Mitochondrial DNA Microsatellites as New Markers for the Quantitative Determination of Hematopoietic Chimerism after Allogeneic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5303-5303
Author(s):  
Myung-Geun Shin ◽  
Hyeoung-Joon Kim ◽  
Hye-Ran Kim ◽  
Il-Kwon Lee ◽  
Je-Jung Lee ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Quantitative Determination ◽  
Cell Transplantation ◽  
Control Region ◽  
Tandem Repeat ◽  
Mtdna Control Region ◽  
Donor Cells

Abstract Variable number tandem repeat (VNTR) and short tandem repeat (STR) markers of nuclear DNA are increasingly used for monitoring the engraftment of donor cells after stem cell transplantation (SCT). Mitochondria are the only organelles of animal cells other than the nucleus that contain DNA as well as their own machinery for RNA and protein synthesis. Mitochondrial DNA (mtDNA) is present in multiple copies (usually 103 to 104 copies per cell) (Blood2004;103:4466–77). We reported new markers using the analysis of mtDNA control region and microsatellites (mtMS) for monitoring donor cell engraftment in marrow transplant recipients. MtDNA control regions (nucleotide position (np) 16024 to 16569 and 1 to 576) and six mtMS (np 303–315 poly C, np 16184–16193 poly C, CA repeat starting at np 514, np 3566–3572 poly C, np 12385–12391 poly C and np 12418–12426 poly A) from total DNA samples from 25 sets of cases (donor, recipient and after SCT) were amplified using designated specific primers and PCR. We directly sequenced the control region which includes hypervariable regions, then carried out a qualitative and quantitative profiling mtDNA length heteroplasmies of six mtMS using size-based PCR product separation by capillary electrophoresis (ABI 3100 Genetic Analyzer and ABI Prism Genotyper version 3.1). The results were compared to those from six VNTR markers (D12S391, D1S80, F13A1, HUM RENA-4, HUM FABP2 and D18S51–5). MtDNA control region polymorphisms lead to the identification of an informative marker in 88% (22 cases) of all cases. Among six mtMS markers, the result of informativeness from np 303–315 and np 16184–16193 poly C mtMSs was 63% and 67% respectively. The combination of mtDNA control region direct sequencing, np 303–315 and np 16184–16193 poly C length heteroplasmies completely distinguished donor cells from recipients. In data from a typical mixing experiment for the determination of sensitivity, the detection limit (DL) of gene scan analysis in a mtDNA mixture was visible at 1% heteroplasmy in np 303–315 poly C mtMS marker; however DL from D12S391 in the same mixing experiment was 5–10% heteroplasmy. MtDNA control region sequencing and the length heteroplasmies of np 303–315 and np 16184–16193 poly C tracts can provide sensitive, accurate and quantitative determination of mixed chimerism after SCT.

Mitochondrial DNA Minisatellites Showed Higher Informativeness and Sensitivity Than the Nuclear DNA Markers for the Quantitative Determination of Chimerism After Allogeneic Stem Cell Transplantation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4004-4004
Author(s):  
Hye Ran Kim ◽  
Eun-Jeong Won ◽  
Hyun-Jung Choi ◽  
Hwan-Young Kim ◽  
James Moon ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Quantitative Determination ◽  
Allogeneic Stem Cell Transplantation ◽  
Tandem Repeat ◽  
Short Tandem Repeat ◽  
Str Markers ◽  
Short Tandem

Abstract Abstract 4004 Background: Mitochondrial DNA (mtDNA) is widely used in forensic identification and anthropologic studies on account of its abundance resulting in preferential amplification, sequencing and inherent variability. We developed mtDNA markers to monitor donor cell engraftment after allogeneic stem cell transplantation(SCT), then compared with nuclear short tandem repeat (STR) markers. Patients and methods: The mtDNA control regions and six mtDNA minisatellites (mtMS) (303 poly C, 16184 poly C, 514 (CA) repeat, 3566 poly C, 12385 poly C and 12418 poly A) from the total DNA samples of 215 cases (donor, recipient and after allogeneic SCT) were amplified using the designated specific primers and PCR. The results were compared with those from the six short tandem repeat (STR) markers (D12S391, D18S51, F13A1, HUM RENA-4, HUM FABP2 and Amelogenin). Results: Polymorphisms in the mtDNA control region identify an informative marker in 88% (189 cases) of all cases. Among the six mtMS markers, the informativeness of 303 poly C and 16184 poly C mtMS was 63% and 67% respectively. A combination of direct sequencing through the mtDNA control region, 303poly C and 16184 poly C mtMS could completely distinguish the donor cells from the recipient cells. The results from a typical mixing experiment to determine the sensitivity revealed a detection limit (DL) of the gene scan analysis in a mtDNA mixture to be visible at 1% heteroplasmy in 303 poly C mtMS marker. However, the DL from D12S391 in the same mixing experiment was 5–10% heteroplasmy. Conclusions: mtMS markers, especially 303 poly C and 16184 poly C markers, can provide a sensitive, accurate and quantitative determination of mixed chimerism after a SCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ex vivo determination of stem cell transplantation graft-versus-HIV reservoir effects

2015 ◽  
Vol 1 ◽  
pp. 19
Author(s):  
L.E. Hogan ◽  
K.S. Hobbs ◽  
D.R. Kuritzkes ◽  
J. Ritz ◽  
T.J. Henrich
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Ex Vivo ◽  
Hiv Reservoir

Second allogeneic stem cell transplantation for relapse after allografting in multiple myeloma using CD 34+ selected donor cells without immunosuppression

2020 ◽  
Vol 55 (9) ◽  
pp. 1817-1820
Author(s):  
Polona Novak ◽  
Evgeny Klyuchnikov ◽  
Ute-Marie von Pein ◽  
Martina Güllstorf ◽  
Maximilian Christopeit ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Cd 34 ◽  
Donor Cells

Quantitative Analysis of Chimerism Using Insertion/Deletion Polymorphisms Is An Useful Predictor of Relapse After Allogeneic Stem Cell Transplantation for Acute Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3541-3541
Author(s):  
Nathalie Jacque ◽  
Nathalie Dhédin ◽  
Jean Louis Golmard ◽  
Madalina Uzunov ◽  
Stéphanie Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Quantitative Analysis ◽  
Stem Cell Transplantation ◽  
Acute Leukemia ◽  
Cell Transplantation ◽  
Tandem Repeat ◽  
Peripheral Blood ◽  
Hazard Ratio ◽  
Mixed Chimerism ◽  
Post Transplant

Abstract Abstract 3541 Poster Board III-478 Introduction Quantitative analysis of chimerism after allogeneic hematopoïetic stem cell transplantation (allo-HSCT) for acute leukemia is routinely used to monitor the kinetic of engrafment. Usually, chimerism is assessed by variable number tandem repeat (VNTR) or short tandem repeat (STR) amplification by polymerase chain reaction (PCR), which have a sensitivity of 1 to 5 %. Many studies have shown that these methods were not sensitive enough to predict relapse. Insertion/Deletion (InDel) polymorphism analysis by real-time quantitative PCR (InDel-QPCR) is a more sensitive method and thus it should be able to predict relapse earlier. Patients and methods We conducted a retrospective unicentric study including all consecutive patients transplanted for acute leukemia from May 2004 to January 2009 in the Pitié Salpétrière Hospital (Paris France). Seventy four patients (53 acute myeloblastic leukemia and 21 acute lymphoblastic leukemia) were included. Median age was 41 years (18-67). Conditionning regimen was myeloablative in 51 patients (69%). The donor was a HLA-identical sibling for 31 patients. The source of stem cell was bone marrow in 35 patients (47%), peripheral blood in 35 (47%) and umbilical cord blood in 4 (6%). Sixty four patients (86%) were in complete remission (CR) at the time of transplantation. InDel-QPCR was performed every month in peripheral blood samples with a reproducible sensitivity of 0.1% at least. An increasing mixed chimerism was defined as a one log increase between two successive chimerism assays when recipient rate was inferior to 0.1% and as any increase beyond this limit (0.1%). Results In this 74 patients population, overall survival was 65% at one year, with a median follow up of the survivors of 651days (129-1809). Thirty six patients developed an acute GVHD (2-4) and 21 a chronic GVHD. Eighteen patients (24%) presented a cytological relapse, at a median time of 203 days (63-1001) after transplant. In two patients, the quantification of recipient DNA rate was constantly superior to 1% at each time point post transplant and these patients presented an early relapse (at 3 and 4 months after transplant). In the 72 remaining patients, DNA rate was inferior to 1%: in 63 patients inferior to 0.1% and in 41 patients inferior to 0.01%. Among the 62 patients who presented an increasing mixed chimerism (IMC) at one point, 18 relapsed. Among the 24 patients who presented an IMC at two successive points, 16 relapsed. In univariate analysis, factors associated with higher risk of relapse were the status of disease at transplant (refractory disease+CR2 versus CR1, p=0.002, hazard ratio=9.54) and 2 successive IMC (p=0.0001, hazard ratio=8.74 (CI: 3.33-22.9). In multivariate analysis both factors remained significantly correlated with the incidence of relapse (status of disease p=0.003, hazard ratio=4.24(CI: 1.6-11.2) and two IMC p<0.0001, hazard ratio=9.20(CI: 3.44-24.57)). The median interval between the first IMC and the diagnosis of relapse was 45 days (7-154). At the time of the first IMC, the peripheral blood cell count was normal in all except one patient who had persistent post-transplant thrombocytopenia. Conclusion Analysis of chimerism by using In/Del polymorphism is a sensitive technique with a reproducible detection threshold inferior to 0.1%. The detection of two successive IMC is highly predictable of relapse in patients transplanted for acute leukemia. Therefore, the detection of an increasing chimerism should incite to perform a rapid control, in order to make a precocious diagnosis of relapse and to provide an early therapeutic intervention. This analysis could help to monitor minimal residual disease in post transplant patients without a more specific molecular marker of malignancy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Diagnostic and Prognostic Value of Mitochondrial DNA Minisatellites after Stem Cell Transplantation

2013 ◽  
Vol 19 (6) ◽  
pp. 918-924 ◽  
Author(s):  
Eun Jeong Won ◽  
Hye Ran Kim ◽  
Hwan Young Kim ◽  
Hoon Kook ◽  
Hyeoung Joon Kim ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Prognostic Value ◽  
Diagnostic And Prognostic Value

scholarly journals Cell Competition between Wild-Type and JAK2V617F Mutant Cells Prevents Disease Relapse after Stem Cell Transplantation in a Murine Model of Myeloproliferative Neoplasm

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1468-1468
Author(s):  
Haotian Zhang ◽  
Melissa Castiglione ◽  
Lei Zheng ◽  
Huichun Zhan
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Disease Relapse ◽  
Wild Type ◽  
Cell Competition ◽  
Donor Cells ◽  
Significant Difference ◽  
Mutant Cells

Abstract Introduction Disease relapse after allogeneic stem cell transplantation is a major cause of treatment-related morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The cellular and molecular mechanisms for MPN relapse are not well understood. In this study, we investigated the role of cell competition between wild-type and JAK2V617F mutant cells in MPN disease relapse after stem cell transplantation. Methods JAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) were crossed with Tie2-cre mice to express JAK2V617F specifically in all hematopoietic cells and vascular endothelial cells (Tie2FF1), so as to model the human diseases in which both the hematopoietic stem cells and endothelial cells harbor the mutation. Results To investigate the underlying mechanisms for MPN disease relapse, we transplanted wild-type CD45.1 marrow directly into lethally irradiated Tie2FF1 mice or age-matched control mice(CD45.2). During a 6-7mo follow up, while all wild-type control recipients displayed full donor engraftment, ~60% Tie2FF1 recipient mice displayed recovery of the JAK2V617Fmutant hematopoiesis (mixed donor/recipient chimerism) 10 weeks after transplantation and developed a MPN phenotype with neutrophilia and thrombocytosis, results consistent with our previous report. Using CD45.1 as a marker for wild-type donor and CD45.2 for JAK2V617F mutant recipient cells, we found that the wild-type HSCs (Lin -cKit +Sca1 +CD150 +CD48 -) were severely suppressed and the JAK2V617F mutant HSCs were significantly expanded in the relapsed mice; in contrast, there was no significant difference between the wild-type and mutant HSC numbers in the remission mice. (Figure 1) Cell competition is an evolutionarily conserved mechanism in which "fitter" cells out-compete their "less-fit" neighbors. We hypothesize that competition between the wild-type donor cells and JAK2V617F mutant recipient cells dictates the outcome of disease relapse versus remission after stem cell transplantation. To support this hypothesis, we found that there was no significant difference in cell proliferation, apoptosis, or senescence between wild-type and JAK2V617F mutant HSPCs in recipient mice who achieved disease remission; in contrast, in recipient mice who relapsed after the transplantation, wild-type HSPC functions were significantly impaired (i.e., decreased proliferation, increased apoptosis, and increased senescence), which could alter the competition between co-existing wild-type and mutant cells and lead to the outgrowth of the JAK2V617F mutant HSPCs and disease relapse. (Figure 2) To understand how wild-type cells prevent the expansion of JAK2V617F mutant HSPCs, we established a murine model of wild-type and JAK2V617F mutant cell competition. In this model, when 100% JAK2V617F mutant marrow cells (from the Tie2FF1 mice) are transplanted alone into lethally irradiated wild-type recipients, the recipient mice develop a MPN phenotype ~4wks after transplantation; in contrast, when a 50-50 mix of mutant and wild-type marrow cells are transplanted together into the wild-type recipient mice, the JAK2V617F mutant donor cells engraft to a similar level as the wild-type donor cells and the recipient mice displayed normal blood counts during more than 4-months of follow up. In this model, compared to wild-type HSPCs, JAK2V617F mutant HSPCs generated significantly more T cells and less B cells in the spleen, and more myeloid-derived suppressor cells (MDSCs) in the marrow; in contrast, there was no difference in T, B, or MDSC numbers between recipients of wild-type HSPCs and recipients of mixed wild-type and JAK2V617F mutant HSPCs. We also found that program death ligand 1 (PD-L1) expression was significantly upregulated on JAK2V617F mutant HSPCs compared to wild-type cells, while PD-L1 expression on mutant HSPCs was significantly decreased when there was co-existing wild-type cell competition. These results indicate that competition between wild-type and JAK2V617F mutant cells can modulate the immune cell composition and PD-L1 expression induced by the JAK2V617F oncogene. (Figure 3) Conclusion Our study provides the important observations and mechanistic insights that cell competition between wild-type donor cells and JAK2V617F mutant recipient cells can prevent MPN disease relapse after stem cell transplantation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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