of 281 patients, of ATP-Binding-Cassette genes selected in the first part of the study. RESULTS: In the first part of the study, six genes (ATP-Binding-CassetteA2, ATP-Binding-CassetteB1, ATP-Binding-CassetteB6, ATP-Binding-CassettC13, ATP-Binding-CassetteG1, and ATP-Binding-CassetteG2) were significantly over-expressed in the resistant group compared with the sensitive group. In the second cohort, overexpression of 5 of these 6 ATP-Binding-Cassette genes was correlated with outcome in univariate analysis, and only the well-known ATP-Binding-CassetteB1 and G2, and the new ATP-Binding-CassetteG1 in multivariate analysis. Prognosis decreased remarkably with the number of these over-expressed ABC genes. Complete remission was achieved in 71%, 59%, 54%, and 0%, (P=0.0011) and resistance disease in 21%, 37%, 43%, and 100% (P<0.0001) of patients over-expressing 0, 1, 2, or 3, ABC genes, respectively. The number of ATP-Binding-Cassette genes expressed, among ATP-Binding-CassetteB1, G1, and G2, was the strongest prognostic factor correlated, in multivariate analysis, with achievement of complete remission (P=0.01), resistant disease (P=0.01), and overall survival (P=0.02). CONCLUSIONS: Using expression profiling, we have emphasized the diversity of ATP-Binding-Cassette transporters that cooperate to promote chemoresistance rather than overexpression of single transporters and the putative role of new ATP-Binding-Cassette tranporters, such as ATP-Binding-CassetteG1. Modulation of these multiple transporters might be required to eradicate leukemic cells.

P-gp activity is a critical resistance factor against AVE9633 and DM4 cytotoxicity in leukaemia cell lines, but not a major mechanism of chemoresistance in cells from acute myeloid leukaemia patients.

BACKGROUND: AVE9633 is a new immunoconjugate comprising a humanized monoclonal antibody, anti-CD33 antigen, linked through a disulfide bond to the maytansine derivative DM4, a cytotoxic agent and potent tubulin inhibitor. It is undergoing a phase I clinical trial. Chemoresistance to anti-mitotic agents has been shown to be related, in part, to overexpression of ABC proteins. The aim of the present study was to investigate the potential roles of P-gp, MRP1 and BCRP in cytotoxicity in AVE9633-induced acute myeloid leukaemia (AML). METHODS: This study used AML cell lines expressing different levels of P-gp, MRP1 or BCRP proteins and twenty-five samples from AML patients. Expression and functionality of the transporter protein were analyzed by flow cytometry. The cytotoxicity of the drug was evaluated by MTT and apoptosis assays. RESULTS: P-gp activity, but not MRP1 and BCRP, attenuated AVE9633 and DM4 cytotoxicity in myeloid cell lines. Zosuquidar, a potent specific P-gp inhibitor, restored the sensitivity of cells expressing P-gp to both AVE9633 and DM4. However, the data from AML patients show that 10/25 samples of AML cells (40%) were resistant to AVE9633 or DM4 (IC(50) > 500 nM), and this was not related to P-gp activity (p-Value: 0.7). Zosuquidar also failed to re-establish drug sensitivity. Furthermore, this resistance was not correlated with CD33 expression (p-Value: 0.6) in those cells. CONCLUSION: P-gp activity is not a crucial mechanism of chemoresistance to AVE9633. For patients whose resistance to conventional anthracycline AML regimens is related to ABC protein expression, a combination with AVE9633 could be beneficial. Other mechanisms such as microtubule alteration could play an important role in chemoresistance to AVE9633.

Quantitative and functional alterations of 6-sulfo LacNac dendritic cells in multiple myeloma.

Multiple myeloma (MM) results from expansion of abnormal plasma cells in the bone marrow (BM). Previous studies have shown that monocytes play a crucial role in MM pathophysiology. A 6-sulfo LacNAc-expressing population of dendritic cells (Slan-DCs) that overlaps with intermediate and non-classical monocytes in terms of phenotype has been described. Slan-DCs represent a circulating and tissue proinflammatory myeloid population which has been shown to play a role in different cancer contexts, and which exhibits a remarkable plasticity. Herein, we studied Slan-DCs from the BM and blood of MM patients. We performed quantitative and functional analyses of these cells from 54 patients with newly diagnosed, symptomatic MM, 21 patients with MGUS and 24 responding MM patients. We found that circulating Slan-DCs were significantly decreased in MM patients as compared to those of healthy donors or patients with MGUS, while CD14+CD16+ intermediate monocytes accumulate in the BM. Moreover, after activation with TLR7/8 ligand R848, IL-12-producing Slan-DCs from the BM or peripheral blood from MM patients were decreased as compared with healthy donors. We show that MM cell lines or MM cells isolated from patients at diagnosis were able to inhibit the production of IL-12 by Slan-DCs, as well as to shift the phenotype of Slan-DCs towards an intermediate monocyte-like phenotype. Finally, Slan-DCs that have been cultured with MM cells reduced their capacity to induce T cell proliferation and Th1 polarization. We conclude that Slan-DCs represent previously unrecognized players in MM development and may represent a therapeutic target.

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13.

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.

Genetic hierarchy and temporal variegation in the clonal history of acute myeloid leukaemia.

In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner.

Endothelial protein C receptor gene 6936A/G single-nucleotide polymorphism as a possible biomarker of thrombotic risk in acute myeloid leukemia.

Protein C (PC) is a natural anticoagulant, which interacts with the endothelial PC receptor (EPCR). EPCR single-nucleotide polymorphism (SNP) 6936A/G results in high levels of a free soluble form of EPCR (sEPCR) and may affect the risk of coagulation. The objective of this study was to assess whether the 6936A/G SNP of the EPCR gene is involved in the procoagulant activity displayed by hematological malignancies. EPCR 6936A/G polymorphism analysis was performed in 205 patients with hematological malignancies and in 63 healthy controls. All the subjects were genotyped for the EPCR 6936A/G SNP (AA, AG and GG genotypes). The 6936A/G polymorphism distribution was similar between healthy donors and patients. The association between EPCR 6936A/G SNP and thrombosis was investigated in 110 patients. The disease-wise break-up revealed that 55 of the patients suffered from acute myeloid leukemia (AML). In AML patients, the incidence of thrombosis was 28.3% and significantly higher in the 6936AG compared with that in the 6936AA genotype (50 vs. 22%, respectively). In conclusion, this study revealed a significant association of the 6936AG genotype of EPCR with thrombotic events in AML. Therefore, the presence of the 6936AG genotype in AML patients may be considered as a risk indicator of thrombosis.

Targeting CD13 (aminopeptidase-N) in turn downregulates ADAM17 by internalization in acute myeloid leukaemia cells.

Secreted matrix metalloproteinases (MMP)-2 and MMP-9 and membrane-anchored aminopeptidase-N/CD13 are abnormally expressed in human acute myeloid leukaemia (AML). We previously showed that CD13 ligation by anti-CD13 monoclonal antibodies can induce apoptosis in AML cells. Here, we assessed ADAM17 expression in primary blood blasts CD13+CD33+ from patients with AML. Primary AML cells expressed ADAM17 transcript and its surface expression was higher in subtype M4 (myelomonocytic) and M5 (monocytic) AML specimens than in M0 and M1/M2 (early and granulocytic) specimens. In AML cell lines defining distinct AML subfamilies (HL-60/M2, NB4/M3, THP-1/M5, U937/M5) and primary AML cells cultured ex vivo, anti-CD13 antibodies downregulated surface CD13 and ADAM17 without affecting MMP-2/-9 release. Knockdown of CD13 by siRNA prevented anti-CD13-mediated ADAM17 downregulation, indicating that CD13 is required for ADAM17 downregulation. Soluble ADAM17 was not detected in the medium of anti-CD13 treated cells, suggesting that ADAM17 was not shed. After ligation by anti-CD13, CD13 and ADAM17 were internalized. Subsequently, we found that ADAM17 interacts with CD13. We postulate that the interaction of ADAM17 with CD13 and its downregulation following CD13 engagement has important implications in AML for the known roles of ADAM17 in tumour-associated cell growth, migration and invasion.

Applying ecological and evolutionary theory to cancer: a long and winding road.

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.

Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis.

The MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34(+)CD38(-)-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34(+) cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.