Autophagy and apoptosis mediated nano-copper-induced testicular damage.

Nano-copper has been increasingly employed in various products. In previous studies, we showed that nano-copper caused damage in the rat testis, but it remains unclear whether the toxic reaction can affect the reproductive function. In this study, following 28 d of exposure to nano-copper at a dose of 44, 88, and 175 mg/kg/day, there was a decrease in sperm quality, fructose content, and the secretion of sex hormones. Nano-copper also increased the level of oxidative stress, sperm malformation rate, and induced abnormal structural changes in testicular tissue. Moreover, Nano-copper upregulated the expression of apoptosis-related protein Bax and autophagy-related protein Beclin, and downregulated the expression of Bcl2 and p62. Furthermore, nano-copper (175 mg/kg) downregulated the protein expression of AMPK, p-AKT, mTOR, p-mTOR, p-4E-BP1, p70S6K, and p-p70S6K, and upregulated the protein expression of p-AMPK. Therefore, nano-copper induced damage in testicular tissues and spermatogenesis is highly related to cell apoptosis and autophagy by regulating the Akt/mTOR signaling pathway. In summary, excess exposure to nano-copper may induce testicular apoptosis and autophagy through AKT/mTOR signaling pathways, and damage the reproductive system in adult males, which is associated with oxidative stress in the testes.

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.

Expression of CD94 by ex vivo-differentiated NK cells correlates with the in vitro and in vivo acquisition of cytotoxic features.

The administration of ex vivo-expanded Natural Killer (NK) cells in leukemia therapy is still challenging, in part due to the difficulty to generate in sufficient quantities fully mature and functional NK cells and Identification of surface markers indicative of NK maturation and functionality is therefore needed. Here, based on the analysis of surface receptors of ex vivo-expanded NK cells, we identified CD94 as a surface marker correlating with high lytic potential against leukemic cell lines and immunological synapse formation. CD94-positive ex vivo-expanded NK cells displayed higher expression of NKG2 receptors and the adhesion molecule LFA-1, as compared with their CD94-negative counterparts. We also tested the in vivo anti-leukemic capacity of ex vivo-expanded NK cells against patient-derived acute myeloid leukemia cells. Although no anti-leukemic effect was detected, we noticed that only CD94-positive ex vivo-expanded NK cells were detected in leukemic mice at the end of the 2-week treatment. Moreover, flow cytometry analysis showed a subpopulation harboring CD94 (NK) and CD34 (leukemic cells) double staining, indicative of conjugate formation. Therefore surface expression of CD94 on ex vivo-differentiated NK cells emerged as an indicator of in vitro and in vivo killer cell functionality.

T-cell prolymphocytic leukemia and tuberculosis: a puzzling association.

T-cell prolymphocytic leukemia can result in severe immune T-cell deficiency. Clinicians should be aware of this complication in this rare lymphoid malignancy, and opportunistic infections should be ruled out before the use of usual immunosuppressive procedures such as alemtuzumab and hematopoietic stem cell transplantation.

Targeting primary acute myeloid leukemia with a new CXCR4 antagonist IgG1 antibody (PF-06747143).

The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow (BM) microenvironment and is overexpressed in 25-30% of patients with acute myeloid leukemia (AML). Here we have shown that a new CXCR4 receptor antagonist IgG1 antibody (PF-06747143) binds strongly to AML cell lines and to AML primary cells inhibiting their chemotaxis in response to CXCL12. PF-06747143 also induced cytotoxicity in AML cells via Fc-effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different patient-derived xenograft (PDX) models: Patient 17CXCR4-low and P15CXCR4-high models, characterized by relatively low and high CXCR4 expression, respectively. Weekly administration of PF-06747143 to leukemic mice significantly reduced leukemia development in both models. Secondary transplantation of BM cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143. Administration of a single dose of PF-06747143 to PDX models induced rapid malignant cell mobilization into the peripheral blood (PB). These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy.

Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells.

Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[1] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2+/+/DR1+/+/H-2-ß2m-/-/IAß-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies.

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.

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.

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.

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.

Acute myeloid leukemia in patients older than 75: prognostic impact of FLT3-ITD and NPM1 mutations.

The benefit associated with chemotherapy in older patients with acute myeloid leukemia (AML) is debated. The prognostic impact of molecular mutations in these patients is unknown. We identified 79 patients with AML aged 75 years or over. Forty-two received chemotherapy and 37 supportive care only. In intensively treated patients, overall survival was longer (p < 0.001). Achieving complete remission was associated with longer survival (p < 0.001). NPM1 mutations tended to be associated with a higher complete remission rate (p = 0.12). In multivariate analysis, FLT3-ITD was associated with poorer survival (p = 0.049). Patients harboring FLT3-ITD and no NPM1 mutation had a poorer prognosis than others (p = 0.02). Intensive treatments can benefit a portion of elderly patients. FLT3-ITD and NPM1 mutational status might be useful for prognosis stratification.

Prognosis of body mass index and chemotherapy dose capping in acute myeloid leukaemia.

Body Mass Index (BMI) prognosis in acute myeloid leukaemia (AML) is unknown. Capping chemotherapy dose at 2m(2) of body surface area (BSA) is used without any rationale. We assessed whether both of them could be correlated with outcome in 233 AML patients. Thirty three percent were overweight, 10% obese and BSA over 2m(2) was observed in 15%. BMI and BSA>2m(2) were not associated with OS (p=0.16; p=0.39), nor with DFS (p=0.18; p=0.42), nor with CR. OS-associated factors were age (p<0.001), cytogenetic (p=0.002), FLT3-ITD (p=0.01). BMI and chemotherapy dose capping are not pejorative factors on intensively treated AML patients.

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.

A subset of bone marrow stromal cells regulate ATP-binding cassette gene expression via insulin-like growth factor-I in a leukemia cell line.

The importance of the insulin-like growth factor, IGF, as a signaling axis in cancer development, progression and metastasis is highlighted by its effects on cancer cells, notably proliferation and acquired resistance. The role of the microenvironment within which cancer cells evolve and which mediates this effect is far from clear. Here, the involvement of IGF-I in inducing multidrug resistance in a myeloid leukemia cell line, grown in the presence of bone marrow-derived stromal cells called 'Hospicells' (BMH), is demonstrated. We found that i) drug sensitive as well as resistant leukemia cells express IGF-I and its receptor IGF-IR. However, the resistant cells were found to secrete high levels of IGF-I. ii) Presence of exogenous IGF-I promoted cell proliferation, which decreased when an inhibitor of IGF-IR (picropodophyllin, PPP) was added. iii) BMH and IGF-I are both involved in the regulation of genes of the ATP binding cassette (ABC) related to resistance development (MDR1, MRP1, MRP2, MRP3 and BCRP). iv) The levels of ABC gene expression by leukemia cells were found to increase in the presence of increasing numbers of BMH. However, these levels decreased when IGF-IR was inhibited by addition of PPP. v) Co-culture of the drug-sensitive leukemia cells with BMH induced protection against the action of daunorubicin. This chemoresistance was amplified by the presence of IGF-I whereas it decreased when IGF-IR was inhibited. Our results underline the role of microenvironment in concert with the IGF-1 pathway in conferring drug resistance to leukemia cells.

Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial.

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

p70S6 kinase is a target of the novel proteasome inhibitor 3,3'-diamino-4'-methoxyflavone during apoptosis in human myeloid tumor cells.

Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce apoptosis in AML cells. Herein, the effects and mechanisms of the novel flavone 3,3'-diamino-4'-methoxyflavone (DD1) on AML cell dysfunction were investigated in AML cells (monoblast U937, myelomonocyte OCI-AML3, promyelocyte NB4, myeloblast HL-60) and blood samples from patients with AML. The administration of DD1 inhibited proliferation and induced death of AML cell lines and reduced the clonogenic activity of AML, but not normal, blood cells. The flavone's apoptotic action in U937 cells was associated with recruitment of mitochondria, Bax activation, Bad dephosphorylation (at Ser(136)), activation of caspases -8, -9, and -3 and cleavage of the caspase substrate PARP-1. DD1 induced a marked decrease in (i) Thr(389)-phosphorylation and (ii) protein levels of the caspase-3 substrate P70 ribosomal S6 kinase (P70S6K, known for its ability to phosphorylate Bad). Caspase-dependent apoptosis and P70S6K degradation were simultaneously prevented by the caspase inhibitors. Importantly, DD1 was shown to directly inhibit the proteasome's chymotrypsin-like activity in U937 cells. Apoptotic activity of the proteasome inhibitor bortezomib was also related to Bax activation and P70S6K downregulation. Accordingly, DD1 failed to induce P70S6K cleavage, Bax stimulation and apoptosis in K562 cells resistant to bortezomib. These results indicate that DD1 has the potential to eradicate AML cells and support a critical role for Bax and P70S6K in DD1-mediated proteasome inhibition and apoptosis of leukemia cells.

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.

Phase I studies of AVE9633, an anti-CD33 antibody-maytansinoid conjugate, in adult patients with relapsed/refractory acute myeloid leukemia.

The efficacy of anti-CD33 immunoconjugates had been previously demonstrated for gemtuzumab-ozogamicin. AVE9633 is an anti-CD33-maytansine conjugate created by ImmunoGen Inc. Phase I trials of AVE9633 were performed in patients with AML to evaluate tolerability, pharmacokinetics and pharmacodynamics. Three phase I studies of AVE9633 were performed in 54 patients with refractory/relapsed AML, evaluating drug infusion on day 1 of a 21-day cycle (Day 1 study), day 1 and 8 (Day 1/8 study) and day 1, 4 and 7 (Day 1/4/7 study) of a 28-day cycle. Toxicity was mainly allergic reaction during infusion (3 grade 3 bronchospasms). DLT was reached for the D1-D7 schedule at 150 mg/sqm (1 keratitis, 1 liver toxicity), and the MTD was set at 130 mg/sqm for this schedule. In the two other phases I, the DLT was not reached. In the Day 1/8 study, CD33 on peripheral blasts was saturated and down-modulated for doses of 75 mg/m(2) × 2 or higher, which was correlated with WBC kinetics and plasma levels of AVE9633. Decrease of DM4/CD33 ratio on the blasts surface between day 1 and 8 was the rational for evaluating day 1/4/7 schedule. This induced relatively constant DM4/CD33 levels over the first 8 days, however no activity was noted. One CRp, one PR and biological activity in five other patients were observed in this study. The Day 1 and Day 1/4/7 studies were early discontinued because of drug inactivity at doses significantly higher than CD33 -saturating doses. No myelossuppression was observed at any trial of AVE9633. The pharmacokinetics/pharmacodynamics data obtained in these studies will provide very useful information for the design of the next generation of immunoconjugates.

Hyperforin induces apoptosis of chronic lymphocytic leukemia cells through upregulation of the BH3-only protein Noxa.

We previously reported that hyperforin, a phloroglucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time- and concentration-dependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis. Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis. They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.

Soluble endothelial protein C receptor (sEPCR) is likely a biomarker of cancer-associated hypercoagulability in human hematologic malignancies.

Elevated plasma level of soluble endothelial protein C receptor (sEPCR) may be an indicator of thrombotic risk. The present study aims to correlate leukemia-associated hypercoagulability to high level plasma sEPCR and proposes its measurement in routine clinical practice. EPCR expressions in leukemic cell lines were determined by flow cytometry, immunocytochemistry, and reverse transcription polymerase chain reaction (RT-PCR). EPCR gene sequence of a candidate cell line HL-60 was also determined. Plasma samples (n = 76) and bone marrow aspirates (n = 72) from 148 patients with hematologic malignancies and 101 healthy volunteers were analyzed by enzyme-linked immunosorbent assay (ELISA) via a retrospective study for sEPCR and D-dimer. All leukemic cell lines were found to express EPCR. Also, HL-60 EPCR gene sequence showed extensive similarities with the endothelial reference gene. All single nucleotide polymorphisms (SNPs) originally described and some new SNPs were revealed in the promoter and intronic regions. Among these patients 67% had plasma sEPCR level higher than the controls (100 ± 28 ng/mL), wherein 16.3% patients had experienced a previous thrombotic event. These patients were divided into: group-1 (n = 45) with amount of plasmatic sEPCR below 100 ng/mL, group-2 (n = 45) where the concentration of sEPCR was between 100 and 200, and group-3 (n = 20) higher than 200 ng/mL. The numbers of thrombotic incidence recorded in each group were four, six, and eight, respectively. These results reveal that EPCR is expressed not only by a wide range of human malignant hematological cells but also the detection of plasma sEPCR levels provides a powerful insight into thrombotic risk assessment in cancer patients, especially when it surpasses 200 ng/mL.