scholarly journals Flow Cytometry Based Identification of CD26+ Leukemic Stem Cells in the Peripheral Blood Has a Potential Role in the Rapid Diagnosis of Chronic Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3611-3611
Author(s):  
Praveen Sharma ◽  
Man Updesh Singh Sachdeva ◽  
Shano Naseem ◽  
Sreejesh Sreedharanunni ◽  
Reena Das ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Stem Cells ◽  
Flow Cytometric ◽  
Custom Made

Abstract Background: Demonstration of t(9;22)(BCR-ABL1) fusion is gold standard for the diagnosis of chronic myeloid leukemia (CML). We performed a flowcytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for the diagnosis of CML and its utility for detection of residual disease in CML patients on therapy. Methods: Patients of CML/ CML on follow-up were included and peripheral (PB) and/or bone marrow (BM) samples were utilized for flowcytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. Under 'lyse-wash-stain-wash' sequence, the sample was incubated with a pre-titrated custom-made antibody cocktail in a 'test' tube containing CD45, CD34, CD38 and CD26 mo-abs. Acquisition was carried out on BD FACS Canto II and analysis was done with Diva Software. Clinical data including demographic details, complete blood count and BM findings were also noted. Results: A total of 104 samples (63 PB and 41 BM) from 64 patients [confirmed & treatment naïve CML (n=30), CML on follow-up (n=15), non-CML (n=19)] were tested. The median (range) time for reporting of PB/BM examination, molecular genetic studies and flow cytometry for CD26+ CML LSCs was 5 (3-11 days), 4 (3-6 days) and 1 (0-1 day) respectively. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (Median=0.07%, range 0.002%-26.79%), and also in 8/15 patients of the follow-up group, who also reported persisting levels of BCR-ABL1. None of the patients in the non-CML group and follow-up CML patients with negative RT-PCR results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r=0.917). Conclusion: Flow cytometric assessment for CD26+ LSCs is quick with reporting time of even less than an hour. Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is independent of BCR-ABL1 transcript type and its role in residual disease monitoring needs further investigation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Flow Cytometry Assessment of CD26 + Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia

2019 ◽  
Vol 96 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Donatella Raspadori ◽  
Paola Pacelli ◽  
Anna Sicuranza ◽  
Elisabetta Abruzzese ◽  
Alessandra Iurlo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Chronic Myeloid Leukemia ◽  
Diagnostic Tool ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Leukemic Stem Cells

scholarly journals 871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A912-A912
Author(s):  
Rebecca Moeller ◽  
Julian Scherer ◽  
Sadik Kassim
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Leukemic Stem Cells ◽  
Cd69 Expression ◽  
Acute Myeloid

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Blood ◽  
2014 ◽  
Vol 123 (25) ◽  
pp. 3951-3962 ◽  
Author(s):  
Harald Herrmann ◽  
Irina Sadovnik ◽  
Sabine Cerny-Reiterer ◽  
Thomas Rülicke ◽  
Gabriele Stefanzl ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Specific Marker ◽  
Leukemic Stem Cells ◽  
Dipeptidylpeptidase Iv ◽  
Key Points

Key Points DPPIV (CD26) is a new specific marker of CML LSC that aids CML diagnostics and the measurement, characterization, and purification of LSC. DPPIV on CML LSC degrades SDF-1 and thereby promotes the niche-escape of LSC, which may contribute to extramedullary myeloproliferation in CML.

scholarly journals Difficulties in collecting peripheral blood stem cells in chronic myeloid leukemia related to persistence of the leukemic cell clone

Stem Cells ◽  
1993 ◽  
Vol 11 (S3) ◽  
pp. 43-47
Author(s):  
Ph. R. Hénon ◽  
J. C. Eisenmann ◽  
M. Becker ◽  
G. Beck‐Wirth ◽  
E. Wunder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Cell Clone ◽  
Leukemic Cell ◽  
Peripheral Blood Stem Cells ◽  
Blood Stem Cells

Imatinib Mesylate Therapy in Late Ph+ Chronic Myeloid Leukemia Patients in Stable Complete Cytogenetic Response after Interferon-Alpha Results in a Very High Complete Molecular Response Rate.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2158-2158
Author(s):  
Giuliana Alimena ◽  
Massimo Breccia ◽  
Luigia Luciano ◽  
Fabrizio Quarantelli ◽  
Daniela Diverio ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Imatinib Mesylate ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Cytogenetic Response ◽  
Molecular Response ◽  
Complete Cytogenetic Response ◽  
Complete Molecular Response

Abstract Imatinib mesylate was given to 26 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients who were in late chronic phase (CP) and in stable complete cytogenetic response (CCR) after interferon-alfa (IFN-α), but showed persistent positive residual disease at PCR analysis under this treatment. At diagnosis median age was 40 years (range 21–64) and according to Sokal’s score, 18 patients were low risk and 8 were intermediate risk. Median IFN treatment was 88 mo.s (range 15–202) and median CCR duration was 73 mo.s (range 10–148). Imatinib was administered at the standard dose of 400 mg/die, after stopping IFN for 1 week. Residual disease was measured on bone marrow (BM) cells at baseline, before starting Imatinib, at 3, 6, 12, 18 mo.s and at the last follow-up (median 32 mo.s, range 21–49), by assaying BCR-ABL transcripts using quantitative PCR (RQ-PCR). The copy number (CN) of BCR/ABL and ABL transcript were derived by the interpolation of CT values to the appropriate standard curve, and the result, for each sample, was expressed as ratio of BCR/ABL mRNA copies to ABL mRNA x 100 (normalized copy number - NCN). Imatinib treatment resulted in a progressive and consistent decline of residual disease in all but one patient, from a median of 0.89 at baseline to 0.01 at the end of follow-up. Major molecular response (BCR/ABL levels <0.1) was reached in 20 patients (77%) and BCR/ABL transcripts were undetectable in 13 (50%). Achievement of molecular response was significantly correlated with post-IFN baseline transcript level (mean 1.194 for patients achieving complete molecular response vs 18,97 for those who did not; p<0.001), but not with other clinical/biological patient characteristics. In all patients, imatinib was well tolerated with no side effects requiring drug dose reduction or dose discontinuation. Albeit obtained from an unusual subset of selected patients with favourable prognosis, and likely particularly sensitive to imatinib, present results confirm the efficacy of combining Imatinib and IFN-α and further support investigating treatment approaches employing these two drugs.

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