Highly Sensitive Patient-Specific Real-Time PCR SNP Assay for Chimerism Monitoring after Allogeneic Stem Cell Transplantation

2006 ◽  
Vol 12 (1) ◽  
pp. 39-46 ◽  
Author(s):  
Rinat Eshel ◽  
Oded Vainas ◽  
Meirav Shpringer ◽  
Elizabeth Naparstek
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Real Time Pcr ◽  
Patient Specific ◽  
Highly Sensitive

Highly Sensitive Real-Time PCR of V617F-JAK2-Mutation To Monitor Minimal Residual Disease and Guide Donor Lymphocte Infusion after Allogeneic Stem Cell Transplantation in Patients with Myelofibrosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 669-669
Author(s):  
Nicolaus Kroeger ◽  
Anita Badbaran ◽  
Ernst Holler ◽  
Joachim Hahn ◽  
Guido Kobbe ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Jak2 Mutation ◽  
Highly Sensitive ◽  
Reverse Primer

Abstract The V617F-JAK2-mutation occurs in about 50 % of patients with myelofibrosis and is a reliable marker to monitor residual disease after allogeneic stem cell transplantation. The establishment of valid complete remission criteria for myelofibrosis especially after allogeneic stem cell transplantation remains a major issue. We developed a new, highly sensitive real-time PCR to monitor and quantify V617F-JAK2-positive cells after dose-reduced allogeneic stem cell transplantation. The mutated differs from the wild-type JAK2 allele by just one nucleotide exchange (G à T) leading to the valine to phenylalanine (V à F) transition. Using PrimerExpress® we designed a TaqMan® PCR where the reverse primer (RP) terminates at the (3′) nucleotide corresponding to this point mutation. Thus, this reverse primer should bind with higher affinity to the mutated than to the wild-type allele. To increase the specificity while conserving optimal sensitivity of the MRD-specific PCR we generated a set of primers shortened each time by one nucleotide at their 5’ end. In parallel, all those shortened primers were designed to contain an additional mutation at the third to last 3’ position. This allowed us to identify the reverse primer combining high specificity with so far not reported sensitivity(0.01 %). After 22 allogeneic stem cell transplantation procedures in 21 JAK2-positive patients with myelofibrosis, 78 % became PCR-negative. In 15 out of 17 patients (88 %), JAK2 remained negative after a median follow-up of 20 months. JAK2-negativity was achieved after a median of 89 days post allograft (range, 19 – 750 days). A significant inverse correlation was seen for JAK2 positivity and donor cell chimerism (r: −0.91, p<0.001). Four of five patients who never achieved JAK2-negativity fulfilled during the entire follow-up all criteria for complete remission recently proposed by the International Working Group, suggesting a major role for JAK2 measurement to determine depths of remission. In one case, residual JAK2 positive cells could be eliminated by donor lymphocyte infusion, supporting the graft versus myelofibrosis effect. In conclusion, allogeneic stem cell transplantation after dose-reduced conditioning induces high rates of molecular remission in JAK2-positive myelofibrosis-patients and quantification. V617F-JAK2 mutation by real-time PCR allows detecting minimal residual disease to guide adoptive immunotherapy.

Monitoring of the JAK2-V617F mutation by highly sensitive quantitative real-time PCR after allogeneic stem cell transplantation in patients with myelofibrosis

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1316-1321 ◽  
Author(s):  
Nicolaus Kröger ◽  
Anita Badbaran ◽  
Ernst Holler ◽  
Joachim Hahn ◽  
Guido Kobbe ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Jak2 V617f ◽  
Highly Sensitive

Abstract The JAK2-V617F mutation occurs in about 50% of patients with myelofibrosis and might be a reliable marker to monitor residual disease after allogeneic stem cell transplantation. We describe a new, highly sensitive (≥ 0.01%) real-time polymerase chain reaction (PCR) to monitor and quantify V617F-JAK2–positive cells after dose-reduced allogeneic stem cell transplantation. After 22 allogeneic stem cell transplantation procedures in 21 JAK2-positive patients with myelofibrosis, 78% became PCR negative. In 15 of 17 patients (88%), JAK2 remained negative after a median follow-up of 20 months. JAK2 negativity was achieved after a median of 89 days after allograft (range, 19-750 days). A significant inverse correlation was seen for JAK2 positivity and donor-cell chimerism (r: −0.91, P < .001). Four of 5 patients who never achieved JAK2 negativity fulfilled during the entire follow-up all criteria for complete remission recently proposed by the International Working Group, suggesting a major role for JAK2 measurement to determine depths of remission. In one case, residual JAK2-positive cells were successfully eliminated by donor lymphocyte infusion. In conclusion, allogeneic stem cell transplantation after dose-reduced conditioning induces high rates of molecular remission in JAK2-positive patients with myelofibrosis, and quantification of V617F-JAK2 mutation by real-time PCR allows the detection of minimal residual disease to guide adoptive immunotherapy.

Kinetics of Plasma-Cell Chimerism after Allogeneic Stem Cell Transplantation by Real-Time PCR and Its Value To Quantify Minimal Residual Disease in Patients with Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2748-2748
Author(s):  
Nicolaus Kroeger ◽  
Maria Zagrivnaja ◽  
Sabine Schwartz ◽  
Anita Badbaran ◽  
Tatjana Zabelina ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Cell Transplantation ◽  
Plasma Cell ◽  
Allogeneic Stem Cell Transplantation ◽  
Real Time Pcr ◽  
Plasma Cells ◽  
Patient Specific ◽  
Donor Plasma

Abstract To investigate lineage-specific chimerism of plasma cells after allogeneic transplantation by real-time PCR based on bi-allelic single-nucleotide polymorphism (SNP) or, in case of female-to-male transplantation, on the detection of the DFFRY-gene, 48 samples from bone marrow samples and peripheral blood from 34 non-myeloma patients were analyzed at different times after transplantation. Plasma cells and T-cells were obtained with CD138- or CD3-microbeads and MiniMACS columns. The median chimerism for T-cells at day +100 was &gt; 99.9 % and remained stable on day +180 and one year after transplantation. In contrast, the median donor-plasma-cell chimerism at day +100 was 95.5 %, at day +180 98,6 %, at day +360 99.8 %, and two or more years after transplantation &gt; 99.9 %. There was a trend for more patients with full donor-plasma-cell chimerism at day 180 who received standard conditioning in comparison to reduced-intensity conditioning (56 % vs. 28 %, p=0.3) and who experienced acute graft-versus-host disease (43 % vs. 18 %, p=0.2). To evaluate whether the quantitative measurement of donor-plasma-cell chimerism after allogeneic stem cell transplantation for patients with multiple myeloma might be a useful tool to determine minimal residual disease, we investigated 48 samples from 21 myeloma patients at different times after allogeneic stem cell transplantation, and we compared it with immunofixation and, in some cases, with molecular methods by using patient-specific primers. All patients with molecular remission measured by patient-specific primers had a plasma-cell chimerism of more than 99.5 %. Furthermore, in all but one patient plasma-cell chimerism of more than 99.3 % was associated with a negative immunofixation. In six patients with positive immunofixation after allogeneic stem cell transplantation treatment with bortezomib or donor-lymphocyte infusion was started and monitored by plasma-cell chimerism and immunofixation. All patients experienced an increase of donor-plasma-cell chimerism with negative immunofixation if chimerism exceeded &gt; 99.3 %. Dilution experiments with the TIB-196 cell line showed sensitivity of 104 using real-time PCR and SNPs, 105 in case of Y-PCR and 105 for nested PCR with patient-specific primers. We conclude that plasma-cell chimerism after allogeneic stem cell transplantation is delayed in comparison to T-cell chimerism, suggesting a reduced graft versus hematopoiesis effect on plasma cells. Quantitative measurement of plasma cells after allogeneic stem cell transplantation with highly sensitive real-time PCR allows monitoring of residual host-tumor cells in patients with multiple myeloma and allows guiding adoptive immunotherapy strategies to enhance remission status and to prevent relapse.

scholarly journals Diagnostic value of highly-sensitive chimerism analysis after allogeneic stem cell transplantation

2018 ◽  
Vol 53 (11) ◽  
pp. 1457-1465 ◽  
Author(s):  
Lea Sellmann ◽  
Kim Rabe ◽  
Ivonne Bünting ◽  
Elke Dammann ◽  
Gudrun Göhring ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Diagnostic Value ◽  
Highly Sensitive ◽  
Chimerism Analysis

scholarly journals Earlier relapse detection after allogeneic haematopoietic stem cell transplantation by chimerism assays: Digital PCR versus quantitative real-time PCR of insertion/deletion polymorphisms

PLoS ONE ◽  
2019 ◽  
Vol 14 (2) ◽  
pp. e0212708 ◽  
Author(s):  
Jennifer Valero-Garcia ◽  
María del Carmen González-Espinosa ◽  
Manuel Barrios ◽  
Greta Carmona-Antoñanzas ◽  
Javier García-Planells ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Cell Transplantation ◽  
Real Time Pcr ◽  
Haematopoietic Stem Cell Transplantation ◽  
Digital Pcr ◽  
Haematopoietic Stem Cell ◽  
Quantitative Real Time Pcr
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