scholarly journals Quantitative Real-Time PCR Compared with pp65 Antigen Detection for Cytomegalovirus (CMV) in 1122 Blood Specimens from 77 Patients after Allogeneic Stem Cell Transplantation: Which Test Better Predicts CMV Disease Development?

2003 ◽  
Vol 49 (10) ◽  
pp. 1683-1685 ◽  
Author(s):  
Andreas Nitsche ◽  
Olivia Oswald ◽  
Nina Steuer ◽  
Johannes Schetelig ◽  
Aleksandar Radonić ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Allogeneic Stem Cell Transplantation ◽  
Real Time Pcr ◽  
Disease Development ◽  
Quantitative Real Time Pcr ◽  
Pp65 Antigen ◽  
Blood Specimens ◽  
Cmv Disease

Determining the Appropriate PCR Trigger for Pre-Emptive Anti-CMV Therapy in Allogeneic Stem Cell Transplant Recipients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5281-5281
Author(s):  
Unmesh Mohite ◽  
Diana Westmoreland ◽  
Una O’Callaghan ◽  
Alan K. Burnett ◽  
Keith M.O. Wilson
Keyword(s):  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Conditioning Regimen ◽  
Cell Transplant ◽  
Quantitative Real Time Pcr ◽  
Cmv Infection ◽  
Appropriate Treatment ◽  
Risk Patients ◽  
Cmv Disease

Abstract Background: CMV-related complications are a major cause of morbidity and mortality post allogeneic stem cell transplantation (ASCT). Where a pre-emptive strategy is used, early detection is crucial as this will minimise the risk of CMV infection progressing to CMV disease. As a result, PCR techniques are increasingly replacing pp65 antigen detection as a means of monitoring CMV viral load in at-risk patients. Initiating treatment at too low a cut-off may result in over-treatment since very low level viraemia may not necessarily represent live or replicating virus. However, using too high a cut-off might lead to unacceptable treatment delay. As a result of these uncertainties the optimal pre-emptive strategy remains unknown. We therefore sought to determine whether, using quantitative real time PCR, the manufacturer’s detection cut-off or the validated lower quantitation limit (LQL) was the more appropriate treatment trigger. Methods: CMV surveillance was done by weekly (twice weekly for positive results) quantitative real time PCR (Artus RealArt™ CMV LC PCR Kit, Hamburg, Germany) until immunosuppression was stopped. The manufacturer’s detection cut-off was 6.5 × 102 copies/ml and the validated LQL was 1 × 104 copies/ml. Treatment was initiated following two consecutive results above the LQL. Below the LQL, treatment was only started if symptoms of CMV infection (e.g. fever, cytopenia) were also present. Results: Between 01/04 and 12/05 fourty four patients, median age 43y (range 18–69), underwent ASCT from sibling (n=27) or unrelated (n=17) donors for AML/MDS (n=22), ALL (n=8), CML (n=5), NHL (n=5), myeloma (n=3) and osteogenesis imperfecta (n=1). The conditioning regimen was myeloablative (Cy-TBI, Bu-Cy) in 21/44 and reduced intensity (Flu-Mel, Flu-Cy) in 23/44. Campath-1H was used in 16/44. The recipient/donor CMV serostatus was R+/D+ (n=11), R+/D− (n=9), R−/D+ (n=4) and R−/D− (n=20). With a median follow up of 10 months (range <1–26), 12/24 (50%) patients at high risk of CMV reactivation (6 of 11 R+/D+, 6 of 9 R+/D−, 0 of 4 R−/D+) had CMV detected compared with 1/20 (5%) low risk R−/D− patients. CMV detection occurred at a median of 21d (range 3–128) post ASCT. 12/13 patients with detectable CMV had initial levels below the LQL. All required treatment, 5 because of co-existent symptoms of CMV infection despite consecutive readings below the LQL and 7 because the second reading was above the LQL. Conditioning therapy, donor type and use of CAMPATH-1H did not appear to influence risk of CMV detection. There was no CMV related death in this cohort. Conclusion: We conclude that, using the above kit, the detection cut-off of 6.5 × 102 copies/ml rather than the manufacturer’s validated LQL of 1 × 104/ml is the more appropriate trigger for initiating pre-emptive anti-CMV therapy in patients undergoing ASCT. Since manufacturers do not consider quantification below the LQL to be reliable, we recommend that where the detection cut-off is significantly lower than the LQL, transplant centres seek to determine their own appropriate treatment trigger.

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&lt;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.

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

Achievement of Sustained Molecular Remission Induces Long-Term Freedom From Disease After Autologous-Allogeneic Tandem Transplantation in Patients with Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 148-148
Author(s):  
Nicolaus Kröger ◽  
Tatjana Zabelina ◽  
Francis Ayuk ◽  
Christine Wolschke ◽  
Haefaa Alchalby ◽  
...  
Keyword(s):  
Stem Cell ◽  
Molecular Markers ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Plasma Cell ◽  
Allogeneic Stem Cell Transplantation ◽  
Real Time Pcr ◽  
Molecular Remission ◽  
Specific Primers

Abstract Abstract 148 Multiple Myeloma (MM) is considered to be an incurable disease, but in some patient long-term survival can be achieved after high dose chemotherapy followed by autologous or allogeneic stem cell transplantation. Those patients, who achieved molecular remission, have a high probability of long-term disease-freedom and of cure. More recently the introduction of dose-reduced conditioning and the use of auto-allo tandem approach has broaden the use of allogeneic stem cell transplantation in MM, but the results of prospective trials are controversial. We here investigated within a prospective protocol the incidence and impact of molecular remission on long-term outcome obtained after auto-allo tandem approach in MM. (study registration NCT 00781170). From 4/2000 to 10/2008, 73 patients with a median age of 50 years (r 29–64) and advanced stage II/III MM were included. The conditioning regimen for autologous transplantation consisted of melphalan 200 mg/m2 given divided over 2 days. After an interval of 2–3 months, the patients received a dose-reduced conditioning consisting of melphalan 140 mg/m2, fludarabine 180 mg/m2 and ATG Fresenius® (Fresenius, Bad Homburg, Germany) at a dose of 10 mg/kg for related and 20 mg/kg for unrelated donors on day -3, -2, and -1 followed by allogeneic stem cell transplantation. Remission was defined according to modified EBMT criteria. In 46 patients with CR or nCR minimal residual disease could be monitored by myeloma-specific primers (allele specific oligonucleotides, ASO) targeting IgH gene rearrangements (n=20) or by highly sensitive plasma cell chimerism using real-time PCR after magnet-activated cell sorting of CD138+ cells (n=26). The sensitivity of plasma cell chimerism was 10−4 using real-time PCR and donor/patient specific polymorphisms and 10−5 in case of Y-PCR. Sensitivity for nested PCR with myeloma-specific primers was 10−5. In 5 patients only 1 MRD measurement was available and those patients were excluded from MRD sub-analysis. Molecular complete remission was defined as by least two negative PCR results with ASO primers or achievement of at least two times ≥99.9 % donor chimerism of plasma cells by real-time PCR from bone marrow samples. Sustained MRD negativity was defined by at least 4 negative molecular results either by ASO primer or plasma-cell chimerism. Mixed MRD was defined by negative molecular markers for at least two times, but intermittent positivity was defined by myeloma-specific primers or plasma cell chimerism. Overall 44 patients (60 %) achieved a CR according to EBMT criteria with negative immunofixation. 8 % achieved a VGPR and 18 % a partial remission. 3 % had progressive disease and 11 % were not evaluable for determination of remission. Molecular remission was observed in 30 patients, in 15 patients the molecular markers were sustained negative while in 15 patients molecular markers were only intermittent negative, resulting in an overall complete molecular remission rate of 46 % and a stable sustained complete molecular remission rate of 23 %. After a median follow-up at 7 years the 5 year progressive-free survival was 29 % (95% CI: 17–41%). Patients without del(13q14) had a significantly better PFS than those with del(13q14) (5 y: 56% vs. 17%, p=0.02). Patients who achieved CR after transplantation had improved PFS in comparison to non-CR patients (5 y: 41% vs. 28%, p=0.008). Patients with sustained negative molecular remission had a 5 year PFS of 85 % vs. 31 % for mixed molecular remission (p= 0.003). The 5 year overall survival was 52 % (95% CI: 40–64%). Patients without del(13q14) had significantly improved 5 year survival (75% vs. 40%. p=0.02). Patients who achieved a sustained molecular remission had a 5 year OS of 91 % while those with mixed molecular remission resulted in a 5 year OS of 87 % (p=0.06). The study underlines the importance of the depth of remission and shows that achieving molecular remission as determined by myeloma-specific IgH gene rearrangements and plasma cell chimerism is associated with long-term freedom from disease and potential cure of MM in an auto-/allo SCT approach. Disclosures: No relevant conflicts of interest to declare.

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