Detection Of Leukemic Stem Cells Research Articles

Leukemic Stem Cells (LSCs) As Peri-Transplant MRD Assessment in AML Patients Undergoing Allogeneic Stem Cell Transplantation in CR

Background: Several studies have shown that the leukemic stem cells (LSCs: CD34 +CD38 -CD45RA +/Combi-6 +) play a crucial role in the development of relapses in AML patients (pts). LSCs are less sensitive to conventional therapy; moreover, their phenotypical characteristics may allow immune evasion promoting relapses after allo-SCT. Although ELN guidelines recommend including LSCs into MRD assessment, this approach has not yet been studied extensively in the allogeneic setting. Here we suggested that peri-transplant LSCs detection may provide additional prognostic information to conventional (c)MRD in AML pts allografted in CR. Methods: 90 pts (male, n=46; median, 60.5 years, 19-76) with AML in CR and available pre-transplant cMRD data (multicolor flow cytometry, “different from normal” approach, according to ELN guidelines), who received allografts (matched, n=73; mismatched, n=17) during 2019-2022 years at the Department of Stem Cell Transplantation at University Medical Centre Hamburg were included. LSCs were assessed according to Zeijlemaker et al. 2019 before and after transplant at days +30 and +100. Both methods (cMRD and LSCs detection) were investigated solely and combined for their prognostic impact. Results: Majority of pts had de novo AML (76%), adverse ELN risk (48%), received matched grafts (related, n=73, 81%; unrelated, n=17, 19%) and MAC regimen (n=55, 61%). ATG was used for GvHD prophylaxis in 70 pts (78%). There were 47 cMRD neg (52%) and 43 cMRD pos (48%) pts. LSCs were detected in 39 (56%) pts with a median proportion of 0.01% (0.005-0.37%) of WBC. There was no correlation between LSCs proportion and cMRD status. The cMRD pos pts were more frequently LSCs pos (67% vs 33%), and cMRD neg were more frequently LSCs neg (33% vs 67%, p=0.002). Before allo-SCT, 34 pts (38%) were cMRD neg/LSCs neg, 26 pts (29%) experienced cMRD pos/LSCs pos, 17 pts (19%) had cMRD neg/LSCs pos and 13 pts (14%) had cMRD pos/LSCs neg. The conformity between both assays was 67%. During a median follow up of 10 months (1-29) there were 17 mortalities, 13 relapses and 11 NRM events. The 1-year OS and LFS were significantly better in LSCs neg than in LSCs pos pts: 91% (77-97%) vs 73% (55-86%, p=0.012) and 88% (72-96%) vs 52% (35-68%, p=0.001), respectively. This was due to a higher relapse rate in LSCs pos group: 33% (18-52%) vs 4% (1-20%, p=0.002). The median time to relapse was 180 days (40-395). The difference in NRM was not significant. The area under the ROC curve for relapses was 0.74 (0.61-0.88, p=0.006). Regarding the combined used of LSCs/cMRD, the best 1-year OS (p=0.038) and LFS (p=0.002) were observed for the double negative group: 94% (77-99%) and 88% (65-97%); followed by cMRD pos/LSCs neg: 86% (59-96%) and 86% (61-97%); cMRD neg/LSCs pos: 77% (43-94%) and 71% (39-90%); and the double positive group: 63% (43-79%) and 42% (25-61%)., respectively ( Figure 1). This was due to in the differing relapse rate that was highest in cMRD neg/LSCs pos group: 47% (17-79%), followed by double positive: 29% (14-50%), double negative: 7% (1-32%), and cMRD pos/LSCs neg group: 0% (p=0.02). Post-transplant cMRD/LSCs data on days +30 and +100 were available in 70 consecutive pts. The post-transplant conversion rates (positive > negative) for LSCs, cMRD and cMRD/LSCs were 49%, 61%, and 63%, respectively. The majority of pts converted at day +30. We observed a higher conversion rate to LSC neg status after MAC vs RIC (12/16, 75% vs 5/11, 45%, p=0.12). Of nine pts who were MRD pos at day +30, six were LSC neg and three were LSC pos. One MRD pos/LSC neg pts died due to NRM while remaining five pts survived without relapse. All three MRD pos/LSC pos pts developed relapses with two mortalities. Conclusion: Peri-transplant detection of LSCs has a strong predictive value for early post-transplant relapses in AML pts. This approach can improve pre-transplant risk assessment for cMRD pos patients by defining a population with better survival and low relapses (cMRD pos/LSCs neg) and one with worse survival and more relapses (cMRD pos/LSCs pos). The post-transplant clearance of residual disease is associated with better survival regardless of the flow strategy being used (cMRD, LSCs, or both). Pts with persisting cMRD early after allo-SCT may be subdivided into different risk groups according to their LSCs status. Figure 1. Leukemia-free survival according to pre-transplant cMRD/LSCs status in 90 AML patients.

Prospective Validation of CD34+CD38- Leukemic Stem Cell Frequency in the HOVON-SAKK 132 Trial: Perspectives for Future Improvements

Clinical implementation of measurable residual disease (MRD) requires accurate MRD detection to assess relapse risk for acute myeloid leukemia (AML) patients. One possibility to further improve the prognostic ability of the current MRD assay is to include the quantification of relapse initiating cells such as the CD34+CD38- leukemic stem cells (LSC) in the assay. In the HOVON-SAKK 102 trial, detection of LSC by multiparameter (MFC) flow MRD at diagnosis and after 2nd chemotherapy cycle (C2) has shown prognostic value (Zeijlemaker et al. Leukemia 2019). In addition, together with MRD detection, LSC could identify a category of patients with very high risk of relapse. Based on those data, improvements for LSC assessment measurements have been suggested, such as measuring more white blood cells (WBC) with the purpose of enhancing the sensitivity for the low frequency of CD34+CD38- cells. In the HOVON-SAKK 132 trial (Lowenberg et al. Blood Adv 2021), the one tube LSC assay was used that included LSC markers CD45RA, CD123, CD33, CD44, CLL-1, TIM-3, CD56, CD7, CD22 and CD11b (Zeijlemaker et al. Leukemia 2016). The one tube approach allows acquiring the suggested 4 million WBC for LSC-load estimation. In the current study, we prospectively validated the prognostic value of LSC burden at the following time points, i.e. at diagnosis (764/905 patients) and after two cycles of chemotherapy (C2; 357/706 patients). The median WBC acquired was 2x106 (range: 3x104- 6x106) at diagnosis and 3x106 (range: 4x105-5x106) after C2. Median percentages of LSC at diagnosis and after C2 were respectively 0.004% (range: 0.0000%-26.85%) and 0.00002% (range: 0.0000%-1.36%). Similar to the HOVON-SAKK102 study, three prognostic groups based on the presence or absence of CD34 and/or LSC, showed significant associations between LSC burden at diagnosis and overall survival (OS) or relapse- free survival (RFS) (Figure 1A): 1. CD34negative patients without any measurable LSCs and Leukemia Associated Immunophenotypes on the CD34+ blasts fraction (CD34negLSC=0 3 years (yrs) OS: 76.4% standard error (SE) 5; median OS not reached). 2. Patients with low percentages of CD34+CD38-LSCs < 0.03% (LSClow; 3 yrs OS 62.8% SE 2; median OS not reached) 3. Patients with high percentages of CD34+CD38-LSCs ≥0.03% (LSChigh, 3 yrs OS 44.5% SE 3; median 25.6 months; 95% confidence interval (CI): 18.7-49.1). Multivariate analysis adjusted for age, WBC at diagnosis, AML type, ELN2017 risk group and -only for RFS- the number of chemotherapy cycles to attain complete remission, showed that LSC burden at diagnosis is a strong independent prognostic factor for OS and RFS (Figure 1B). After C2, before the consolidation treatment, a cut-off value of 0.000075% of LSC showed the best prognostic value of LSC (univariate, LSCneg versus LSCpos: OS, HR 1.79 95% CI: 1.25-2.56, P < 0.01; RFS, HR 1.55, 95% CI: 1.12-2.13, P < 0.01). This cut-off appeared also highly significant as a prognostic factor in former trials (Terwijn et al. PLoS One 2014; Zeijlemaker et al. Leukemia 2018). Multivariate analysis showed that also LSC status after C2 was an independent prognostic factor for OS and RFS (Figure 1B). By combining the LSC status after C2 (≥0.000075%) together with the MRD status as used in the HOVON-SAKK 132 (based on ≥0.1% MFC-MRD and/or NPM1-MRD), MRDpos patients who are LSCpos have worse prognosis in OS and RFS (univariate: MRDnegLSCneg vs MRDposLSCpos; OS HR 3.2, 95% CI: 1.94-5.28, P < 0.01, RFS: HR 2.61 95% CI: 1.65-4.12, P < 0.01, multivariate: Figure 1B). Further analyses of the data are now directed towards potential influences of the cell amount of acquisition, ELN risk groups and MRD guidance in this trial. In conclusion, this prospective validation confirmed that assessing LSC burden, at diagnosis and after C2, remains to have additional prognostic value to MRD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microfluidic chip with reversible interface for noninvasive remission status monitoring and prognosis prediction of acute myeloid leukemia

Acute myeloid leukemia (AML) requires close monitoring of remission status for timely disease management. Liquid biopsy serves as a noninvasive approach for evaluating treatment response and guiding therapeutic modifications. Herein, we designed a non-invasive Leukemic stem cell Specific Capture Chip (LSC-Chip) with reversible recognition interface for AML remission status monitoring and prognosis prediction. A stem cell marker CD34 antibody coated herringbone chip with disulfide linkers was designed to capture and release leukemic stem cells (LSCs) in peripheral blood for efficient LSC enumeration and downstream single-cell analysis. Samples from 32 AML patients and 10 healthy donors were recruited for LSC enumeration and prognosis-associated subtyping with panels of official LSC markers (CD34+/CD123+/CD38-) and (Tie2+/CD34+/CD123+/CD38-), respectively. A cutoff value of 2.5 LSCs per milliliters of peripheral blood can be used to precisely distinguish non-remission AML patients from complete remission group. Moreover, single-cell RNA-seq of LSCs was performed to check different transcriptional profiles of LSC subtypes. Overall, the LSC-Chip with reversible recognition interface enabled reliable detection of LSCs from AML patient samples for noninvasive remission status monitoring and prognosis prediction in clinical AML management.

CD96 as a Leukemic Stem Cell Marker in Acute Myeloid Leukemia Patients: Relation to Remission Induction Chemotherapy

Abstract Background: Leukemic stem cells (LSCs) play a crucial role in chemotherapy resistance in acute myeloid leukemia (AML), the classical LSCs phenotype is CD34+/CD38-. Successful eradication of LSCs requires combination of different strategies including targeting LSC specific surface molecules. Aim of Study: To investigate the role of CD96, as a stem cell marker, in AML and its relation to induction therapy outcome. Material and Methods: The current study was conducted on 50 patients with de-novo AML, in addition to 20 controls. Using multicolor flow cytometry, we analyzed the expression of CD96, a potential marker for LSCs among CD34+/CD38- cell population in AML patients at initial diagnosis and correlate it to the response of induction therapy. Results: Percentage of CD96 among CD34+/CD38- was significantly higher in AML patients compared to control group (p < 0.001). However, no statistical significant difference was observed between complete remission (CR) and non-remission (NCR) groups. Also the correlation studies of LSC markers with laboratory findings revealed no significant correlation. Conclusions: CD96 expression among CD34+/CD38- cells may be used as a useful marker for detection of LSCs in AML patients. However its expression is not correlated to post induction therapy outcome.

OMIP 072: A 15-color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia.

This panel was designed to identify, quantify and phenotypically characterize putative leukemic stem cells (LSCs) in bone marrow (BM) samples from individual pediatric patients diagnosed with acute myeloid leukemia (AML). Based on an aberrant expression on immunophenotypically defined hematopoietic stem cells (HSCs), several antigens have been proposed as LSC markers in AML research, using healthy adult BM samples as reference material. Generally, these antigens have been evaluated individually in smaller panels (e.g. 8-color panels). This necessitates several tubes to characterize the LSC phenotype and compromises the ability to evaluate LSC heterogeneity. The present 15-color OMIP incorporates nine suggested LSC markers to comprehensively capture LSC immunophenotypes and to explore heterogenic marker-patterns within LSC populations in a single tube. Importantly, this single tube approach requires less input material, which is essential when sampling BM aspirates from pediatric patients where sample volumes often are sparse. As knowledge on normal expression levels of the included LSC markers in HSCs from hematologically healthy children are a prerequisite for labelling a phenotype as abnormal, we have evaluated the applicability of the panel on cryopreserved mononuclear cells (MNCs) isolated from BM samples from pediatric patients without hematological disorders as well as pediatric AML patients. The panel is optimized for cryopreserved BM MNCs, but could in principle, be utilized for LSC detection in any biological material containing human hematopoietic cells.

Multicolor Immunophenotyping of Candidate Leukemic Stem Cell Markers in CD34+CD38- Chronic Myeloid Leukemia Stem Cells

Introduction: Detection of leukemic stem cells (LSCs) may represent a new potential prognostic parameter in chronic myeloid leukemia (CML) and a tool for minimal residual disease monitoring in combination with standard qPCR method. To date CD26 is studied as a most specific marker for CML LSC detection. Several other candidate LSC markers have been reported, such as IL1-RAP, CD25 and CD93, however a side-by-side testing of their specificity is lacking. Recently, we have identified CD69 molecule to be overexpressed in CD34+CD38-CD26+ cells, which makes this antigen another candidate marker for LSC. Aim: To compare the surface expression of LSC markers CD69, CD26, CD25, CD56, IL1-RAP, CD56, CD93 in bulk CD34+CD38- population at diagnosis using a multicolor phenotypization assay Methods: In total, 44 patients were analyzed at diagnosis of chronic phase CML before administration of any treatment. Fresh (n=38) or cryopreserved (n=6) leukocytes obtained by erythrolysis were stained with CD45, CD34, CD38, CD25, CD26, CD56, CD69, CD93, IL1-RAP antibodies and 7-AAD for selection of live cells. Analysis was performed on FACSAria Fusion (BD Biosciences). Acquisition of live mononuclear cells ranged from 2×104 to 2.5×106. Results: Expression of candidate LSC markers CD26, CD25, CD56, IL1-RAP, CD56, CD93 was analyzed in BM of 35 patients who carried at least 30 CD34+CD38- cells. Median percentage of marker positive cells was 34% for IL1-RAP, 31% for CD25, 23% for CD26, 16% for CD56 and 2% for CD93, from the parent CD34+CD38- population. Next we analyzed the overlap and combination for the three best markers - IL1-RAP, CD25 and CD26. The 3-combination (defined as IL1-RAP or CD25 or CD26 expression) identified 40% of CD34+CD38- positive cells, which was more than any of the markers alone. Expression of the three markers showed good overlap and ruled out mutually exclusive expression of the markers. This was demonstrated by median difference of 0.4% of CD34+CD38- cells (range: 0-18%) and a correlation coefficient r2=0.9914, when comparing the 3-combination and the best performing marker in each patient. In contrast, in 12/35 (34%) of patients, one of the three markers failed to identify at least half of the cells positive for another marker. In 21/35 patients, we also analyzed the expression of CD69 in the CD34+CD38- compartment. The CD69 showed similar performance as the 3-combination of CD26, CD25, and IL1-RAP, 59% vs 54% positive cells, respectively. We observed excellent overlap between CD69 and 3-combination expression in individual patients with median difference of 0% of CD34+CD38- cells (range 0-15%) and correlation coefficient r2=0.9890. Furthermore, we compared marker positivity in BM vs PB in 19 paired samples. Both, sample types showed similar frequency of CD34+CD38- cells (5×10-3 in BM, and 2×10-3 for PB), but PB carried higher percentage of LSCs identified by the 3-combination - median 76 vs 52% cells. Conclusions: We show an overlap in surface expression of three previously reported CML LSC markers - IL1-RAP, CD25 and CD26. Nevertheless, a combination of these markers can detect more positive cells than any of the markers alone. Moreover, we demonstrate that CD69 identifies the same cells within the CD34+CD38- compartment as the combination of three above mentioned markers, which makes CD69 the best candidate for routine CML LSC quantification. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia.

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post‐treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38− leukemic stem cells

Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs. CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812. In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.

A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Identification of a Neoplastic Stem Cell in Human Mast Cell Leukemia

Leukemic stem cells (LSCs) have recently been identified as an important target of therapy in various human leukemias and related blood cell disorders. Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of mast cells (MCs) in various organ systems, including the bone marrow (BM). Whereas patients with indolent SM (ISM) have a normal life-expectancy, patients with more advanced forms of SM have a poor prognosis. In these patients, neoplastic MCs are usually resistant against conventional drugs and various targeted drugs. MC leukemia (MCL) is the rare leukemic variant of advanced SM, defined by a rapidly devastating expansion of immature MCs in various hematopoietic organs and a poor prognosis with short survival times. Although MCL is considered a stem cell disease, little is known about the origin and phenotype of MCL-initiating LSCs. We examined the phenotypic and functional characteristics of putative LSCs in patients with aggressive SM (ASM, n=12) and MCL (n=6). Putative LSCs were identified and characterized phenotypically by flow cytometry. Highly enriched, sorted LSCs were injected into NOD-SCID-IL-2Rγ-/- mice exhibiting a 220 amino acid isoform of human membrane-bound hSCF (NSGSCF). We found that disease-initiating and propagating LSCs reside within a CD34+ fraction of the MCL clone. Whereas cell fractions containing CD34+ cells as well as highly enriched CD34+ cells produced engraftment in NSGSCF mice with a MCL-like disease (43-77% human MCL cells in mouse BM after 10-22 weeks), no substantial engraftment was produced by MC-rich but stem cell-depleted, KIT+/CD34─ cell fractions obtained from the same patients (<1% engraftment in mouse BM). In dilution experiments, engraftment of CD34+ cells was documented down to a minimum of 50 cells per mouse. The identity of engrafting MCL cells was confirmed by morphology, phenotyping and molecular studies demonstrating the presence of KIT mutations that were initially detected in the primary MCL samples used. Moreover, we were able to confirm long-term engraftment by successful serial transplantations into secondary recipient mice. In consecutive experiments, we were able to show that CD45+/CD34+/CD38─ cells also produce leukemic engraftment in NSGSCF mice. As assessed by flow cytometry, these CD34+/CD38─ MCL LSCs were found to express several stem cells markers, including aminopeptidase-N (CD13), leukosialin (CD43), Pgp-1 (CD44), the IL-3R alpha-chain (CD123), AC133 (CD133) and CXCR4 (CD184). In addition, in most patients examined, MCL LSCs were found to display IL-1RAP, a surface antigen that is otherwise expressed in CML LSCs but is not expressed in normal stem cells. In addition, MCL LSCs were found to express various cell surface targets, including CD33 and CD52. By contrast, MCL LSCs did not express CD2, CD25, CD26 and CLL-1. The more mature progenitor cell fractions (CD34+/CD38+) were found to stain positive for CD13, CD33, CD43, CD44, CD90, CD117, CD123, CD133 and CD184. Mature clonal MCs expressed a similar phenotype, including molecular markers and targets, such as CD13, CD30 CD33, CD52 and CD184. In patients with ISM and aggressive SM (ASM), the CD34+/CD38─ stem cells exhibited a similar surface marker profile compared to MCL, but expressed lower levels of CD133 and did not express IL-1RAP. In the validation phase of our study, we examined the effects of target-specific antibodies. As assessed by flow cytometry, the CD52-targeting antibody alemtuzumab was found to induce complement-dependent lysis of CD34+ and CD34+/CD38─ cells in all MCL samples analysed. Furthermore, pre-incubation of MCL cells with alemtuzumab prior to injection into NSGSCF mice resulted in a significantly reduced engraftment (2.7±4.1%) after 22 weeks. In conclusion, our data show that the MCL clone originates from a primitive hematopoietic stem cell that co-expresses CD34, CD123, CD133 and IL-1RAP but lacks CD25 and CD26. In addition, our data show that MCL LSC express a number of clinically relevant surface targets, including CD33, CD52 and CD117 (KIT). These observations may facilitate LSC detection and isolation in MCL and may lead to the development of novel LSC-eradicating treatment concepts in this highly aggressive and drug-resistant form of leukemia. DisclosuresValent:Novartis: Consultancy, Honoraria, Research Funding.

Detection of Leukemic Stem Cells in Patients with De Novo Acute Myeloblastic Leukemia

ABSTRACT Aim: Defining the self-renewing [Thy1-, CD34 + , CD38-] LICs in AML cases before and after induction chemotherapy as a predictor for relapse and to determine how CSF1R (Fms) and CD34 markers affect the growth and survival of human leukemic cells in the CD38- Thy1- population. Methods: This study was carried out on 30 samples from the peripheral blood of adult patients with de-novo acute myeloid leukemia. The majority of the patients were monocytic AML Samples were sorted into four populations (Fms + CD34-, Fms + CD34 + , Fms-CD34+ and Fms-CD34-) according to the surface markers of the cells. Cells were cultured on mouse stromal cells transfected with a plasmid containing human CSF-1. Samples were cultured using Iscove's modified Dulbecc's medium (IMDM).The cultures were assessed for survival of leukemic cells in days. Results: The mean survival in days of the cells was 13.9 before chemotherapy and 14.1 after chemotherapy. The difference in growth was insignificant (p > 0.05). The Fms-CD34+ population in all but two samples tested had the longest survival time in culture. Conclusions: Our results suggest that leukemic stem cells may survive chemotherapy mainly due to their quiescence. Human CSF-1 was shown to increase the number of leukemic cells in co-culture with mouse stroma after 5 weeks. A novel leukemic stem cell (Fms-CD34+) has been identified and is the cell responsible for the growth and maintenance of the leukemic bulk. Disclosure: All authors have declared no conflicts of interest.

Morphology Antibody Chromosome Technique for Determining Phenotype and Genetic Status of the Same Cell

The morphology antibody chromosome (MAC) technique is a combination of methods that permits analysis of both phenotypic and genetic evaluation on a single interphase or mitotic cell as a basis for lineage analysis of neoplastic and normal cells. This unit describes MAC with sequential phenotypic analysis using antibody and an alkaline phosphatase anti-alkaline phosphatase (APAAP) complex and genotypic analysis using in situ hybridization with either enzymatic or fluorescence detection. Alternate methods for phenotypic analysis are also described, which include the use of horseradish peroxidase-conjugated antibodies, fluorochrome-conjugated antibodies, May-Grunwald-Giemsa cytochemical staining, and Sudan black B cytochemical staining. An additional protocol describes G- and C-banding as alternatives to in situ hybridization (ISH) for genotyping MAC specimens. Support protocols describe methods for preparing specimens, cytospin preparations, in situ cultures, paraffin-embedded or cryostat sections, and blood and bone marrow smears. Also described is a procedure for chromosome painting of previously GTG-banded slides. An additional protocol is included for FISH analysis on sorted hematopoietic stem cells and its application in the detection of leukemic stem cells. For overcoming the drawbacks of scarcity and variability from case to case of malignant plasma cells in multiple myeloma FISH analyses, a protocol is included for the enrichment of plasma cells by immunomagnetic beads.

Identification of leukemia stem cells in acute myeloid leukemia and their clinical relevance

Acute myeloid leukemia (AML) is considered to be a disease of stem cells. A rare defective stem cell population is purported to drive tumor growth. Similarly to their normal counterparts, leukemic stem cells (LSC) divide extreme slowly. This may explain the ineffectiveness of conventional chemotherapy in combatting this disease. Novel treatment strategies aimed at disrupting the binding of LSC to stem cell niches within the bone marrow might render the LSC vulnerable to chemotherapy and thus improving treatment outcome. This review focuses on the detection of LSC, our current knowledge about their cellular and molecular biology, and LSC interaction with the niche. Finally, we discuss the clinical relevance of LSC and prospective targeted treatment strategies for patients with AML.

Flow cytometric detection of leukemic stem cells (LSCs) in Egyptian pediatric B-acute lymphoblastic leukemia

Identifying leukemia stem cells (LSCs) is a challenge and a critical step in understanding their respective biology and may provide insights into a more efficient treatment of acute lymphoblastic leukemia (ALL). This cohort study aimed at detecting LSCs expressing CD19, CD34, and CD38Low in the bone marrow of 20 de novo Egyptian pediatric B-ALL patients at the time of the diagnosis and after the induction of chemotherapy. LSCs were detected in 80% of cases at the time of diagnosis and in 70% of the cases after the induction of chemotherapy. Patients were followed up for 12–18 months. No statistically significance difference was encountered between LSC-positive and LSC-negative patients as regards their clinical and laboratory data at the time of diagnosis. Furthermore, there was no statistically significant difference between the percentages of LSC-positive patients before or after induction of therapy. On follow-up, two patients died; they had residual LSCs after the induction therapy. However, the existence of LSCs did not affect the patient's 1-year disease-free survival rate. LSC may resist the traditional lines of treatment applied for childhood B-ALL; it may be used in minimal residual disease monitoring as well as being a target for new therapeutic lines to improve the outcome of treatment especially in patients with high-risk and relapsed ALL.

Accurate Detection of Residual Leukemic Stem Cells In Remission Bone Marrow Predicts Relapse In Acute Myeloid Leukemia Patients

AbstractAbstract 759Detecting leukemia initiating cells or leukemic stem cells (LSCs) in acute myeloid leukemia (AML) is a challenge. AML is a heterogeneous disease and since the first studies on LSCs, different immunophenotypic cell compartments have been reported to contain LSCs. For CD34 positive (CD34+) patients, the most important LSC compartment is most likely CD34+CD38-. However, both LSCs and normal hematopoietic stem cells (HSCs) reside in this compartment. Therefore, LSC detection requires the ability to accurately discriminate between LSCs and HSCs. Previous research showed that LSCs, in contrast to HSCs, expressed aberrant markers (AM, including lymphoid lineage markers and the LSC marker CLL-1). Although AM- cells are often presumed to be normal HSCs, AM expression can be absent or very weak on LSCs and AM- cells can therefore be malignant too. We previously described that in 30% of the patients, besides the AM expression, additional FACS parameter (forward scatter (FSC) and sideward scatter (SSC) and/or CD34 expression levels) could also be used to detect LSCs (Terwijn et al., ASH 2009 abstract 399). To prove that the CD34+CD38- fraction contains both normal and malignant SCs, and in addition, to prove that AM- cells can be malignant too, we studied molecular aberrancies (3 cases of FLT3-ITD positive AML) in the relevant cell fractions. 1: CD34+CD38-AM+ cells proved to be FLT3-ITD positive in all three cases. In addition, these cells initiated leukemic engraftment in NOD-SCID IL-2Rγ -/- mice in 2/2 cases. 2: The CD34+CD38-AM- cells with a FSC/SSC low or CD34 high character were FLT3-ITD negative in 3/3 cases and initiated multilineage engraftment in mice in 2/2 cases, indicating that these are HSCs. 3: The CD34+CD38-AM- cells with a FSC/SSC high or CD34 low phenotype were FLT3-ITD positive in 2/2 cases and therefore malignant, indicating that AM expression alone is not always sufficient in discriminating HSCs from LSCs. Since therapy surviving LSCs are thought to be responsible for relapses, we hypothesized that LSC frequency, as a fraction of total WBC, detected in remission bone marrow (BM) would provide prognostic information. Using AM as a primary gating strategy and, when applicable, FSC/SSC and/or CD34 expression, as a secondary gating step, LSC analysis was performed in 112 CD34+ AML patients (<60 years). In 76/112 patients, LSCs were detected using the most specific AM (CLL-1 (n=45), CD7 (n=19), CD11b (n=4), CD56 (n=3), CD22 (n=2), CD19 (n=2) and CD36 (n=1)) as a primary gating strategy. In 20 of these patients, a secondary gating step in FSC/SCC (n=16) and CD34 expression (n=4) refined the malignant population. In 36/112 patients, no AM was expressed on the CD34+CD38- cells, but in 25 cases, FSC/SSC could be used to gate the malignant fraction, in 6 cases combined with CD34 expression. Thus, in total, 101/112 patients were monitored for LSC frequency. After first cycle of chemotherapy, using a cut-off 5 × 10−6, patients with LSC frequency above cut-off (LSC+) showed shorter overall survival (OS, p=0.06) and especially relapse-free survival (RFS, p=0.001) as compared to LSC- patients. Median RFS was 11 months vs >36 months, respectively (fig A). Also after second induction cycle, high LSC frequency (>5 × 10−6) predicted shorter OS and RFS (both p<0.001). Median RFS of LSC+ patients was 8 months, that of LSC- patients >36 months (fig B). After consolidation therapy, 2 × 10−6 was the most optimal cut-off to define LSC+ and LSC- patients. OS and RFS were significantly shorter in LSC+ patients (p=0.037 and p=0.001, respectively), with a median RFS of 13 months vs. > 36 months (figure C). The relative risk of relapse was 5.0 (95% C.I 1.8–14.0) after first induction cycle, 4.7 (95% C.I.2.2-10.1) after second cycle and 8.5 (95% C.I 1.8– 41.4) after consolidation therapy. Multivariate analysis including WBC count, cytogenetic risk profile and the number of cycles needed to reach CR showed that LSC frequency is an independent factor for RFS (1st cycle p=0.01, 2nd cycle p<0.001, consolidation p=0.038). With this gating strategy, 90% of CD34+ AML patients could be monitored for residual LSCs, resulting in accurate prognostic information which is valuable for risk stratification-based treatment. In addition, the prospective isolation of LSCs and HSCs from diagnosis AML BM might identify more selective therapies that eradicate LSCs, while leaving HSCs intact.This work was supported by Netherlands Cancer Foundation KWF. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

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

Abstract 399In 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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Specific Detection of Aberrant and Normal Stem Cells in Acute Myeloid Leukemia Patients Opens the Way for Defining Highly Specific Targets for Stem Cell Therapy.

In acute myeloid leukemia (AML), a small fraction of blast cells contains the tumor initiating cells, further referred to as leukemic stem cells (LSCs). LSC resemble hematopoietic stem cells (HSCs) with respect to self renewal capacity and quiescence. Therefore, LSCs are proposed to be therapy resistant. In order to optimally target LSCs and sparing HSC and to monitor therapy, discrimination between LSC is HSC is required. We showed that within the CD34+CD38− stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers, including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen et al., Leukemia 2007 and Blood 2007). Too low aberrant marker expression, however, hampers discrimination in part of the cases. Therefore, we investigated additional parameters that would allow to distinguish LSCs from HSCs in CD34 positive AML patients. In 14 out of 48 cases studied, flow cytometry revealed a double population within the CD34+CD38− compartment, characterized by a small but clear difference in forward scatter (FSC, reflecting cell size) and sideward scatter (SSC, reflecting granularity). In 7/14 cases with high marker expression, FSChighSSChigh population coincided completely with the population with aberrant marker expression. In the other cases, marker expression was too moderate to show a complete overlap. The FSClowSSClow population within the CD34+CD38− stem cell compartment is the minor population at diagnosis (median 16%, range 0.2%–92%; n=14), had no expression of aberrant markers and, moreover, closely resembled the FSC/SSC characteristics of normal BM HSCs. In addition, in these patients, the normality of the FSClowSSClow population was also supported by the fact that the CD34 and CD45 antigen density was similar to that of normal BM HSCs. Altogether, this enabled to use FSC/SSC characteristics together with aberrant CD34 and CD45 expression to discriminate between LSC and HSC in cases with low or absent aberrant marker expression (8/48). In addition, the malignant character of the FSChighSSChigh population and the normal character of the FSClowSSClow population could be proven in three AML patients with cytogenetic aberrancies. Patient 1 had a t(8;21) translocation and presented with a CD34+CD38−- population that was CD19 positive (81% of the stem cell compartment) and had FSChighSSChigh properties. FACSsorted cells contained the translocation in 90% of the cells. The CD19 negative population (19% of the stem cell compartment) had FSClowSSClow characteristics and contained 0% t(8;21) cells. In two other AML cases with a cytogenetic aberrancy (t(8;21) and t(15;17), respectively), FSC/SSC characteristics, CD34/CD45 antigen density and aberrant marker expression (CD56 in one case and CLL-1 in the other) were partly overlapping (estimated LSC contribution to the CD34+CD38− compartment was 85% in both cases). Cell sorting on the highest FSC/SSC and marker expression nevertheless resulted in enrichment of cytogenetically aberrant cells (63% and 73%, respectively), while the corresponding FSClowSSClow cells, which missed CD56 and CLL-1 expression, were enriched for cytogenetically normal HSCs (87% and 67%, respectively). The marker and scatter parameters discussed above have generated the possibility to discriminate between LSCs and HSCs and now allows specific detection of LSC in >75 % of the patients. Discrimination between LSCs and HSCs in AML might not only facilitate to establish the therapeutic window of current therapies in terms of LSC specificity, but also allow the identification of new highly AML stem cells specific therapeutic targets. This should ultimately result in more selective therapies, which would be highly effective for AML stem cells, while leaving the normal HSC intact. This work was supported by Netherlands Cancer Foundation KWF.