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Increased caspase-3 activity in refractory anemias: lack of evidence for Fas pathway implication

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoietic stem cells (HSCs) characterized by ineffective hematopoiesis. MDSs are responsible for 1 or several peripheral cytopenias. The evidence accumulated in recent years demonstrates that in addition to HSC defects, a particular role is also played by stromal microenvironment dysfunctions, which mediate the direct contact with hematopoietic precursor cells (HPCs). These interactions help regulate different adhesion-related processes, such as progenitor cell proliferation, apoptosis, clonogenic growth, and maintenance in in vitro cultures. As previously reported, these interactions are responsible for altering the microenvironment in MDS. Herein, we present a novel selection protocol for obtaining a standards-compliant mesenchymal stromal cell (MSC) preparation. This method allowed us to comparatively analyze 2 subpopulations of bone marrow MSCs (BM-MSCs) in terms of their adhesion profiles and growth abilities: BM-MSCs selected from MDS settings and their normal counterparts. Functional assays revealed that the MSCs from MDS are intrinsically pathological, thus showing a continuous decline of proliferation and a reduced clonogenic capacity during 14 days of culture and in the absence of signals from hematopoietic cells. The MSC growth defects were significantly correlated with decreases in CD44 adhesion molecules and CD49e (α5-integrin).

The BCL-2 proto-oncogene encodes a mitochondrial protein that blocks programmed cell death. High amounts of bcl-2 protein are found not only in lymphoid malignancies, but also in normal tissues characterized by apoptotic cell death, including bone marrow. Using a monoclonal antibody to bcl-2 protein, we analyzed 82 samples of newly diagnosed acute myeloid leukemia. The number of bcl-2+ cells in each sample was heterogeneous (range, 0% to 95%), with a mean of 23%. The percentage of bcl-2+ cells was higher in M4 and M5 types, according to French- American-British classification, and in cases with high white blood cell counts. bcl-2 expression was also correlated with that of the stem cell marker CD34. In vitro survival of leukemic cells maintained in liquid culture in the absence of growth factors was significantly longer in cases with a high percentage of bcl-2+ cells. High expression of bcl-2 was associated with a low complete remission rate after intensive chemotherapy (29% in cases with 20% or more positive cells v 85% in cases with less than 20% positive cells, P < 10(-5)) and with a significantly shorter survival. In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.

In 1992, we reported the results of the first randomized trial comparing the combination of cyclophosphamide (CY) (120 mg/kg) and total body irradiation (TBI) (CYTBI) versus CY and busulfan (BU) (BUCY) as preparation for an allogeneic bone marrow transplantation (BMT) for adult patients with acute

Background The median survival of patients with chronic myelogenous leukemia in myeloid blast crisis (MBC-CML) is poor even for patients treated with tyrosine kinase inhibitors (TKIs). Design and methods We conducted a dose-escalating study of daunorubicin combined to fixed doses of IM (imatinib mesylate, 600 mg/d) and cytarabine (200 mg/d for 7 days), followed by hematopoietic stem cell transplantation or maintenance therapy with single-agent IM in patients with MBC-CML at onset or after failure of therapy excluding TKIs. Results Thirty-six patients were evaluated. Median follow-up is 6.1 years. Daunorubicin was escaladed up to 45 mg/m 2 /d 3 days. Twenty eight patients (77.7%) had hematologic response including 20 patients (55.5%) in complete hematologic response (CHR). Patients who received daunorubicin at 30–45 mg/m 2 /d had higher CHR rates compared to other patients. Median overall survival was 16 months. Overall survival in patients with hematological response was 35.4 months. Better results were observed in patients diagnosed with MBC-CML at onset. Conclusions The combination of IM with a standard “3 + 7” regiment was well tolerated and provided a high response rate. More than 55% of the patients achieved CHR and hematopoietic stem cell transplantation (SCT) was feasible in half of the cases. This trial was registered at www.clinicaltrials.gov as # NCT00219765 .

Thirty-four patients with acute myeloid leukemia (AML) in complete remission (CR), 30 of them aged over 60, received maintenance therapy scheduling four courses of low-dose cytarabine (LDA) 20 mg/m2/day in two subcutaneous injections for 3 weeks every 6 weeks. Each course was stopped when hematologic toxicity occurred, and doses of LDA were subsequently reduced by 50% for the following courses. During the first course of LDA, 15 patients needed blood and four patients platelet transfusions. Overall, 28 patients received four courses of LDA: 11 did not require any dose reduction, while 14 required one dose reduction and three neeeded two successive dose reductions. Two patients were hospitalized during maintenance. Median disease-free survival (DFS) is 308 days, with 16% of patients surviving at 5 years. Seven patients relapsed during the 168 days of maintenance, while ten of the 27 patients remaining at risk on day 169 relapsed during the 168 days following maintenance. We conclude that in AML in CR, the maximal dose of LDA tolerated by ambulatory patients is 10 mg/m2/day for 3 weeks. LDA seemed to delay relapse; however, precise assessment of the efficacy of this approach would require a randomized trial.

To evaluate the clinical value of the expression of multidrug resistance P-glycoprotein (P-170) on the surface of acute nonlymphoblastic leukemia (ANLL) cells, we analyzed specimens from 150 newly diagnosed patients for staining with MRK16, a monoclonal antibody (MoAb) that binds to an external epitope of P-170. Other surface markers (CD13, CD14, CD15, and CD34) were studied by the same technique. A marker was considered positive when 20% or more cells were stained. Of 150 samples, 71 were P-170-positive. These cases did not differ from P-170-negative cases with regard to age, sex, initial white blood cell (WBC) counts, or French-American-British (FAB) type (except for M3 ANLL, which were more frequently negative). However, leukemias arising from previous myelodysplastic syndrome (MDS) and therapy- induced leukemias were more frequently P-170-positive. CD34 and P-170 expression were significantly associated. All patients were treated by intensive chemotherapy. Complete remission (CR) rates were significantly lower in P-170-positive (23/71, 32%) than in P-170- negative cases (64/79, 81%) (P less than 10(-5)). CD34 positivity was also associated with a low remission rate (P less than 10(-5)). Survival was shorter for P-170- and CD34-positive patients (P less than 10(-5)). The prognostic value of both markers was confirmed in multivariate analysis. CR duration was also shorter for P-170-positive cases, but the difference is less significant (P = .05). It is concluded that P-170 analysis may be an important tool for predicting the outcome of intensive chemotherapy in ANLL patients.

Variant translocations (2;18 and 18;22) are described in this review. The chromosomal and molecular findings of these translocation of BCL2 and their effect on possible BCL2 gene activation is discussed. Unanswered questions still remain and these include why this is so rare compared to the 25% incidence recorded for translocations in Burkitt's lymphoma. Further studies are obviously still needed in order to determine the true frequency of these findings and their distribution in the various B-cell disorders.

10–20% of APL patients relapse and the challenge remains to early identify these patients to improve survival rate. We report PML-RARα transcript detection by RQ-PCR in 260 consecutive APL patients ( n = 970 samples). 223 patients with samples of sufficient RNA quality to demonstrate they reached molecular remission were monitored for MRD. During follow-up, 38 of these patients were tested positive for PML-RARα mRNA. 13 out of the 38 patients (34%) effectively developed hematological relapse. In the first positive sample, specific PML-RARα NCN thresholds over which, or under which, patients could effectively be predicted to relapse or not, were identified and subsequently validated in a second cohort.

Toxoplasmosis is a rare but well recognized opportunistic infection that can occur after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Besides encephalitis, other common presentations of Toxoplasma gondii infection are interstitial pneumonitis and myocarditis. Because of its non-specific clinical and biological signs and its lethal outcome, toxoplasmosis is often misdiagnosed and only revealed at autopsy. We report a case of a postmortem diagnosis of disseminated toxoplasmosis associated with hemophagocytic syndrome, which underlines the value of necropsy in cases of death after transplantation. We also discuss clinical presentations and risk factors that lead to toxoplasmosis in allo-HSCT recipients.

Background: The knowledge of normal marrow is mandatory to assess the malignant counterpart of normal cells and define leukemia-associated immunophenotypes (LAIPs). In this study, the expression of a variety of antigens expressed in normal and postchemotherapy bone marrow (BM) was analyzed to provide a frame of reference for the identification of myeloid LAIPs. Methods: Multiparameter four- and six-color flow cytometry was used to define antigen combinations totally absent or present at very minimal levels in marrow cells of normal individuals (n = 20) and patients receiving chemotherapy for acute lymphoblastic leukemia (n = 20). Immature (blast) cells were gated according to CD45/SSC properties. Fifty-three acute myeloid leukemia (AML) samples were studied in six-color combinations. Results: In six-color flow cytometry, 47 phenotypes were totally absent from blast gate in all normal samples. Forty-one other phenotypes were identified in less than 0.05% of blast cells. There was no difference between normal and postchemotherapy BMs. The four-color panel allowed to identify only 30 phenotypes present at a frequency

The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of a variety of client proteins with receptor and signal transduction functions. The objective of this study was to assess its expression in primary acute myeloid leukemia (AML) cells and to evaluate its biological and clinical significance. The in vitro effects of 17-AAG, a selective inhibitor of HSP90, was also evaluated. Cells from 65 patients with newly diagnosed AML were studied. The expression of HSP90 correlated with that of CD34, p170, and bcl-2 proteins but not with white cell counts, FAB or WHO subtype, or cytogenetics. HSP90 levels were also higher in samples exhibiting an autonomous growth in liquid culture or forming spontaneous colonies. A concomitant constitutive activation of the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/AKT pathways was observed in a majority of samples and was significantly correlated with HSP90 expression. All patients received induction chemotherapy. The percentages of HSP90-, CD34-, bcl-2-, and p170-positive cells were higher in patients who did not attain complete remission. Survival was also shorter in patients with high levels of HSP90. In vitro exposure of leukemic cells to 17-allylamino-demethoxy geldanamycin (17-AAG) resulted in inhibition of growth in liquid and clonogeneic cultures and in apoptosis, at concentrations which in most cases were not toxic for normal CD34-positive or progenitor cells. The concentration inhibiting 50% growth at 72 h in liquid culture correlated with HSP90 expression. Our study suggests that HSP90 is overexpressed in poor-prognosis AML cells and plays a role in cell survival and resistance to chemotherapy. Targeted therapy with 17-AAG represents a promising antileukemic strategy in adult AML.

Using flow cytometry, we investigated the clinical and hematologic relevance of expression of heat-shock proteins (HSP) HSP27, HSP60, HSP70, HSP90 and HSP110 in bone marrow of 142 patients with newly diagnosed myelodysplastic syndromes, together with that of the membrane differentiation antigen CD34 and the drug-resistance related protein, P170 (Pgp).

The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene. TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and promote DNA demethylation. TET2 somatic mutations occur in about 25% AML, distributing across the whole coding sequence without obvious hot spots. These mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. TET2 exon 2 is spliced in a mutually exclusive manner with exon 1 yet it is used as an alternative promoter (https://fasterdb.lyon.unicancer.fr/). However, TET2 exon 2 is not translated into protein and its role in TET2 regulation is still unknown. Having found that AraC sensitive cells harbor the spliced TET2 isoform, we investigated whether or not skipping of TET2 exon 2 correlate with disease outcome in AML patients treated with AraC-based intensive chemotherapy (AraC-IC). The discovery cohort included 106 consecutive AML patients treated with AraC-IC (median age 57.91, 64 males). RNA was extracted from bone marrow MNCs and assayed for TET2 exon 2 skipping through real-time quantitative ESRTPCR (qESRTPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2 exon 2 skipping was quantified by calculating the ratio E1E3/E2E3. For statistical analysis, the ratio E1E3/E2E3 was dichotomized using median value as the cutoff value. Skipping of TET2 exon 2 was associated with a significantly lower response rate: 65% vs 92%, p = 0.001 but with a significantly lower relapse rate: 39% vs 85%: p In conclusion TET2 exon 2 skipping is associated with a low relapse rate and possesses a strong favorable prognostic impact in AML treated with AraC-CTI. The mechanism linking this alternative exon usage with resistances to AraC are currently investigated while our results suggest that the determination of TET2 exon 2 splicing status might assist risk stratification. Disclosures Nicolini: Novartis: Consultancy.

The important metastatic potential of lung cancers is directly correlated with cell adhesion. Cell-extracellular matrix interactions occur in specialized structures termed focal adhesion (FA) complexes. Our aims were to investigate: (i) the expression of the major FA components in three lung cancer cell lines (non metastatic: A549, or metastatic: Calu-1 and H460), (ii) the modifications of the FA complex occurring when apoptosis was induced by Vinorelbine in the A549 cells.The FA complex was characterized by flow cytometry, immunocytochemical staining and Western blot.The expressions of alpha3, betsaP, paxillin, p-paxillin and Grb2 varied depending on the histological type of the tumor. In apoptotic cells, the expressions of the PYK2, p-p38, PI3K and Grb2 adhesion proteins were increased.Our data suggest that these adhesion proteins may be implicated in the transduction of death signals.

Heat shock proteins (HSPs) acts as molecular chaperones by helping in the refolding of misfolded proteins and assisting in their elimination if they become irreversibly damaged. HSPs induced by stress treatment have a role in the modulation of apoptosis. The reduction in protein expression levels was correlated with an increased susceptibility to drug-induced apoptosis. HSPs have also been implicated in the resistance of leukemia cells to potential therapeutic agents. The mechanisms of cellular protection used by HSPs have yet to be fully defined. HSPs were shown highly expressed by acute myeloid leukemia (AML) cells as well as by acute lymphoblastic leukemia (ALL) cells. HSP expressions were correlated with that of differentiation antigens and that of drug-resistance and apoptosis proteins. Complete remission (CR) rates were higher in patients with lower expression of HSPs. Overall survival (OS) was significantly longer in patients with lower expression of HSPs. Therapeutically, inhibition of inducible HSP expression or activity should not cause any undesired side effects. HSPs emerge as novel therapeutic targets in anticancer protocols. Early results of phase I studies indicate that 17-allylamino-17-demethoxygeldamycin (17-AAG), capable of binding and disrupting the function of HSP90, results in an acceptable toxicity profile while achieving in vivo disruption of multiple oncogenic client proteins.

BACKGROUND Acute leukemia (AL) requiring cytotoxic treatment occurring during pregnancy poses a very difficult therapeutic dilemma. METHODS By means of a mail questionnaire, information on a series of 37 patients with a diagnosis of AL during pregnancy was collected from 13 French centers between December, 1988 and November, 2003. RESULTS Thirty-one patients had acute myeloid leukemia (AML), and 6 patients had acute lymphoblastic leukemia (ALL). Nine patients were diagnosed during the first trimester, 10 patients were diagnosed during the second trimester, and 18 patients were diagnosed during the third trimester. Fifteen pregnancies ended with therapeutic or spontaneous abortion. There were 13 normal deliveries, including 1 gemellary pregnancy, and 9 Cesarean sections. Twenty-three healthy babies survived from the 37 pregnancies, of whom 15 babies had been exposed to chemotherapeutic agents. A complete remission was achieved in 34 patients. Eleven women had severe extrahematologic complications during the induction remission course. The median disease-free survival (DFS) was not reached, with a 5-year DFS of 54%. Ten patients developed recurrent disease. Overall, 12 of 37 pregnant women died from leukemia. CONCLUSIONS Pregnancy does not affect the course of AL. In the first trimester, termination of pregnancy should be discussed because of the potential fetal consequences of chemotherapy. Chemotherapy treatment during the second or third trimester may not require termination of pregnancy, because as remission of AL and delivery of a normal infant are likely to be obtained. Cancer 2005. © 2005 American Cancer Society.

PURPOSE. Eliminating fetal calf serum (FCS) from corneal organ culture (OC) media has long been a challenge. This study was an assessment of a new animal compound–free (ACF) medium for corneal storage and of its combination with poloxamer for end-of-storage corneal deswelling. METHODS. A randomized controlled study with masked assessment compared the ACF medium to standard commercialized media containing 2% FCS and their combination with dextran for deswelling. Paired human corneas were randomly allocated at procurement, one to the ACF medium and the other to the FCS media, and then assessed at day (D)2 and D30 of OC storage and after 48 hours of deswelling. Comparison criteria were endothelial cell density (ECD) and morphometry by a corneal analyser, quality of endothelial visualization (using saline), EC mortality (trypan blue), corneal thickness, corneal transparency, and folding. Fifty-six corneas (28 pairs) with ECD of 2000 cells/mm 2 or more were enrolled. Data were compared using paired tests with P 0.01 deemed significant. RESULTS. Parameters were similar at baseline (D2) between groups. Daily EC loss during the 30 days of storage was reduced with the ACF compared with standard (0.31% 0.30% vs. 0.88% 0.38%, P 0.001). With poloxamer 188 (Lutrol F68; BASF, Ludwigshafen, Germany), EC loss was substantially reduced (1.43% 3.60 vs. 15.41% 10.13%, P 0.001) and morphometry better preserved, despite thickness reduction, transparency improvement and folding reduction comparable to dextran. After 30 days of storage in ACF medium and deswelling in poloxamer 188, ECD was 30% higher (2466 447 cells/mm 2 vs. 1729 281 cells/mm 2 , P 0.001). ACF medium alone and combined with poloxamer 188 considerably facilitated EC visualization at D30 and after deswelling. CONCLUSIONS. The ACF medium combined with poloxamer 188 for deswelling showed superiority over standard FCS medium in its ability to preserve EC viability and facilitate endothelial visualization. This innovative use of poloxamer for deswelling appears far less toxic than does dextran. (Invest Ophthalmol Vis Sci. 2005;46:816 – 822) DOI:10.1167/iovs.04-1078

To identify prognostic factors alternative or additional to drug-resistance and apoptosis proteins, we studied the impact of the expression of heat-shock proteins (HSPs) in 98 newly diagnosed acute myeloid leukemia (AML). HSP27 was expressed by 39%, HSP60 by 26%, HSP70 by 58%, HSP90 by 41%, and HSP110 by 30% of cases. HSP expressions were correlated with that of differentiation antigens (CD34, CD14, CD15, CD33) and that of drug-resistance (MRP, MRK) and apoptosis (Bcl-2) proteins. HSP90 and HSP110 were correlated with FAB subtype and karyotypic grouping. Complete remission (CR) was obtained in 68 cases (69%). Median disease-free survival (DFS) of the 68 remitters was 18.1 months with a 3-year DFS rate of 41%. CR rates were higher in patients with lower expression of HSPs. Overall survival (OS) was significantly longer in patients with lower expression of HSPs. Cytogenetics, CD34 positive expression, MRK positive expression, and HSP110 positive expression remained as pejorative prognostic factors for OS in the multivariate analysis. When considering patients with intermediate risk cytogenetics, HSP110 and MRP positive expressions and CD33 negative expression were of poor outcome, while HSP27 and HSP60 positive expressions appeared of pejorative prognostic value in patients with unfavorable karyotypes.

Various transplantation strategies have been designed to improve the poor prognosis of adult (ages 15 to 60 years) acute lymphoblastic leukemia (ALL). The GOELAL02 trial evaluated the impact of early allogeneic bone marrow transplantation (alloBMT) or delayed unpurged autologous stem cell transplantation (ASCT) for patients who had no human leukocyte antigen (HLA)-matched sibling donor or who were older than 50 years. Inclusion criteria included at least one of the following: age older than 35 years; non-T-ALL; leukocytosis greater than 30 x 10(9)/L; t(9;22), t(4;11), or t(1; 19); or failure to achieve complete remission (CR) after one induction course. Among 198 patients, the median age was 33 years. The CR rate was 80% with vincristine, idarubicine, L-asparaginase, and randomized intravenous injection or oral steroids (P = nonsignificant [ns]). AlloBMT was performed after 2 consolidation courses while ASCT was delayed after 1 additional reinduction. Intensified conditioning regimen before transplantation included etoposide, cyclophosphamide, and total body irradiation (TBI). Median follow-up was 5.1 years. The median overall survival (OS) was 29 months, with a 6-year OS of 41%. On an intent-to-treat analysis for patients younger than 50 years, alloBMT significantly improved the 6-year OS (75% versus 40% after ASCT; P = .0027). Randomized interferon-alpha maintenance had no effect on relapse or survival after ASCT. In conclusion, the outcome of adult ALL is better after early alloBMT than after delayed ASCT.

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase localized to regions called focal adhesions. Many stimuli can induce tyrosine phosphorylation and activation of FAK, including integrins and growth factors. The major site of autophosphorylation, tyrosine 397, is a docking site for the SH2 domains of Src family proteins. The other sites of phosphorylation are phosphorylated by Src kinases. Phosphorylated FAK binds proteins of focal adhesion and can activate them directly or indirectly by phosphorylation. These activated proteins forming the FAK complex facilitate the generation of downstream signals necessary to regulate cell functions, like motility, survival and proliferation. Dysregulation of FAK could participate in the development of cancer. This review will focus upon the mechanisms by which FAK transmits biochemical signals and elicits biological effects.

Allogeneic peripheral blood stem cell transplantation (PBSCT) has emerged as an alternative to bone marrow transplantation. PBSCT can be associated with a higher incidence of chronic graft-versus-host disease (cGVHD). In this study, we investigated whether there was a correlation between the composition of PBSC grafts (CD34+ and CD3+ cells) and hematological recovery, GVHD, relapse, and relapse-free survival (RFS) after myeloablative HLA-identical sibling PBSCT. The evolution of 100 acute or chronic leukemia patients was analyzed. Neither hematological recovery, acute or cGVHD, nor relapse, was significantly associated with CD3+ cell dose. Increasing CD34+ stem cells was associated with faster neutrophil (P=0.03) and platelet (P=0.007) recovery. Moreover, 47 of the 78 patients evaluable for cGVHD (60%; 95% CI, 49-71%) developed extensive cGVHD. The probability of extensive cGVHD at 4 years was 34% (95% CI, 21-47%) in patients receiving a 'low' CD34+ cell dose (8.3 x 10(6)/kg), as compared to 62% (95% CI, 48-76%) in patients receiving a 'high' CD34+ cell dose (8.3 x 10(6)/kg) (P=0.01). At a median follow-up of 59 months, this has not translated into a difference in relapse. In patients evaluable for cGVHD, RFS was significantly higher in patients receiving a 'low' CD34+ cell dose as compared to those receiving a 'high' CD34+ cell dose (P=0.04). This difference was mainly because of a significantly higher cGVHD-associated mortality (P=0.01). Efforts to accelerate engraftment by increasing CD34+ cell dose must be counterbalanced with the risk of detrimental cGVHD.

Excessive apoptosis is implicated in the pathogenesis of myelodysplastic syndromes (MDS). We assessed by flow cytometry the expression of several members of the Bcl-2 family in bone marrow mononuclear cells (BMMNC) of 168 MDS samples at diagnosis. The proteins studied were Bcl-2, Bcl-xL (anti-apoptotic), Bax, Bad, Bak, and Bcl-xS (pro-apoptotic). The percentage of BMMNC expressing Bcl-2 and Bcl-xL was higher in refractory anemia with excess of blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) than in refractory anemia (RA) and RA with ringed sideroblasts (RAS). Conversely pro-apoptotic proteins Bad, Bak, and Bcl-xS were detected in a higher percentage of cells in RA and RAS. RA and RAS were associated with an increased Bcl-xS/Bcl-xL ratio. The expression of anti-apoptotic proteins was also correlated with that of CD34 and P170 and with the percentage of blast cells. Two-color analyses demonstrated that CD34 and Bcl-2 were usually expressed in the same cells. No significant correlation was found with cytogenetic abnormalities. Higher expression of pro-apoptotic Bcl-2-family proteins (Bak, Bad, Bcl-xS) and higher Bcl-xS/Bcl-xL ratio were associated with longer survival and decreased risk of leukemic transformation in univariate analysis, whereas expression of anti-apoptotic proteins was associated with decreased survival. Consequently Bcl-2 proteins expression was well correlated with the International Prognostic Scoring System (IPSS). Our data confirm that the control of apoptosis is deregulated in MDS cells. Moreover, the study of markers such as CD34 (or Bcl-2), Bcl-xL, and Bcl-xS provides additional prognostic information.

Background: Philadelphia chromosome-positive chronic myelogenous leukemia (CML) is a rare disease in children, and the optimal therapy is not clearly defined in these patients when a human leukocyte antigen-identical donor is not available. The present work focuses on the therapeutic efficacy and the toxicity of interferon (IFN) a2b in combination with cytosine arabinosine (Ara-C) in patients younger than age 18 years enrolled in the randomized trial CML 91, which compared the efficacy of IFN and cytosine arabinoside (Ara-C) with IFN alone in 810 patients with CML in the chronic phase. Patients and Methods: Twelve patients younger than age 18 years were enrolled in the randomized trial CML 91. Hydroxyurea and IFN (5 million units/m 2 , once a day) were given as initial treatment in all patients. After randomization, six patients received IFN (5 million units/m 2 , once per day) and Ara-C (20 mg/m 2 for 10 days each month) (IFN plus Ara-C group), and six patients received IFN alone (5 million units/m 2 once per day) (IFN group). Results: Six months after the beginning of the treatment, a complete hematologic response was obtained in all the patients in the IFN plus Ara-C group and in four patients in the IFN group. A major cytogenetic response was observed in three patients in the IFN plus Ara-C group and in two patients in the IFN group. Five patients from the IFN group who crossed over to receive Ara-C did not experience additional hematologic toxicity. Three patients in the IFN plus Ara-C group and two from the IFN group are alive, in major cytogenetic response, with a follow-up of 18 to 48 months. Conclusion: The combination of IFN and Ara-C induces complete hematologic and major cytogenetic responses and is well tolerated in patients younger than age 18 years with CML. This combination may offer an alternative to bone marrow transplantation in children in the chronic phase of CML without a histocompatible donor.

Acute Myeloid Leukemia (AML) is characterized by the expansion and resistance to apoptosis of myeloid cells blocked in early stages of differentiation. A subset of stem or progenitor cells, termed leukemic stem cells (LSC), which retain or reacquire self-renewing properties, as well as the capacity to remain in a poorly differentiated stage, give rise to the leukemic clone. The identification and purification of the LSC can provide a powerful tool for diagnosis, prognosis and therapy. Several studies suggested that LSC belong to the CD34+ CD38- compartment. Self-renewal and lack of differentiation are properties of embryonic and induced pluripotent stem cell which specifically express a set of differentiation antigens (SSEA1 and SSEA3) and transcription factors (OCT¾, SOX2, and NANOG), designated as ESCA. We postulate that an epigenetic reprogramming could induce the ESCA expression on HSC. The aim of our study was to investigate the expression ESCA in the CD34+CD38- cells from normal bone marrow (NBM) and from 50 AML BM patients. Then compare ESCA's expression between CD34+CD38- and CD34+CD38+ cells. We studied the expression in 5 cell lines (NTERA-2 which is control cell line, KG1a, U937, THP-1 and HL60 leukemic cell lines) and their potential involvement in myeloid differentiation in 2 cell lines (HL60 and THP-1). Thereafter, we inhibited the expression of ESCA in 3 leukemic cell lines (HL60, KG1a and U937) to better understand their role in abnormal proliferation or differentiation. The preliminary experiments showed an important expression of ESCA on all 5 cell lines by Multicolor Flow Cytometry (MFC), confirmed by RT-PCR. Then, we compared the expression of ESCA in CD34+CD38- cells between normal and leukemic marrow. We observed an up-regulation of two transcription factors OCT3/4 and SOX2 and the protein SSEA3 with 2-fold higher expression in AML cells compared to normal HSC. In addition, we found the down regulation of SSEA1 protein involved in cell adhesion, migration and differentiation. We also compared the expression of ESCA between the CD34+CD38- and CD34+CD38+ AML population. We observed a higher expression of OCT3/4 and SSEA3 (1.3-fold) in CD34+CD38-. We found a higher expression of SSEA1 (1.9-fold), NANOG and SOX2 (1.2-fold) in AML CD34+CD38+ population compared to CD34+CD38- population. We investigated whether there was a relationship between the overexpression of ESCA and a cytogenetic subgroup of AML. We found that AML t(15;17) expressed OCT3/4 at higher level than the other ESCA. Finally, the inhibition experiments showed that Oct3/4 was strongly inhibited in the KG1a cell line. In conclusion, these results suggest that the deregulation of ESCA may have a potential role in leukemogenesis by maintaining the LSC properties. This prompts us to test the relevance of these markers for LSC identification and as therapeutic strategies. Citation Format: Lydia Campos, TIPHANIE PICOT, CARMEN AANEI, PASCALE FLANDRIN-GRESTA, EMMANUELLE TAVERNIER, DENIS GUYOTAT. Embryonic stem cells antigens: expression in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1961. doi:10.1158/1538-7445.AM2014-1961

Cytarabine ocfosfate (YNK01) is a prodrug analogue of cytarabine which is resistant to systemic deamination after oral administration. Following initial studies indicating significant anti-tumour activity of YNK01 a phase II trial was initiated in order to assess the tolerability and efficacy of a combination of this agent with interferon alpha-2b (IFN-alpha2b) in recently diagnosed chronic phase CML patients (n = 98). The treatment was subdivided into cycles consisting of 4 weeks of continuous administration of IFN-alpha-2b (3 MU/m(2)/day 1st week and then 5 MU/m(2)/day) and 14 days of oral YNK01 (600 mg/day 1st cycle). At the end of each cycle the dose of YNK01 was adjusted according to the blood count observed during the previous 4 weeks. The median time from diagnosis to inclusion in the trial was 2 months (range 6 days to 7.5 months). At 12 weeks, 62 patients (63%; 95% CI, 54-73) achieved a complete hematological response. At 24 weeks, of 98 patients, two achieved a complete cytogenetic response, 14 a partial response (16% major cytogenetic response rate; 95% CI, 9-24) and 34 a minor response; 19 patients were not evaluable for cytogenetic response. During the trial, 20 patients progressed to accelerated (6) or blastic phases (14). The median time to progression was 15 months (range 2-38 months). At 3 years the overall survival was 79% (95% CI, 70-88). Although the complete hematological response rate compared favorably with the 40% response rate previously obtained with the subcutaneous formulation of Ara-c, the cytogenetic response rate was less than expected. Most of the patients experienced side-effects and all permanently stopped YNK01. Although the combination seems attractive the initial dose of 600 mg per day is probably too high and should be reconsidered in further trials.

In chronic myelogenous leukemia (CML), autologous stem cell transplantation could be a promising new approach for patients with no cytogenetic response after interferon alpha (IFN-alpha) therapy. We report data on 28 CML patients autotransplanted in chronic phase with peripheral blood progenitor cells mobilized with G-CSF (5 microg/kg/day x 5 days) given subcutaneously while continuing IFN-alpha therapy. At mobilization, 23 patients (82%) were in complete hematological remission (CHR), 16 (57%) achieved a minor cytogenetic response (mcr). We obtained, after stimulation, a median of 37.4 x 10(9)/l (6.9-108) white blood cells, 7.2 x 10(8)/kg (2.2-16.6) mononuclear cells, 39 x 10(4)/kg (4.8-403.5) CFU-GM and 4.2 x 10(6)/kg (0-58.6) CD34+ cells. Six patients received GM-CSF after transplantation. All patients engrafted, with no significant influence stemming from the Sokal index score and pretransplantation IFN-alpha therapy duration. The first cytogenetic evaluation after transplantation showed 11 (39%) major cytogenetic response (Mcr), and nine (32%) mcr with no significant correlation between these responses, the Sokal index score, and pretransplantation IFN-alpha therapy duration, although there was a significant impact from GM-CSF administration (P=0.01). After transplantation, 26 patients received IFN-alpha alone or associated with hydroxyurea. The median follow-up was 12 months after transplantation and 57 months after diagnosis. At the time of follow-up, nine patients were in CHR, six remained stable in chronic phase, three presented an mcr and one remained in Mcr. At the last follow-up, 22 patients were alive. We conclude that the results of this strategy are encouraging in poor IFN-alpha responders but that other prospective studies that try to maintain the cytogenetic responses obtained immediately after transplantation are needed.

To assess the place of allogeneic hematopoietic stem cell transplantation (HSCT) in the advanced stage of acute myeloid leukemia (AML), we retrospectively analyzed 379 consecutive patients who underwent allogeneic HSCT for advanced AML. The median follow-up of the entire cohort was 7.5 years. Sixty-nine patients (18%) were transplanted with primary resistant disease. Three hundred and ten (82%) were relapsed patients, 94 (30%) of whom were in untreated relapse, 67 (22%) in refractory relapse and 149 (48%) in 2nd or 3rd complete remission at time of transplantation. The 5-year probabilities of overall survival (OS), disease-free survival (DFS), and transplant-related mortality (TRM) were 22 ± 4% 20 ± 4%, 45 ± 6%, respectively. In multivariate analysis, we demonstrated the favorable impact on OS, DFS and TRM of two factors over which we have no control (age

To the editor: A family history of chronic lymphocytic leukemia (CLL) is one of the best characterized risk factors for the development of CLL. First-degree relatives of individuals with CLL have a 3- to 8-fold increased risk for CLL.[1][1],[2][2] CLL often has an indolent behavior, but some

Philadelphia (Ph) or BCR/ABL-negative cells with immature phenotype (CD34-positive, DR-negative) can be recovered from patients with chronic myeloid leukemia (CML) in chronic phase. We used the technique described by Berardi et al (Science 1995; 267: 104-108) to select stem cells from marrow or blood of CML patients at diagnosis or during treatment with alpha-interferon. Mononuclear cells (MNC), and in some experiments CD34+ cells, were maintained for 7 days in the presence of 5-fluorouracil (5-FU), stem cell factor and interleukin-3. The number of viable cells recovered after culture was between 7.4 and 70.2 for 10(6) cells plated. These cells exhibited the following phenotype: CD34+, CD117+, CD38-, lineage-, and were able to generate cobblestone areas and secondary colonies in long-term culture (LTC), with a frequency similar to that of cells selected from normal marrow. Study by fluorescence in situ hybridization of LTC cells or secondary colonies showed no evidence of BCR/ABL rearrangement. Reverse transcriptase polymerase chain reaction studies on pooled LTC cells or secondary colonies were also negative. By contrast, LTC cells or secondary colonies obtained from CML CD34+ cells without culture in the presence of 5-FU were always positive for BCR/ABL rearrangement. Finally, 5-FU selected cells were able to engraft NOD/SCID mouse, as human cells were detected in blood and marrow 10 weeks post transplantation, which were BCR/ABL negative by RT-PCR. This method of culture makes it possible to select constantly BCR/ABL-negative cells with capacities of development in LTC assay and of NOD/SCID mouse engraftment.

Objective To evaluate the effectiveness and the safety of danaparoid sodium in the treatment of critically ill patients with standard unfractionated heparin-induced thrombocytopenia (HIT) or low-molecular-weight HIT. Setting University hospital. Patients and methods Retrospective analysis of 42 consecutive critically ill patients who were admitted for HIT between October 1992 and February 1997 and were treated either with therapeutic or prophylactic doses of danaparoid sodium. Results Among the 26 patients treated with therapeutic doses, neither new thrombotic complications nor thrombosis extension was clinically suspected. Two deaths were directly related to lower limb acute arterial thrombosis associated with HIT. Two major hemorrhagic complications were observed when aspirin in addition to danaparoid sodium was administered. When danaparoid sodium was used in prophylactic doses (20 courses of treatment) to prevent either postsurgical or medical thrombotic complications, no thrombotic event was observed. No death related to HIT or danaparoid sodium treatment was observed. One aggravation of a postsurgical cerebral lesion was observed. During danaparoid sodium treatment, a persistence or a recurrence of thrombocytopenia was observed in 6.5% of patients without thrombotic complications. Conclusion Danaparoid sodium appears to be an efficient and safe treatment in critically ill patients with HIT. The concomitant use of aspirin in addition to danaparoid sodium seems to represent an important additional hemorrhagic risk that should be avoided in patient management.

Objective—To investigate the role of activated protein C resistance (APCR, factor V Leiden) in coronary artery thrombosis. Methods—The prevalence of APCR and of congenital deficiencies of antithrombin III, protein C, protein S, plasminogen, and factor XII was investigated in adult patients under 45 years of age with acute myocardial infarction. The results were compared with those of a group of 53 age and sex matched control subjects. Results—Among 75 patients under the age of 45 years who were admitted from November 1994 to April 1996 for acute myocardial infarction, 22 (29.3%) had normal coronary arteriography (group I) and 53 (70.7%) had significant coronary artery disease (group II). Inherited thrombophilia was more often found in group I (4/22, 18.2%) than in group II (4/53, 7.5%) but the difference was not significant (F test: p = 0.22). The prevalence of APCR was 9.1% (2/22) in group I, 3.8% (2/53) in group 2 (p = 0.57), and 3.8% (2/53) in the normal control group (p = 0.57). Conclusions—The prevalence of congenital thrombophilias, including APCR, does not seem to be increased in young patients with myocardial infarction and normal coronary angiograms, compared with young patients with coronary atherosclerosis and with normal control subjects. However, the statistical power of the study is too low to detect a significant difference and these results are published to allow a meta-analysis of this problem in the future. Keywords: myocardial infarction; factor V Leiden; coagulation factors; inherited thrombophilia

The effectivness of interferon alpha (IFN) for the treatment of chronic myelogenous leukemia (CML) during the chronic phase is now well established. Several studies have shown that IFN produces hematologic remission in 60% to 80% of patients [1]. In some studies the achievement of complete hematologic response within 3 months seems to represent a significant factor which correlates with a cytogenetic response [2]. Also, the treatment with IFN resulted in suppression of the Ph-positive cells population which was complete in 7% to 25% of the patients [3, 4]. A major cytogenetic response (i.e., less than 35% Ph-positive cells) was noted in 35 to 45% of the patients. Such cytogenetic response was not only complete but also prolonged for periods ranging between 2 and 8 years [5].

Allogeneic BMT for patients with acute myeloid leukemia (AML) is presently a reference therapy. The indications for this therapy may likely rely upon prognostic factors, and their importance are constantly investigated. To examine the impact of time from diagnosis to transplant on survival and Leukemia Free Survival (LFS), we analyzed 109 patients from the data base of the SFGM, patients who all had received an HLA-identical allogeneic BMT for a diagnosis of AML in First Complete Remission (CR1) between January 1987 and December 1992. All patients were prepared with cyclophosphamide (Cy) and Total Body Irradiation (TBI) (Cy — TBI), and Methotrexate (MTX) + Cyclosporine A (CSA) was used as Graft-vs.-Host Disease (GVHD) prophylaxis. Eleven patients needed 2 courses of induction to achieve CR. Time between diagnosis and BMT was 120 (64–287) days. Forty nine patients developed a grade 2 acute GVHD (Actuarial probability = 46%). With a median follow-up of 52 months (30–100), the 5 year probabilities for transplant related mortality (TRM), relapse, overall survival and LFS are respectively 25, 26, 59%, and 55%. A multivariate analysis showed that survival is adversely influenced by 3 independent factors: time to transplant (>120 days vs.≤120 days), acute GVHD (grade 2–4 vs. grade 0–1) and age (>33 vs.≤33). LFS is influenced by only the first two of these factors. The favorable impact of less time from diagnosis to transplant should lead to proceeding to the transplant as soon as possible. Practically speaking, this means that when such therapy is chosen for a patient with CR1 AML, the search for an allogeneic donor should be immediately investigated and transplant performed as soon as possible.

High expression of the multidrug resistance gene product P170, and of the oncoprotein bcl-2 have been associated with in vitro resistance to chemotherapeutic agents and with poor clinical outcome in acute myeloid leukemia (AML). More recently, it has been shown that autonomous proliferation of blast cells in liquid culture was also predictive of poor prognosis. In a series of 72 adult AML cases at diagnosis, we studied by flow cytometry the expression of P170 and bcl-2 proteins, together with autonomous growth of leukemic cells in liquid culture. Cases were classified as exhibiting no proliferation (N = 29), intermediate proliferation (N = 25) and high proliferation (N = 18). We observed a significant correlation between the percentage of cells in each sample expressing P170 and bcl-2. This was confirmed by double staining techniques showing that both antigens were present in the same cells. We also observed a significant association between growth pattern and P170 or bcl-2 expression. All patients were treated by intensive chemotherapy including an anthracycline drug and cytarabine. The blasts of patients achieving complete remission (N = 47) were less frequently positive for CD34, P170 and bcl-2 than those from patients who did not. Growth pattern also influenced significantly CR. In univariate analysis, CD34, P170 and bcl-2 expression, as well as growth pattern, significantly influenced survival. However, in multivariate analysis P170 expression remained the only significant factor, bcl-2 (or proliferation) having no independent value. Our study confirms the prognostic value of P170 and bcl-2 expression as well as the value of spontaneous proliferation and suggests that several drug-resistance mechanisms are implicated concomitantly in AML.

Discrepancies in the literature on acute leukemia blast cell immunophenotypes are sometimes related to differences between the epitopes recognized by various monoclonal antibodies (MoAb) in the same cluster of differentiation. CD15 is one example of such a variation. CD15 expression has been reported in 1.6% to 39% of acute lymphoblastic leukemias (ALL). We studied the expression of CD15 using 10 different commercially available anti-CD15 MoAbs and we observed three different expression patterns using anti-CD15 MoAbs by flow cytometry in 158 cases of ALL: Smy15c was found in 70% of B lineage ALLs, Smy15a and FMC-13 in 30 to 40% of cases and all others in less than 9% of B-ALL cases (p < 0.0001). In T lineage ALLs, Smy15c, Smy15a and FMC-10 identified CD15 in 30% of the cases and all others in less than 8% of the cases. Logistic regression revealed that Smy15a, CD34 and CD14 correlated significantly with Smy15c expression. We conclude that CD15 MoAbs have to be chosen carefully when ALL immunophenotype and subsequent studies of prognostic significance are performed particularly in assessing multiphenotypic ALLs.

A double-blind randomized trial was conducted to research a hypercoagulable state rebound after abrupt versus gradual withdrawal of acenocoumarol, 20 patients were included: 10 in the abrupt withdrawal group (AW) and 10 in the gradual withdrawal group (GW). Between days 1 and 15,F1 + 2 was higher in group AW (P < 0.002). A significant increase of D-dimer with time was found (P < 0.001) without difference between the two groups, tPA and PAI-1 levels remained stable throughout without difference between the two groups. No rebound phenomenon was observed. Four thrombotic recurrences were observed: group AW: 1, group GW: 3 (P = 0.29). There is neither clinical nor biological support for a gradual anticoagulation withdrawal.

From November 1987 to April 1994, 522 adult (15-50 years) patients with de novo acute myeloblastic leukemia (AML) were included in the GOELAM 1 protocol comparing allogeneic bone marrow transplantation (BMT), intensive consolidation chemotherapy (ICC) and autologous BMT (ABMT). For induction treatment, patients were randomized to receive a combination of cytosine-arabinoside (ara-C) (200 mg/m2 per day continuous infusion on days 1-7) and either idarubicin (IDR) (8 mg/m2 per day on days 1-5) or rubidazone (RBA) (200 mg/m2 per day on days 1-4). After achievement, of complete remission (CR) an allogeneic BMT was proposed to patients up to the age of 40 with an HLA-identical sibling. Other patients in CR had to receive a first course of ICC (ICC1) with highdose ara-C (3 g/m2 every 12 by 3-h infusion on days 1-4, eight doses) and either IDR (10 mg/m2 per day on days 5-6) or RBZ (200 mg/m2 per day on days 5-6). Bone marrow was collected after ICC1 and cryopreserved without any in vitro manipulation. If the hematopoietic quality of the collected marrow was adequate, patients were then randomly assigned to receive a second course of ICC (ICC2) with m amsacrine (AMSA); (150 mg/m2 per day on days 1-5) and etoposide (VP-16) (100 mg/m2 per day on days 1-5) or an ABMT after a preparative regimen with busulfan (4 mg/kg per day for 4 days) and cyclophosphamide (50 mg/kg per day for 4 days). As of July 1, 1994 490 patients were evaluable and 361 (74%) achieved CR with no significant difference between IDR and RBZ. An allogeneic BMT was planned in 83 cases and was actually performed in 67. Out of the 278 other patients 227 did receive ICC1. The median duration of neutropenia after ICC1 was 19 days and there were nine toxic deaths (4%). A total of 171 patients were randomized between ICC2 (84) and ABMT (87), and 128 have currently been analyzed (61 ICC2, 67 ABMT). The main reasons for exclusion were toxicity, refusal, poor hematologic reconstituion post ICC1, and relapse. With a median follow up of 44 months, the overall survival of the entire cohort of patients is 37% at 6 years (median 22 months) with no difference between the two induction treatment arms. The 4-year disease-free survival (DFS) of the patients in CR who actually received the assigned treatment was 45% for allogeneic BMT, 53% for ICC2, and 47% for ABMT. When considering intention to treat there was no significant difference in DFS between allogeneic BMT and other forms of post-remission therapy or between ICC2 and ABMT. We conclude that (a) a significant improvement of survival can be obtained for patients with de novo AML up 50 years of age with three different modalities of intensive consolidation; (b) the three approaches give comparable results—after ICC1, ICC2 appears to be as effective as unpurged ABMT and is easier to perform; (c) new strategies are needed to reduce the exclusion rate.

Introduction Chimerism analysis after allogeneic hematopoietic stem cells transplantation (HSCT) allows documentation and understanding of important clinical events such as engraftment, graft failure, and/or relapse. Few data are available on whether T-cell chimerism might be more informative than global chimerism. In this study, we focused on selective CD3+ T-cell chimerism and its interest to predict events after allogeneic HSCT. Methods This is a single center retrospective study. Only patients with a survival more than 3 months were included. Information concerning donors, recipients, conditioning regimen, graft harvesting and follow-up were collected in our center using prospective forms from Promise database. Evaluation of T-cell chimerism was performed on CD3+ blood cells after immunomagnetic sorting, by PCR-STR (short-tandem repeats) multiplex technique using 15 different markers. Analysis of the tandem repeat polymorphism was performed by sequencer and Genscan software (Applied Biosystem Inc.). Chimerism was evaluated in 2 ways: positive or negative, and % of recipient. An analysis with repeated measures was also performed to determine the utility of a positive measure of chimerism, independently of the date of sample after allograft, to predict a relapse. Results Between January 2006 and December 2011, 148 patients (pts) were admitted in our unit for allogeneic HSCT (32% with myeloablative conditioning and 68% with reduced conditioning). Median age was 54.3 years, 59 male, 89 female. Diagnosis included AML/MDS (n=77), ALL (n=23), Aplastic anemia (n=4), lymphoma and CLL (n=23), myeloma (n= 8) and myeloproliferative disorders (n=13). The donor was matched sibling (n=51), matched unrelated (10/10) (n=74) or mismatched unrelated (9/10)(n=23). Graft source was PBSC (n=97), BM (n=33) or CB (n=18). At transplant, 108 pts (73%) were in complete remission, 19 (13%) presented partial response and 21 (14%) have progressive disease. At time of analysis, median follow-up was 1.75 years. Median overall Survival (OS) was estimated at 2 years. Forty-seven pts (32%) presented relapse. Seventy pts (47%) are still alive. Main causes of death were HSCT related (n=38), relapse or progression (n=33) and other (n=7). Acute GVHD grade II-IV incidence was 32% (13.5% grade III-IV) and 58% for chronic GVHD. At day +30, 56% of pts presented a mixed T-cell chimerism with 36 (24.3%) pts with a chimerism higher than 25% recipient. At day +90, 41% have remained positive. For NRM, aGVHD and age were the only prognostic parameters statistically identified. For PFS, only T-cell chimerism with a positive 25% cut-off at D+30 was identified as a prognostic factor (p=.0017, HR(IC 95%) 2.52(1.39; 4.58). PFS was not reached if T-cell chimerism was inferior to 25% at D+30 versus 1.35 years (figure 1). The results were concordant in a subgroup analysis performed according to the myeloid or lymphoid neoplasia status. Otherwise, cGVHD incidence was higher if T-cell chimerism was less than 25% at D+30 (70% vs. 18%). Finally, the analysis with repeated measures confirmed that positive T-cell chimerism is strongly correlated with relapse (p=.001; HR (CI95%) 90.18(30.11; 270.13)). Conclusion After allogeneic HSCT, mixed T-cell chimerism higher than 25% at day +30 is strongly correlated with poor PFS, independently of source of cells, donors, intensity of conditioning, diagnosis or disease status at transplant. Considering this high-risk population, early intervention should probably be considered. Disclosures: No relevant conflicts of interest to declare.

From December 1987 to June 1992, 251 patients aged 50–65 with de novo acute myelogenous leukaemia (AML) were recruited to a multi-institutional randomized clinical trial. Induction therapy consisted of Ara-C (200 mg/m2, continuous infusion, days 1–7) with either zorubicin (ZRB) (200 mg/m2, i.v., days 1–4) or idarubicin (IDR) (8 mg/m2, i.v., days 1–5). Consolidation therapy consisted of a single course of intensive chemotherapy with high-dose Ara-C (3 g/m2, 3 h infusion, q 12 h, days 1–4) and m-Amsa (100 mg/m2/d, i.v., days 5–7). The complete remission (CR) rate was (73%) with Ara-C/IDR versus (60%) with Ara-C/ZRB (P = 0.033). In multivariate analysis, factors found to be significant in predicting CR were normal karyotype and treatment with IDR. With a median follow-up of 73 months, the median disease-free survival (DFS) duration of all CR patients and the probability of CR at 6 years were 17 months and 29%. In multivariate analysis the only factor associated with an increased DFS duration was a normal karyotype. The median event-free survival (EFS) duration for all evaluable patients and the median overall survival duration for all eligible patients were respectively 7 and 12 months without any difference between induction arms. The study shows that in patients aged 50–65 idarabicin is more effective than zorubicin for remission induction. However, the type of anthracycline did not influence overall survival duration. Using a unique consolidation course, we observed a prolonged DFS which compares favourably with results obtained with more prolonged consolidation therapy or maintenance treatment.

Objective: to evaluate whether long travel in sitting position is associated with an increase of coagulation activation and/or a decrease of fibrinolytic activity. Design : Comparison of blood coagulation and fibrinolysis parameters before and after two pleasure trips by bus organized in winter period (600 km in 8 hours) and in summer period (1200 km in 16 hours). Subjects: 31 and 23 healthy elder volunteers for the winter and the summer trip respectively. Nine other elder volunteers were selected as a control group for the winter study. Main outcome measures: prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III (TAT), D-dimers (D-D), factor VII activated, plasminogen activator inhibitor (PAI), tissue-type plasminogen activator (t-PA), plasma albumin. Results: A significant difference before and after the travel was only observed for TAT in the summer period. However all values of TAT were in the normal range. No volunteer presented with thromboembolic disease during the month following the travel. Conclusion: In the condition of our study, long travel in sitting position does not lead to an enhanced procoagulant state for elderly with varicose veins. These results suggest that there is no biological support to propose heparin prophylactic therapy for the elderly with varicose veins wishing to travel by bus.

We demonstrated previously that sera from quinine-treated patients reversed the MDR phenotype in vitro. Then, the combination of quinine with mitoxantrone (MTX) and cytarabine (Ara-C) was shown to be well-tolerated in patients with acute leukemias. To answer the question whether the response rate could be improved by quinine, we designed a phase III multicentric study. During the first year, the trial involved 112 adult patients (age 18–65y) with either relapsed or refractory acute myeloblastic or lymphoblastic leukemia or secondary leukemia or blastic transformation of myelodysplastic or myeloproliferative syndrome. All patients were treated with a combination of MTX (12 mg/m2/day — 4 days) and Ara-C (1 g/m2/12h — 5 days — 3 hrs iv infusion). After randomisation, 55 patients also received quinine (30 mg/kg/d — 5 days — beginning 24 hrs before MTX infusion). A 20% dose decrease was necessary in 14 patients due to vertigo or tinnitus and quinine was discontinued in 1 patient, due to excessive QT extension The remaining 57 patients received the chemotherapy alone. Toxic death was observed in 2 patients (one in each group) and ten patients (7 in quinine-treated group) died in aplasia before day 30. Response was assessable in 106 patients. As compared with the patients treated with MTX-Ara-C only, the patients treated with MTX-Ara-C plus quinine had a higher overall rate of response [29 / 52 (56%) versus 26 / 54 (48%)]. However, this difference is not significant. Quinine increased the duration of neutropenia (28,3 vs 23,9 days; p < 0.05) and thrombopenia (35 vs 28,3; p = 0.02). The incidence of nausea, vomiting and mucositis was also significantly higher in the quinine-treated group. This partial analysis confirmed the feasibility of using quinine in combination with MTX and Ara-C and suggested a trends for quinine to improve the response rate although these results have to be confirmed by the extended study.

Abstract 2841 Introduction: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, splenomegaly, cytopenias and constitutional symptoms. Ruxolitinib was recently approved by the FDA for the treatment of MF in the USA; its approval in Europe is still pending. However, EU patients may access to ruxolitinib through compassionate programs. In France, health authorities opened a compassionate patient-named program (Authorization for Temporary Utilization [ATU] program. Methods: 241 French patients (pts) with MF, including primary (PMF), post-polycythemia vera (PPV) and post-essential thrombocythemia (PET) MF were granted ruxolitinib therapy through ATU program, independently of their JAK2 mutational status, between April 15, 2011 and May 31, 2012. Physicians were asked to provide information on disease characteristics, treatment history, constitutional symptoms, spleen size, platelet and neutrophils count, as well as the ruxolitinib dose prescribed and adverse events (AE). Request forms had to be submitted at the time of initial application and every 3 months upon drug resupply or in case of treatment discontinuation. This analysis has been performed based on data available at baseline (n= 241), after 3 months (n= 101), 6 months (n= 57), 9 months (n= 21) and 12 months (n= 4). Results: In the entire cohort, 138 pts were men and 103 women. Median age was 68.3 years. 51.5% of pts had PMF, 22.8% PPV-MF and 23.8% PET-MF. 99.2% of pts had received ≥1 lines of therapy for MF prior to ruxolitinib (hydroxyurea: 56%; pipobroman: 15.4%; iMIDs: 13.7%; interferons: 13.7%; erythropoietins: 6.6%; spleen irradiation: 6.6%; anagrelide: 5.8%; corticosteroids: 4.9%). Despite these therapies, 93.7% had constitutional symptoms and 94.2% of patients presented a palpable splenomegaly (median 15 cm below costal margin) at inclusion. Efficacy: According to the baseline platelet count, ruxolitinib therapy was initiated at 15 mg BID in 132 pts (54.8%) or 20 mg BID in 103 pts (42.7%), or other doses in a minority of pts (n=6). Among the pts who were evaluable after 3 and 6 months of therapy, 96.5% and 90% presented a mean reduction in the spleen size (by palpation) by 47.2% and 46% from baseline, respectively. Constitutional symptoms resolved in 65.3% and 70.2% of pts at the aforementioned time points, respectively. In pts who completed 9 months follow-up (n=21), benefits in spleen size reduction and symptoms resolution were durable (95% and 71.4%). Safety: Since the beginning of the ruxolitinib ATU program, 83 pts presented at least one AE, including 27 pts with serious AE (SAE), with or without causal relationship to ruxolitinib therapy. AE, all grades, were essentially hematologic abnormalities (51.6%), gastro-intestinal 6.3%, cardiac 3.1%, musculoskeletal 3.1%, hepatic 2.3%, infection 2.3%. Dose adjustments were reported in 60 pts, mainly due to thrombocytopenia (n=36) and anemia (n=13). However, no patient discontinued ruxolitinib therapy because of cytopenia. Treatment was discontinued in 11 patients after a median duration of 2.6 months (range 0.3–7.9 months). Reasons for discontinuation were death (n=6), AE (n=2), inefficacy (n=2), and patient decision (n= 1). Conclusion: In this large, unselected population of heavily pretreated MF pts, ruxolitinib therapy appeared to be effective in treating both constitutional symptoms and splenomegaly, in line with previously reported efficacy in clinical trials. The safety profile seems also comparable. Comprehensive data and updated follow-up of the cohort will be presented. Disclosures: Off Label Use: compassionate use of ruxolitinib for myélofibrosis in France (indication not yet approved by EMEA). Rey:Novartis: Consultancy. Nicolini:novartis, Bristol myers Squibb, Pfizer, Ariad and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Recher:Celgène, Genzyme, Sunesis, Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel to ASH Other. Ranta:Novartis: Membership on an entity's Board of Directors or advisory committees. Legros:Novartis, Bristol Myers-Squibb: Research Funding, Speakers Bureau, Travel to meeting Other. Viallard:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dupriez:novartis: Membership on an entity's Board of Directors or advisory committees. Coiteux:Novartis, Bristol Myers-Squibb: Speakers Bureau. Demory:Novartis: Honoraria. Giraudier:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ugo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel to ASH Other. Kiladjian:Incyte: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding. Roy:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel to ASH Other.

Objectives: The recent evidences demonstrate that in myelodysplastic syndromes (MDS) a particular role is played by stromal microenvironment dysfunctions, which mediate the direct contact with hematopoietic precursor cells (HPC). The aims of our study were to assess the putative growth deficiencies of mesenchymal stromal cells (MSC) selected from MDS individuals, with regard to their ability to generate a microenvironment suitable to HPC development. Then, we intended to decode the focal adhesion (FA) signalling pathways and to understand whether adhesion-mediated processes contribute to transduction of intrinsic proliferative signals, as well as their impact on HPC-to-MSC interactions. Methods: To this end, we imagined a selection procedure using MSC specific markers expression (STRO-1 and CD73), we performed their phenotypic evaluation, and we conducted the functional assays on MSC and HPC selected from MDS patients vs. healthy volunteers. Finally, we have used the immunofluorescence microscopy to characterize the FA proteins (paxillin and focal adhesion kinase [FAK]), and of their regulators, p130CAS and HSP90. Results: The MSC production in STRO-1+ and CD73+ cell cultures from refractory cytopenia (RC) marrows was deficient, and, in addition, the clonogenic ability of these fractions was strongly diminished. The relative proliferation in MSC cultures from RC is the result of a continuous division process occurring at a low rate and lacking the ability to generate the normal functional progenitors required to form colonies. By contrast, in refractory anemia with excess of blasts (RAEB) settings, the proliferation rate is moderately improved due to the reduced doubling time of STRO-1 cells. However, this was not accompanied, at the end point, by complete functional maturity as reflected in the CFU-F number. Likewise, we have to point out the diminution of CFU-F capacity of CD73+ fractions in MDS that directly correlates with the CD44 mitigate on their surface. In addition, the doubling time of MSC from MDS inversely correlate with their expression for CD49e (Δ5-integrin). The proliferation differences occur in MDS cultures compared to normal settings can be attributed equally to the qualitative defects of FA proteins (FAK, and paxillin). The MSC from RAEB cultures highlight a strong complexation of FA proteins to HSP90 in nuclear area, which support a proliferative behaviour of these cells. Moreover, this high colocalisation to HSP90 indicates the cessation of proteasome-mediated recycling of these proteins. Furthermore, the preliminary results indicate the fact that the clonogenic potential of HPC is controlled by adhesion mechanisms dependent on stroma, and FAK is one of the molecules involved in this process. Conclusions: These data prove that MSC selected from MDS patients are intrinsically pathological and they could influence HSC behaviour by their direct interactions via FA proteins signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2470. doi:1538-7445.AM2012-2470

Endothelial cell activation during allogeneic bone marrow transplantation, mainly in acute graft-versus-host disease (aGvHD) was studied in 23 recipients and 5 controls using anti-von Willebrand factor (vWF) antibody, antibodies to endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and anti-HLA-DQ antibody, by immunohistological staining of skin. vWF extravasation, ELAM-1 and VCAM-1 expression were present in most recipients with a cutaneous rash which was confirmed as an aGvHD by histological examination (documented aGvHD) (p = 0.005 for vWF extravasation and ELAM-1 expression and p = 0.03 for VCAM-1 expression in comparison with the controls). In recipients with a rash, the cases displaying vWF extravasation and ELAM-1 expression were significantly more numerous in those with a documented aGvHD than in those without histological features of aGvHD (p = 0.01). vWF extravasation and ELAM-1 occurred concomitantly (p

Pentosan polysulfate is a low-molecular-weight sulfated polysaccharide used as an antithrombotic drug. We present two patients who developed thrombocytopenia and venous thrombosis during treatment with pentosan polysulfate. The relationship between pentosan polysulfate and thrombocytopenia is supported by platelet aggregation and serotonin release tests. In the light of the literature and our two cases, it appears that pentosan polysulfate alone as standard heparin and low-molecular-weight heparin can induce thrombocytopenia and thrombosis. Platelet counts should therefore be periodically monitored during pentosan polysulfate treatment. In the case of pentosan polysulfate-induced thrombocytopenia, it seems that heparin or low-molecular-weight heparin should not be instituted during the acute phase even if platelet aggregation studies are negative, because of their low sensitivity. After remission of thrombocytopenia, whether or not glycosaminoglycans can be reinstituted, at least temporarily, after antibody had disappeared is still an open question.

Abstract 2747 Introduction Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML), patient response is heterogeneous and it is generally accepted that residual disease and relapse are due to persistent CML cells, considered as leukemic stem cells. Their resistance has been related to lower TKI uptake. The amount of drug penetrating the targeted cells is most likely a major parameter of targeted therapy efficacy since it is essential that the therapeutic molecule be as close as possible to the target molecule. We developed a flow cytometry technique to analyze primary cells. Method To evaluate intracellular imatinib (ICIM) uptake, we developed a patented method based on natural UV fluorescence related to chemical structure. Consequently, since the difference in UV fluorescence units between treated and control cells is proportional to the amount of intra-cellular drugs, we validated this method after incubating K562 and KCL22 cell lines with TKI. The flow cytometry technique was standardized by using Flow-Check Fluorosphere calibrated beads immediately before, and at the end of, each series of analyses with a Coulter Epics Elite™ flow cytometer (Beckman Coulter) equipped with an Innova I90C-4 UV laser (Coherent). Then we analyzed primary blood cells from CML patients in chronic phase before any treatment. After lysis of erythrocytes, nucleated cells were incubated at 1.106 cells/ml with different doses of imatinib (IMA) (n=22), Nilotinib (NIL) (n=20) and Dasatinib (DAS) (n=20) at different times. Whenever possible, CML stem cells were analyzed using CD34-FITC staining. Results In preliminary assays, we checked that there was a significant correlation between additional fluorescence measured by flow cytometry and the amount quantified by physico-chemical analysis after lysing a known number of cells (n=57, r2=0.73, p We then applied our method to primary CML blood cells in comparison with normal blood cells. TKI penetrated all cell subsets, but amounts varied depending on cell sizes (FS/SS characteristics). The first data obtained with IM showed ICIM levels in CML cells that were relatively heterogeneous from one patient to another, ranging from 0.9 to 4 pg/cell for an extracellular concentration of 5 μM, i.e. a higher concentration (x 300) than in culture medium. The ability of the granulocyte cell lineage to store IMA was related to the Sokal prognostic index (p=0.05). We detected variable ICIM levels in CML CD34+ cells from 10/16 patients (0.04–0.7 pg/cell) and no signal for 6/16 patients. Surprisingly, the ability of CD34+ to store second generation TKIs is variable and not necessarily correlated to IMA uptake. Discussion We developed a simple, rapid flow cytometry method directly applicable to primary cells and requiring only few cells which makes it possible to identify target cell subsets, such as CML stem cells. The strong correlation between the ICIM amount and the sensitivity of CML cell lines to TKIs validated the method and suggested that ICIM could be a relevant biomarker for predicting the sensitivity of the CML clone. In our CML series, we observed striking inter-patient variability of the capacity of primary CML cells to store TKI. A correlation with the Sokal score suggests possible predictive value with regard to in vivo CML response to IMA, which could be taken into account when choosing TKI for first-line therapy. Furthermore, we observed marked heterogeneity between CML CD34+ cells for storing TKI that could partially explain the heterogeneity of in vivo response. The relationship between the ability of untreated CML CD34+ cells to store TKI and complete molecular response has to be established. Disclosures: No relevant conflicts of interest to declare.