Stem cells in liver repair

The liver can be subjected to many damaging insults, usually from toxins, viral infections, immune or metabolic diseases, during its lifetime. Normal restoration of liver tissue occurs via division of mature functional hepatocytes. In addition, a liver-stem-cell compartment, lying deep within the intrahepatic biliary tree, can be activated during severe or iterative stress. Recent studies have suggested that the bone marrow (BM) may also contribute to liver regeneration, although these observations remain controversial.

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The Novel AML Stem Cell Associated Antigen CLL-1 Discriminates between Normal and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v106.11.4.4 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4-4 ◽

Cited By ~ 1

Author(s):

Anna van Rhenen ◽

Nicole Feller ◽

Angèle Kelder ◽

Guus Westra ◽

Lex Bakker ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Clinical Outcome ◽

Stem Cell Marker ◽

Cell Detection ◽

The Novel ◽

Antibody Treatment ◽

Cell Compartment ◽

Stem Cell Compartment

Abstract In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event likely takes place in the CD34+CD38- stem cell compartment. Survival of these cells after chemotherapy hypothetically leads to minimal residual disease (MRD) and relapse. We have previously shown that a high CD34+CD38- frequency correlates with both MRD frequency, especially after the third course of chemotherapy and poor survival (Clin Cancer Res, in press). Furthermore, we have shown that a monoclonal antibody against the novel cell surface marker C-type lectin-like molecule-1 (CLL-1), directed against myeloid cells, stains 92% of diagnosis AML (Bakker et al., Cancer Res.64:8443, 2004). In the present study we investigated whether this antibody can be used to identify AML stem cells in remission bone marrow. Such would offer opportunities for MRD stem cell detection and stem cell-directed therapy. We found that anti-CLL-1 antibody homogeneously stained the CD34+CD38- compartment in 77/89 cases (median expression of 33.3% in all 89 cases, range 0–100%). The median stem cell expression of CLL-1 in control bone marrow was 0% ranging from 0–11% (n=11). Furthermore, CLL-1 expression on AML stem cells is highly stable: no differences between paired diagnosis and relapse samples (p=0.9, n=12). Like most antigens CLL-1 is expressed on part of the CD34+CD38+ compartment, but expression is absent on megakaryocytic precursors, which for therapeutic use would circumvent delayed platelet recovery. For antibody-mediated therapy it is crucial that normal stem cells remain negative throughout treatment of the disease. Therefore we tested bone marrow regenerating after high dose chemotherapy, obtained from either non-AML hematological patients or CD34 negative or CLL-1 negative AML patients. In those patients complete absence of CLL-1 expression was found in CD34+CD38− cells (n=4). Under MRD-conditions CLL-1 staining thus enables to accurately discriminate between normal and malignant CD34+CD38− stem cells. In agreement with this, the different ratios of AML and normal stem cells that were found in a number of patients, paralleled clinical outcome in terms of probability of relapse. For comparison, the stem cell marker CD123 was studied. Although anti-CD123 antibody homogeneously stained CD34+CD38− cells with high intensity in almost all AML samples studied (35/36 cases) with also no differences between diagnosis and relapse (p=0.6, n=6) and with low expression in normal bone marrow (median 14.9%, range 0–18.8%, n=5), a high expression was found in regenerating bone marrow (median 60%, range 53–84%, n=4). The latter suggests that anti-CD123 antibody is not AML stem cell specific under all conditions of disease. In conclusion, our data provide strong evidence that a large CD34+CD38− population at diagnosis reflects a higher percentage of chemotherapy-resistant cells, which, in remission, will lead to the outgrowth of MRD, thereby affecting clinical outcome. The specificity of anti-CLL-1 antibody under all conditions of disease enables both reliable detection and quantification of the stem cell compartment for prognostic use under MRD conditions, as well as characterization. Moreover, it shows that AML stem cell targeting using antibody treatment at different stages of disease has now become an option in the treatment of AML patients.

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Leukemic Stem Cell Assessment in Remission Bone Marrow of Acute Myeloid Leukemia Patients Is a New Prognostic Parameter.

Blood ◽

10.1182/blood.v114.22.399.399 ◽

2009 ◽

Vol 114 (22) ◽

pp. 399-399 ◽

Cited By ~ 3

Author(s):

Monique Terwijn ◽

Angèle Kelder ◽

Arjo P Rutten ◽

Alexander N Snel ◽

Willemijn Scholten ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Prognostic Information ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Acute Myeloid

Abstract Abstract 399 In acute myeloid leukemia (AML), relapses originate from the outgrowth of therapy surviving leukemic blasts know as minimal residual disease (MRD). Accumulating evidence shows that leukemia initiating cells or leukemic stem cells (LSCs) are responsible for persistence and outgrowth of AML. Monitoring LSCs during and after therapy might thus offer accurate prognostic information. However, as LSCs and hematopoietic stem cells (HSCs) both reside within the immunophenotypically defined CD34+CD38- compartment, accurate discrimination between LSCs and HSCs is required. We previously showed that within the CD34+CD38- stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers (leukemia associated phenotype, LAP), including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen, Leukemia 2007, Blood 2007). In addition, we reported that flowcytometer light scatter properties add to even better detection of LSCs, allowing LSCs detection in AML cases lacking LAP (ASH abstract 1353, 2008). Using this gating strategy, we determined LSC frequency in 64 remission bone marrow samples of CD34+ AML patients. A stem cell compartment was defined as a minimum of 5 clustered CD34+CD38- events with a minimal analyzed number of 500,000 white blood cells. After first cycle of chemotherapy, high LSC frequency (>1 × 10-3) clearly predicted adverse relapse free survival (RFS, figure 1a). LSC frequency above cut-off led to a median RFS of 5 months (n=9), while patients with LSC frequency below cut-off (n=22) showed a significantly longer median RFS of >56 months (p=0.00003). In spite of the relatively low number of patients, again a high LSC frequency (>2 × 10-4) after the second cycle and after consolidation therapy predicted worse RFS: after second cycle, median RFS was 6 months (n=9) vs. >43 months for patients with LSC frequency below cut-off (p=0.004). After consolidation, these figures were 6 months (n=7) vs. >32 months (n=6, p=0.03). Although total blast MRD (leukemic blasts as % of WBC) is known to predict survival (N.Feller et al. Leukemia 2004), monitoring LSCs as compared to total blast MRD has two major advantages: the specificity is higher (van Rhenen et al. Leukemia 2007) and well-known LSC makers like CLL-1, CD96 and CD123 can in principle be used for LSC monitoring, but not for total blast MRD detection since these markers are also expressed on normal progenitor cells. On the other hand, LSCs constitute only a small fraction of all leukemic blasts and therefore monitoring total blast MRD may have the advantage of a higher sensitivity. We thus tested the hypothesis that even more accurate prognostic information could be obtained by combining LSC frequency with total blast MRD. Total blast MRD after first cycle was predictive for survival with borderline significance (p=0.08): a cut-off of 0.3% resulted in two patient groups with median RFS of 9 months vs. >56 months. Figure 1b shows the result of the combined data of LSC and MRD frequency after first cycle therapy. We used the terms LSC+ and MRD+ for cell frequencies above cut-off and LSC- and MRD- for those below cut-off. We could clearly identify that apart from LSC+/MRD+ patients, LSC+/MRD- patients too have very poor prognosis, while MRD+/LSC- patients show an adverse prognosis as compared to LSC-/MRD- patients. These results from the first study on the in vivo fate of LSCs during and after therapy, strongly support the hypothesis that in CD34+ AML the leukemia initiating capacity originates from the CD34+CD38- population and is important for tumor survival and outgrowth. These results show that LSC frequency might be superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency, while the combination of both may offer the most optimal parameter to guide future intervention therapies. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

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Stress Response Gene Egr-1 As Tumor Suppressor in BCR-ABL Mediated Leukemia

Blood ◽

10.1182/blood.v120.21.33.33 ◽

2012 ◽

Vol 120 (21) ◽

pp. 33-33

Author(s):

Silvia Maifrede ◽

Dan Liebermann ◽

Barbara Hoffman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Tumor Suppressor ◽

Tyrosine Kinase ◽

Wild Type ◽

Response Gene ◽

Cell Compartment ◽

Stem Cell Compartment ◽

Novel Targets

Abstract Abstract 33 Chronic Myelogenous Leukemia (CML) is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. In the late 1990s a Tyrosine Kinase Inhibitor (TKI), imatinib mesylate (Gleevec, Novartis) started to be effectively used on CML patients. However, imatinib, therapy suppresses rather than eliminates the disease, and resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. The transcription factor early growth response 1 (Egr-1) was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There's evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). Therefore, we chose to investigate if Egr-1 can act as a suppressor of BCR-ABL driven leukemia. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, we investigated the stem cell compartment of both Egr-1 WT and Egr-1−/− BM, by determining the percentage of stem cells (Lin−Sca+c-Kit+, LSK), before infection with BCR-ABL; no statistically significant difference in the percentage of LSK cells was observed between Egr-1 WT and Egr-1−/− BM. Thus, the BM stem cell compartment of the Egr-1−/− mice does not offer a quantitiative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. Furthermore, when BM of transplanted mice was analyzed we observed an increased population of lineage negative cells in Egr-1−/− BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1−/− BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Additional data from our animal model, as well as analysis of human leukemia samples will be presented, further corroborating that Egr-1 functions as a suppressor of BCR-ABL driven CML. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics for CML, as well as for other leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic stem cells: effect of preirradiation, bleeding, and erythropoietin on thrombopoietic differentiation

Blood ◽

10.1182/blood.v49.2.253.253 ◽

1977 ◽

Vol 49 (2) ◽

pp. 253-261 ◽

Cited By ~ 4

Author(s):

R Goodman ◽

H Grate ◽

E Hannon ◽

S Hellman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Proliferative Activity ◽

Bone Marrow Cells ◽

Cell Compartment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Cell Assays

Abstract A method of measuring differentiation of stem cells towards platelets is described using syngeneic bone marrow injected into lethally irradiated mice. Fourteen days after such injection, the platelet counts are found to be proportional to the number of bone marrow cells injected and can be used as a measure of platelet progenitors. Perturbation of the milieu in which the transplanted marrow is placed by host preirradiation, bleeding, or erythropoietin administration leads to enhanced thrombopoiesis. It has been shown previously that similar perturbation favors erythropoiesis at the expense of granulopoiesis. The data from these and other experiments appear to be consistent, with a model of the stem cell compartment as a continuum with proliferative activity increasing as commitment is restricted. These functions vary inversely with the capacity for self-renewal. The various stem cell assays measure different ranges of stem cells, but overlap within this continuum.

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Effect of graft-versus-host disease on hematopoiesis after bone marrow transplantation in mice

Blood ◽

10.1182/blood.v78.10.2773.2773 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2773-2779 ◽

Cited By ~ 14

Author(s):

PJ van Dijken ◽

J Wimperis ◽

JM Crawford ◽

JL Ferrara

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Cell Compartment ◽

Host Disease ◽

Murine Bone Marrow ◽

Graft Versus Host ◽

Stem Cell Compartment

Abstract We have examined the effect of graft-versus-host disease (GVHD) on the reconstitution of donor hematopoiesis in a murine bone marrow transplant (BMT) model of GVHD to minor histocompatibility antigens. GVHD had no effect on peripheral blood counts, which normalized by 1 month after BMT, and did not affect numbers of hematopoietic progenitors in the BM, which remained decreased in all transplant recipients. Donor stem cells (colony-forming unit-spleen day 8) and stem cell self-renewal remained low in all mice for 5 months after transplant, but GVHD further damaged the stem cell compartment. Peripheral counts 1 month after transplant were supported by increased numbers of stem cells in cycle and increased splenic hematopoiesis. However, GVHD altered the pattern of extramedullary hematopoiesis, causing dramatically decreased activity in the spleen and increased activity in the liver. We conclude that GVHD further decreases hematopoietic reserve and causes damage to the donor stem cell compartment during hematopoietic reconstitution after transplant, despite unaffected progenitor frequencies and peripheral blood counts.

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Studies of hematopoietic stem cells spared by 5-fluorouracil.

Journal of Experimental Medicine ◽

10.1084/jem.159.3.679 ◽

1984 ◽

Vol 159 (3) ◽

pp. 679-690 ◽

Cited By ~ 136

Author(s):

G Van Zant

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Developmental Maturation ◽

Marrow Cells

Mouse marrow cells were exposed to 5-fluorouracil (FU) either in vivo or in vitro and the effects on the hematopoietic stem cell compartment were studied. The drug was highly toxic to bone marrow cells including the spleen colony-forming unit (CFU-S) population. The small population of stem cells surviving FU, however, caused a different pattern of spleen colony growth when injected into lethally irradiated mice. Whereas numbers of spleen colonies caused by normal marrow cells remained constant during an 8-14 d period after transplantation, spleen colonies derived from FU-treated marrow cells increased by as much as 100-fold during this time. This effect on stem cells was dose dependent both in vitro and in vivo. When FU was given in vivo, the day 14/day 8 ratio of colonies was greatest 1 d after injection and, over the next 7 d, returned to a near-normal value, that is, unity. A number of studies have shown that the stem cell compartment is heterogeneous with respect to self-replicative capacity and developmental potential. An age structure for the stem cell compartment has been proposed wherein cells with a short mitotic history are more likely to self-replicate than they are to differentiate; hence they are more primitive. 'Older' stem cells with a longer mitotic history are, according to the hypothesis, more likely to differentiate. 5-fluorouracil may be toxic to the older stem cells and selectively spare the more primitive subpopulation. Although the surviving cells may not themselves be able to form spleen colonies, they may give rise to an older cohort of cells more likely to differentiate and form spleen colonies. It is the requisite developmental maturation within the stem cell compartment that may be responsible for the delay in appearance of spleen colonies derived from FU-treated marrow. Our results support this explanation and identify the locus of at least part of this activity as the bone marrow. We found that the FU-treated marrow did not cause an increase in spleen colony numbers between 8 and 14 d in hosts with a long-standing marrow aplasia, due to the incorporation of 89Sr into bone. I propose that the delayed spleen colony appearance in normal hosts is the result of developmental maturation of the primitive stem cell compartment that survives FU and is responsible for spleen colonies arising around day 14. This maturation, at least initially, occurs in the marrow and leads to the replenishment of the more differentiated CFU-S subsets ablated by FU, which are normally responsible for spleen colonies appearing earlier after transplantation.

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Hematopoietic stem cells: effect of preirradiation, bleeding, and erythropoietin on thrombopoietic differentiation

Blood ◽

10.1182/blood.v49.2.253.bloodjournal492253 ◽

1977 ◽

Vol 49 (2) ◽

pp. 253-261

Author(s):

R Goodman ◽

H Grate ◽

E Hannon ◽

S Hellman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Proliferative Activity ◽

Bone Marrow Cells ◽

Cell Compartment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Cell Assays

A method of measuring differentiation of stem cells towards platelets is described using syngeneic bone marrow injected into lethally irradiated mice. Fourteen days after such injection, the platelet counts are found to be proportional to the number of bone marrow cells injected and can be used as a measure of platelet progenitors. Perturbation of the milieu in which the transplanted marrow is placed by host preirradiation, bleeding, or erythropoietin administration leads to enhanced thrombopoiesis. It has been shown previously that similar perturbation favors erythropoiesis at the expense of granulopoiesis. The data from these and other experiments appear to be consistent, with a model of the stem cell compartment as a continuum with proliferative activity increasing as commitment is restricted. These functions vary inversely with the capacity for self-renewal. The various stem cell assays measure different ranges of stem cells, but overlap within this continuum.

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Effect of graft-versus-host disease on hematopoiesis after bone marrow transplantation in mice

Blood ◽

10.1182/blood.v78.10.2773.bloodjournal78102773 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2773-2779

Author(s):

PJ van Dijken ◽

J Wimperis ◽

JM Crawford ◽

JL Ferrara

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Cell Compartment ◽

Host Disease ◽

Murine Bone Marrow ◽

Graft Versus Host ◽

Stem Cell Compartment

We have examined the effect of graft-versus-host disease (GVHD) on the reconstitution of donor hematopoiesis in a murine bone marrow transplant (BMT) model of GVHD to minor histocompatibility antigens. GVHD had no effect on peripheral blood counts, which normalized by 1 month after BMT, and did not affect numbers of hematopoietic progenitors in the BM, which remained decreased in all transplant recipients. Donor stem cells (colony-forming unit-spleen day 8) and stem cell self-renewal remained low in all mice for 5 months after transplant, but GVHD further damaged the stem cell compartment. Peripheral counts 1 month after transplant were supported by increased numbers of stem cells in cycle and increased splenic hematopoiesis. However, GVHD altered the pattern of extramedullary hematopoiesis, causing dramatically decreased activity in the spleen and increased activity in the liver. We conclude that GVHD further decreases hematopoietic reserve and causes damage to the donor stem cell compartment during hematopoietic reconstitution after transplant, despite unaffected progenitor frequencies and peripheral blood counts.

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Blockade of the Hedgehog Signaling Pathway by the Novel Agent NVP-LDE225 Induces Differentiation, Prevents De-Differentiation and Inhibits Proliferation of Multiple Myeloma Stem Cells in Vitro

Blood ◽

10.1182/blood.v120.21.3950.3950 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3950-3950

Author(s):

Shihong Yang ◽

Ross D Brown ◽

Yen S Loh ◽

Hayley Suen ◽

James Favaloro ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Bone Marrow ◽

Stem Cell ◽

Cell Lines ◽

Side Population ◽

Cell Compartment ◽

Stem Cell Compartment ◽

Significant Difference

Abstract Abstract 3950 Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells. NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent. CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient's CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC50 was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p<0.0275) and de-differentiation was blocked. The studies confirm there is flux, to and from the SP and presumably MM stem cell compartment, that the canonical Hh pathway is upregulated in MM SP cells, and that the Hh pathway inhibitor NVP-LDE225 can induce SP differentiation, block de-differentiation, inhibit SP proliferation and at high concentrations is cytotoxic to all cells including MM stem cells. NVP-LDE25 could synergise with other maintenance therapy to reduce drug resistance by inducing the differentiation of chemo-resistant MM stem cells. Disclosures: No relevant conflicts of interest to declare.

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High Fractions of CD34+CD38- Cells with Aberrant hMICL Expression Predicts Shorter Overall- and Progression Free Survival in Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v124.21.3235.3235 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3235-3235

Author(s):

Marie Toft-Petersen ◽

Line Nederby ◽

Peter Hokland ◽

Anne Stidsholt Roug

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Myelodysplastic Syndrome ◽

Progression Free Survival ◽

Cell Compartment ◽

Aberrant Expression ◽

Free Survival ◽

Stem Cell Compartment

Abstract Introduction Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal diseases marked by dysplasia in the bone marrow (BM) causing cytopenias in the peripheral blood and with an inherent tendency to progress to acute myeloid leukemia (AML). Myelodysplastic syndrome has generally been regarded a stem cell disease, which very recent research has emphasized to hold true also for low-risk MDS (Woll, P. S. et al Cancer Cell. 2014). In AML, we have identified the human Myeloid Inhibitory C-type lectin-like receptor (hMICL) (also named CLL-1 or CLEC12A) as a stable and reliable marker at diagnosis and as a tool for assessing minimal residual disease (Roug, A. S. et al Br. J. Haematol. 2014). Furthermore, hMICL has been proposed as a marker of the leukemic stem cell (LSC) since it is present on CD34+CD38- cells in the majority of CD34-positive AMLs, while it is absent on the CD34+CD38- cell compartment in normal controls (van Rhenen, A. et al Leukemia. 2007). Previous studies have shown aberrant marker-expression on CD34+CD38- cells in MDS, e.g. CD7 or CD2, but only in minor fractions of patients (Xie, W. et al. Cytometry A. 2010). Using multicolor flowcytometry, we have examined the aberrant expression of hMICL on the CD34+CD38- stem cell compartment from 19 untreated MDS patients. We correlated the aberrant hMICL positive cells as a percentage of the total CD34+CD38- compartment (CD34+CD38-hMICL+/CD34+CD38-) to follow-up data. Materials and methods Bone marrow (BM) samples were obtained from 19 MDS patients diagnosed and followed at the Department of Hematology, Aarhus University Hospital from January 2010 to January 2011. Diagnosis was determined by morphology according to the 2008 WHO classification. Risk stratification of patients was done using the International Prognostic Scoring System (IPSS). All samples were collected before treatment with a cytoreductive agent or 5-azacytidine. Normal bone marrow (NBM) from 11 healthy volunteers served as controls. Following lysis of red blood cells, BM was stained with the following monoclonal antibodies: anti-CD34 APC, anti-CD38 FITC, anti-CD45 PerCP-Cy5.5 and anti-hMICL PE. Data acquisition was performed on a FACSCanto II and subsequently analyzed using FACSDiva Software. Using the back-gating strategy, the CD34+CD38-hMICL+ cell subset was shown to cluster together in an FSC-SCC plot. A total number of 5 clustered events were considered significant for further analyses. Results and perspectives The median age at sampling was 69 years (range 44-83 years). According to IPSS, 1 patient was classified as low-risk, 11 patients as intermediate-1 and 3 patients as intermediate-2. Four patients could not be classified. Consistent with previous reports, hMICL was not found on CD34+CD38- cells in any of the 11 NBM examined (0.0%) whereas aberrant hMICL expression was present on the CD34+CD38- stem cells in 16/19 (84%) of MDS cases. In MDS, the CD34+CD38-hMICL+ cells amounted a median of 5.68% (range 0,0-56.94%) of the CD34+CD38- subset, which was significantly different from NMB (p<0.0001) (Figure 1A). The median follow-up period for the cohort was 3.7 years. Patients with fractions of hMICL positive CD34+CD38- cells above the median (5.68%) showed a significantly shorter progression free survival (PFS) compared to patients with hMICL+ fractions below the median (p=0.01) (Figure 1B). The same trend was evident with regards to overall survival (OS), although borderline significant (p=0.06) (Figure 1C). The aberrant expression of hMICL on CD34+CD38- stem cells from MDS patients belonging to both low- and intermediate risk groups identifies hMICL as a potential LSC-marker in this heterogeneous group of diseases. A considerable advantage of hMICL is its complete absence on normal hematopoietic stem cells and the fact that it was present in the majority of MDS cases studied. Despite the small patient-number in our cohort, the impact of a high hMICL+ stem cell fraction on PFS and OS is evident. Thus, hMICL marking of the CD34+CD38- stem cell compartment might serve as a prognostic indicator in the future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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