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Introduction:

Background:Measurable Residual Disease (MRD) assessments are gaining increasing acceptance as a prognostic factor for tailoring treatment in hematological malignancies. Acute Myeloid Leukemia (AML) is a heterogeneous disease with high relapse rates and presents a high unmet need for effective treatment options. Measurement of residual disease after therapy reflects a combination of all resistance mechanisms and is currently used for guiding treatment options.

Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci: GBT: Membership on an entity's Board of Directors or advisory committees; Addmedica: Research Funding; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Low-molecular-weight heparins (LMWHs) can be administered in fixed subcutaneous doses permitting predictable parenteral anticoagulation in an out-of-hospital setting. The renal clearance of LMWHs leads to uncertainty about the safety of use of LMWHs in patients on hemodialysis given the potential for bioaccumulation of anticoagulant effect which may lead to bleeding.We conducted a randomized open label trial to investigate the feasibility and safety of a perioperative anticoagulation protocol in hemodialysis patients comparing two LMWHs preparations as outpatient bridging therapy when warfarin is interrupted for invasive procedures. The primary objective of the study was to compare if tinzaparin and dalteparin differentially bioaccumulate in hemodialysis (HD) patients after three therapeutic doses. Warfarin therapy was discontinued in these patients 5 to 6 days prior to surgery. Patients were randomized to either 3 doses of tinzaparin (175 IU/kg/day) or dalteparin (200 IU/kg/day) with two dialyses in the interval between the first dose of study drug and inavsive procedure. Primary outcome was pre-dialysis anti-Xa levels 20-24hrs post third therapeutic LMWH dose to determine if these LMWHs accumulated in HD patients, and if they differentially accumulated. After procedure, patients received a daily prophylactic dose of tinzaparin (4500 IU) and dalteparin (5000 IU) for 3 to 4 days along with dialysis. Post- procedure pre- and post- dialysis anti-Xa levels were also monitored.Of 29 eligible and consenting patients, 17 patients (58.6%) received tinzaparin sodium and 12 patients (41.4%) received dalteparin sodium. Two patients were withdrawn prior to invasive procedure and the invasive procedures were canceled (1 for important bio-accumulation in the dalteparin arm; 1 for major bleed in the tinzaparin arm). Primary outcome anti-Xa samples were not analyzable in 2 patients (1 contaminated with heparin; 1 drawn too early). There was important and significant bioaccumulation in both study drug arms. The mean primary outcome pre-dialysis anti-Xa level 20-24hrs post third tinzaparin dose was 0.40 IU/ml (SD= 0.21). The mean primary outcome pre-dialysis anti-Xa level 20-24hrs post third dalteparin dose was 0.57 IU/ml (SD= 0.43). There was no statistically significant difference between the two study drug groups (student's t-test (p value = 0.33). Postoperatively 16/21 (76%) of patients tested had undetectable pre-dialysis anti-Xa levels 20-24hrs post prophylactic dose, with the remaining 5 patients having a mean anti-Xa level of 0.23 IU/ml (SD = 0.25). During subsequent follow-up, 2 patients experienced serious adverse events (one non-STEMI in patient who died of sepsis (tinzaparin group) and one upper extremity DVT (dalteparin group)).Tinzaparin and Dalteparin significantly accumulate at therapeutic doses in HD patients with no statistically significant difference detected in accumulation between the drugs. Neither drug importantly accumulates at prophylactic doses in HD patients. “Bridging therapy” with LMWHs at therapeutic doses in patients on hemodialysis who require temporary interruption of warfarin for invasive procedures is risky and of uncertain risk benefit.Rodger: Bayer: Research Funding; Leo Pharma: Research Funding; Pfizer: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Biomerieux: Research Funding; GTC Therapeutics: Research Funding.

No relevant conflicts of interest to declare.

It is generally accepted that myelodysplastic syndromes (MDS) most often originate in a multipotent, myelorestricted progenitor population, although primary transformation may occur at the hematopoietic stem cell level. MDS can be classified into low risk and high risk with evolution to acute myeloid leukemia (AML) predominantly in the latter cases. In AML, survival of leukemia-initiating cells, often referred to as leukemic stem cells, after chemotherapy is thought to lead to minimal residual disease and relapse. Hence, in de novo AML a larger size of the stem cell compartment is predictive for poor survival. [Van Rhenen et al.,Clin Cancer Res 2005,11] The monoclonal antibody against the cell surface antigen C-type lectin-like molecule-1, CLL-1, together with lineage infidelity markers enables discrimination of normal and malignant stem cells. [Van Rhenen et al.,Blood 2007,110; Van Rhenen et al.,Leukemia 2007,21] It could be hypothesized that CLL-1 and aberrant marker expression on MDS stem cells together with size of the stem cell compartment may predict leukemic evolution. Therefore, stem cells, defined as CD45dimCD34+CD38−, were analyzed for expression of CLL-1 and aberrant lineage markers in bone marrow samples from 88 MDS patients classified by WHO as 16 RA w/o RS, 42 RCMD w/o RS, 3 MDS-U, 5 hypoplastic MDS, 6 MDS/MPD and CMML, 15 RAEB-1 and 2, 20 AML patients with a known MDS history and 26 healthy controls. Analysis of the CD34+CD38−frequency in all MDS patients and normal controls revealed no significant differences (median 0.0061% vs. 0.0074%, respectively), whereas the frequency of CD34+CD38−cells was 17-fold higher in high risk MDS (RAEB-1 and 2, median: 0.076%) as compared to low risk MDS (median: 0.0046%, p<0.001). Similar as in AML, stem cells were significantly more prevalent within the blast cell fraction (CD45dimSSCint/low) of high risk MDS as compared to low risk MDS (median 0.77% and 0.25%, respectively), reflecting the differences in clinical course in these patients (p=0.040). Regarding CLL-1 expression, a reliable number of stem cells (>20) could be tested in 11/15 high risk RAEB-1 and 2 cases and in 16/73 of the remaining low risk MDS cases. In these cases, median CLL-1 expression on the CD34+CD38−cells was 1.6% (range 0–50) in low risk and 2.0% (range 0–27) in high risk MDS. Median CLL-1 expression on stem cells was 0.0% (range 0–4.7) in normal controls. Nevertheless, expression of lineage infidelity markers, such as CD5, CD7 and CD56, on CD34+CD38−stem cells in MDS strongly suggests that a considerable part of these stem cells is malignant (median 35% in 7/16 patients tested). Our data show that CLL-1 is virtually absent on stem cells in MDS. Remarkably, median CLL-1 expression on stem cells in AML cases that evolved from MDS (7%, range 0–53, n=9) was manifold lower than in de novo AML (median 45% when excluding non de novo AML [Van Rhenen et al.,Blood 2007,110], p=0.034). Detailed analysis of CLL-1 expression in AML had already revealed that CLL-1 expression increases with differentiation (CD34−> CD34+CD38−> CD34+CD38+). [Bakker et al.,Cancer Res 2004,64;Van Rhenen et al.,Blood 2007,110] Thus, our data suggest that the CD34+CD38−cells in high risk MDS and AML with antecedent MDS are more immature than in most de novo AML, which might explain poor prognosis of AML cases with MDS history. To conclude, our data indicate that CLL-1 is a specific marker of de novo AML, while CLL-1-negative AML may have been evolved from a MDS pre-phase that is further characterized by an increasing size of the stem cell compartment upon progression towards AML.

Acute myeloid leukemia (AML) likely originates from the CD34+CD38- hematopoietic stem cell (HSC). The so-called side population (SP), defined by Hoechst 33342 dye, might offer an alternative/supplementary stem cell compartment. The relationship between both compartments is largely unknown. We found that the CD34+CD38- compartment can be subdivided in an AML and normal compartment, based on expression of AML stem cell specific antigen CLL-1: CLL-1 positive CD34+CD38- cells carry AML specific cytogenetic aberrations and initiate leukemia in NOD/SCID mice (van Rhenen, Blood 2007, in press). Lineage aberrancies including CD7, CD19, CD56 and aberrant myeloid aberrancies, enabled to further define AML and normal CD34+CD38- sub-compartments (van Rhenen et al., Leukemia21:1700, 2007). This led us to investigate whether SP too have aberrancies and whether these define primitive stem cells. SP cells were detected in 40 of 47 AML patients with median frequency of 0.02% (range 0.002–7.6%). In the majority of cases there was also a CD34+CD38- compartment with a median frequency of 0.44% (range 0–27%), which is 22 fold higher (in all individual cases >1-fold) than SP frequency. The median frequency of CD34+CD38- cells within SP compartment was only 2.5% (range: 0–49%). SP cells were partly or completely positive for CLL-1 (median 53%), CD123 (27%), CD7 (35%), CD19 (20%) and CD56 (53%). SP cells in NBM (n=12; median frequency 0.12%, range 0.008–4.1%) were completely negative for CLL-1 and lineage markers (median 0%, ranges 0–4% at maximum), but not for CD123 (median expression 27%, range 1–82%). These results strongly suggest that considerable part of SP cells are malignant, which was confirmed by FISH analysis. The whole SP fraction was remarkably heterogeneous with at least 4 different subpopulations present: 3 with lymphoid characteristics, ie CD7+ (median 7% of total SP), CD19+ (2%) and CD56+ (4%), all 3 CD45high and CD48+; a myeloid population (median 54% of SP population; range 4–91%), CD45low and CD48-, with relatively high forward and sideward scatter (FSChigh/SSChigh) and high CD38 expression (median 82%) and usually with aberrant marker expression; a low-frequency FSClow/SSClow myeloid fraction, CD45low and CD48-, with lower CD38 expression (median 48%), and negative for aberrant markers and a similar population but with aberrant markers present. The latter, presumably primitive, malignant population had median frequency (of WBC) of 0.0018% (range 0.00016–0.0056%). The CD34+CD38- content herein was 20% at maximum. NBM too had the 3 lymphoid populations (median 8%, 2% and 5%, resp), the FSChigh/SSChigh myeloid population (in 9/12 cases) and the FSClow/SSClow myeloid population (12/12 cases). The putative primitive character of the AML FSClow/SSClow SP subpopulation was substantiated by suspension culture for 5 weeks with subsequent CFU assay (14 days): FSClow/SSClow cells had >200 fold clonogenic ability compared to FSChigh/SSChigh cells. In conclusion, the AML SP compartment is highly heterogeneous and contains a low-frequency (median 1:50,000) subpopulation, defined by aberrant markers and with primitive characteristics, as a likely candidate stem cell population. In a stem cell concept integrating the CD34+CD38- and SP compartment, the presumed AML stem cell frequency would be in the order of 1:250,000. which probably is close to the presumed frequency of leukemia initiating cells in diagnosis AML.

Acute myeloid leukemia (AML) is generally regarded as a stem cells disease. A large proportion of remission patients ultimately relapses, indicating ineffectiveness of current therapies in eradicating leukemia stem cells (LSCs). Additional to the CD34+CD38− compartment, the so-called side population (SP) exists. In order to trace residual SP stem cells under remission conditions, allowing adapted risk stratification or more accurate prediction of relapses, we sought for leukemia stem cell specific immunophenotypic markers i.e. not staining the normal SP stem cells. We used C-type Lectin-like molecule-1 (CLL-1, van Rhenen, Blood106: 6a, 2005) and IL-3 receptor α-chain CD123, previously shown to be stem cell markers. Lastly, several markers making up the so-called leukemia associated phenotypes (LAP), used for MRD detection (Feller, Leukemia18:1380, 2004), were tested for their ability to stain the AML SP stem cell as well. Using Hoechst 33342 dye, PI and monoclonal antibodies against CD7, CD19, CD56, CD123 and CLL-1, SP immunophenotyping using FACS analysis was performed on bone marrow (BM) samples of 17 AML patients at diagnosis, 8 healthy donors (nBM) and 4 relevant donors with BM regenerating after chemotherapy (rBM). SP cells were detected in 13 of 17 AML patients with median frequency of 0.01% (% of WBC, range 0.00002-0.17%). In all 13 cases, SP cells were partly or completely positive for CLL-1 and/or CD123. In 10/13 cases SP cells were partly or completely positive for LAP markers These results strongly suggest that the majority of SP cells are malignant, which was confirmed by FISH analysis [n=2; t(8;21)]. Marker positive SP cells as a fraction of total SP was 0.66 (range 0.22–1.0). Furthermore, SP cells from control nBM and rBM were completely negative for CLL-1 and LAP markers, but not for CD123 (40%-82%). Remarkably, 11/13 AML samples revealed two subpopulations within the SP, in terms of sideward scatter (SSC): high side scatter (HSSC) SP cells, with median frequency of 46 % (% of whole SP compartment, range 21–86%) and low side scatter (LSSC) SP cells (median frequency 54 %, range 14–79%). In the 2 remaining cases only LSSC SP was present. HSSC and LSSC populations were also seen in 5/8 nBM SP cells, with only LSSC SP present in the remaining 3 cases. HSSC AML SP cells had a more differentiated phenotype (high SSC, high CD38 expression) and were all malignant in terms of LAP marker positivity and CLL-1 positivity. LSSC SP cells were more primitive (low SSC, mostly CD38 negative or low), only partly expressing CLL-1 or LAP. FISH analysis for a patient with t(8;21) confirmed the malignant nature of both the HSSC and LSSC LAP marker (CD19 in this case) positive cells and the normal character of the CD19 negative LSSC cells. Marker positive, primitive LSSC SP cells (n=13) had median frequency of 14.2% of total SP (range 4.6–48.9%). The presumed median frequency of these LSSC SP cells at diagnosis is thereby close to 1: 105, (% of WBC) which is close to the presumed AML stem cell frequency in diagnosis AML. In conclusion, diagnosis AML SP cells can be discriminated from normal SP cells based on expression of CLL-1 and LAP markers. SP cells show a primitive low-frequency sub fraction as a likely candidate to contain the leukemia initiating cell. Future studies include functional characterization of this LSSC SP subfraction for purposes of risk stratification and therapeutic intervention.

In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event is thought to occur in the CD34+CD38− stem cell compartment. Survival of these cells after chemotherapy hypothetically results in minimal residual disease (MRD) and leads to relapse. C-type lectin-like molecule 1 (CLL-1) could serve as a possible target for therapy, as we previously showed that it is expressed on malignant CD34+CD38− cells at diagnosis and not on normal CD34+CD38− cells (van Rhenen et al., Blood106: 6a, 2005). CLL-1 expression is also present on malignant CD34+CD38− cells after chemotherapy. In the present study we investigated whether other antigens are also aberrantly expressed on CD34+CD38− cells in AML patients at diagnosis which would allow detection of AML CD34+CD38− cells in remission bone marrow. Such would offer opportunities for patient risk stratification and guidance of therapeutic intervention. Flowcytometry was performed on bone marrow CD34+CD38− cells in AML at diagnosis, after chemotherapy and in normal (NBM) and regenerating (RBM) bone marrow. Antibodies were anti-CD34, anti-CD45 and anti-CD38 together with antibodies against CLL-1, CD2, CD5, CD7, CD11b, CD19 and CD56, apart from CLL-1 all used as leukemia-associated phenotypes (LAPs) in immunophenotypic MRD detection (Feller et al., Leukemia8:1380, 2004; further referred to as “whole blast” MRD). In diagnosis AML marker expression on CD34+CD38- cells was scored as <50% or >50%, because at least 50% expression is needed for accurate measurements of malignant CD34+CD38- cells after chemotherapy. At diagnosis, 60/77 AML samples were CD34-positive. A reliable number of CD34+CD38- events (>20) could be measured in 56/60 cases. CLL-1 expression >50% was observed in 15/60 cases, LAP expression in 9/60 cases and both CLL-1 and LAP in 8/60 cases. Altogether, in 32/60 CD34-positive cases, detection of malignant CD34+CD38- cells was possible. In NBM (n=4) as well as in RBM, the CD34+CD38- cells did show low CLL-1 (n=6) and low LAP (n=2, for all antigens) expression (all <3%). For three AML samples in which a leukemia-specific translocation was present, FISH analysis showed the translocation in CD34+CD38-LAP+ cells. Therefore, CLL-1 and/or LAP staining might enable to accurately discriminate between residual normal and malignant CD34+CD38- cells after chemotherapy. Firstly, we found that irrespective of the aberrant phenotype or the time point of analysis the frequency of residual malignant CD34+CD38- cells significantly correlated with “whole blast” MRD (r=0.42, p=0.008, n=38). Secondly the ratio between malignant and normal CD34+CD38- cells (stem cell ratio) was found to parallel both CD34+CD38- cell frequency and “whole blast” MRD. Both parameters, similar to “whole blast” MRD, parallelled clinical outcome with decreases when entering remission, no changes in non-responders and increases with forthcoming relapses. In 3/60 cases in which “whole blast” MRD could not be used, stem cell MRD could be performed. Since the populations are so well defined [CD34+/CD38−/CD45dim/aberrant marker] stem cell MRD will require less extensive experience than currently used MRD frequency assessment. These advantages are expected to increase as the use of 5 or more fluorescence channels will improve the success of stem cell MRD. In conclusion it is possible to detect malignant stem cells in AML patients at diagnosis and after chemotherapy, which would offer opportunities for future patient risk stratification and guidance of therapeutic intervention.

A high relapse rate in AML suggests that present therapies are ineffective in eliminating leukemic stem cells. These may be present in the side population (SP) cells, which are able initiate leukemia in NOD/SCID mice. LSC detection may offer opportunities for detection of stem cells under conditions of minimal residual disease (MRD). Separately (Moshaver, this meeting) we show that leukemia associated immunophenotypes (LAP; used for MRD detection, Feller, Leukemia18:1380, 2004) and CD34+CD38- stem cell marker C-type Lectin-like molecule-1 (CLL-1, van Rhenen, Blood 106: 6a, 2005), and Il-3 receptor a-chain CD123, are able to discriminate between normal and leukemic SP stem cells at diagnosis. In addition, LAP marker and CLL-1 expression, but not CD123 expression, on SP cells in control bone marrows (n=12) was lacking. SP cells could be detected in 13/17 diagnosis AML patients. The contribution of the AML SP to total SP was calculated based on expression of above-mentioned AML markers and checked with FISH analysis. We further use “AML SP frequency” (AML SP as % of WBC) and “AML SP fraction” (AML SP as fraction of total SP). We obtained 12 follow-up samples of 6 of these patients. At diagnosis median AML SP frequency was 0.015 and median AML SP fraction was 0.7. At follow-up, median total SP frequency was 0.01% (0.0007%–2.0 %). AML SP frequency herein was median 0.004% (<0.00001 – 1.05). AML SP frequency detection was compared with MRD detection method, known to predict prognosis (Feller, 2004). In 5/6 patients with low AML SP frequencies (< 0.004%) MRD was undetectable (=0.01%). In 4/6 patients with high frequencies, MRD>0.01% was found. 3/6 patients showed a remarkable phenomenon: AML SP frequency was relatively high (0.006%, 0.007% and 0.014%, respectively) with apparently non-matching, relatively low MRD frequencies (0.02%, 0.02% and 0.01%, resp.), but with a drastic increase of MRD frequency at the next sampling time point (to 0.17%, 0.11% and 0.21%, resp). Such increases as well as the absolute percentages (>0.1%) inevitably lead to relapse (Feller 2004), which at present short follow up time already occurred for one patient. In addition, AML SP fraction was compared with MRD: 5/5 with low AML SP fraction (<0.21) had no detectable MRD (MRD=0.01%). For high AML SP fraction (=0.21) 4/6 had MRD>0.01%. AML SP fraction identified the same 3 special patients as did AML SP frequency: the AML SP fraction was high (0.33, 0.64 and 1.0, resp.) with the low MRD frequencies that subsequently strongly increased. Both stem cell parameters thus add additional prognostic value to MRD assessment. In conclusion, AML and normal stem cells can be detected with great specificity in AML remission bone marrow using LAP markers and CLL-1, that specifically stain AML SP stem cells and not normal SP stem cells. Additional predictive power for forthcoming relapses comes from the selective sparing of AML SP stem cells after therapy compared to both the more mature AML cells and the normal stem cells. The introduction of such stem cell parameters may thus contribute to MRD based risk stratification during treatment, offer guidelines for future clinical intervention time points, while studying cellular characteristics of both AML and normal stem cells may allow to design stem cell directed therapies that are effective for eradication of AML stem cells with minimal toxicity towards normal stem cells.

Immunophenotypical assessment of minimal residual disease (MRD) has been shown by us (Feller et al, Leukemia 18:1380, 2004) and others (San Miguel et al., Blood 98: 1746, 2001, Venditti et al., Blood 96: 3948, 2000) to be predictive for clinical outcome in AML both in patients treated with standard chemotherapy (SD) and high dose chemotherapy followed by autologous (AutoTQ) or allogeneic (AlloTQ) transplantation. Directly after consolidation therapy using a cut-off of 0.11% in the high MRD group the risk of relapse was 7.2 fold than in the low MRD group (Feller et al., 2004). Using that cut-off level, in the present study another important issue, i.e. sequential MRD assessments after end of treatment were done to i) prove consistancy of low MRD% in patients that remain in CR, ii) prove the ability to predict relapse using increase of MRD% and iii) assess the minimally required inter-sampling period to make MRD assessment clinically useful. To enable such 124 samples of 33 patients (<60 years) were included: 16 after SD, 8 after AutoTQ and 9 after AlloTQ. All time points refer to post-treatment periods, time point zero reflecting end of therapy.