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

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.

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The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation

Leukemia ◽

10.1038/leu.2012.66 ◽

2012 ◽

Vol 26 (8) ◽

pp. 1821-1828 ◽

Cited By ~ 57

Author(s):

T Lion ◽

◽

F Watzinger ◽

S Preuner ◽

H Kreyenberg ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Chimerism Analysis

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Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation

Leukemia ◽

10.1038/sj.leu.2402311 ◽

2001 ◽

Vol 15 (12) ◽

pp. 1976-1985 ◽

Cited By ~ 94

Author(s):

J Mattsson ◽

M Uzunel ◽

L Tammik ◽

J Aschan ◽

O Ringdén

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Myelodysplastic Syndrome ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Myeloid Leukemia ◽

Chimerism Analysis ◽

Acute Myeloid

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The EuroChimerism Concept for a Standardized Approach to Chimerism Analysis Following Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v108.11.621.621 ◽

2006 ◽

Vol 108 (11) ◽

pp. 621-621

Author(s):

Thomas Lion ◽

Marcel Tilanus ◽

Hélène Cavé ◽

Mark Lawler ◽

Anna Serra ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Mixed Chimerism ◽

Concerted Action ◽

Hematopoietic Stem ◽

Marker Panel ◽

Post Transplant ◽

Chimerism Analysis

Abstract Transplantation of hematopoietic stem cells from related and unrelated donors is becoming an increasingly important approach in the successful treatment of different malignant and non-malignant disorders. There is thus growing demand for clinical diagnostic methodologies permitting the surveillance of chimerism during the post-transplant period. The techniques currently employed are rather heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from ten European countries have therefore performed a collaborative study supported by a grant from the European Commission, the EuroChimerism Concerted Action. The aim of this Concerted Action was the development of a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. A set of microsatellite markers, selected on the basis of their excellent performance in chimerism analysis at the reference centers involved, have been carefully evaluated and optimized for quantitative chimerism testing under standardized experimental conditions. A EuroChimerism panel designed to optimally meet the specific requirements of quantitative chimerism analysis was established utilizing 13 markers (D2S1360, D7S1517, D8S1132, D9S1118, D10S2325, D11S554, D12S1064, D12S391, D17S1290, D19S253, MYCL1, P450CYP19 and SE-33), which best satisfied the criteria of the EuroChimerism consortium. Individual microsatellites from the panel can be analyzed in multiplex reactions to facilitate the identification of one or more markers optimally suited for the monitoring of chimerism during the post-transplant period. Extensive testing of related individuals (>500 pairs) revealed that the EuroChimerism marker panel will provide at least two informative markers which meet the stringent criteria of eligibility defined by the EuroChimerism consortium (Watzinger et al., Leukemia 2006) in >99% of all patient/donor constellations. In addition to the outstanding informativeness of the marker panel, chimerism testing by singleplex assays permitted sensitive detection of residual cells of patient or donor origin at levels ranging between 0.8–1.6% in about 90% of instances. The assay also facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells in peripheral blood or bone marrow specimens and in specific cell lineages isolated by flow-sorting. The precision in determining the relative contribution of donor/recipient cell populations to mixed chimerism was high within the range between 10–90% donor- or recipient-derived cells, while there was a tendency to overestimate the percentage of the subdominant cell population within the range between 1–10%. Wide use of the EuroChimerism assay will provide a basis for international standardization of chimerism testing, which will ultimately contribute to improved clinical management of patients undergoing allogeneic stem cell transplantation.

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