Augmentation of Sensitivity of FLT3/ITD Assay Allows Detection of Minimal Residual Disease In Stem Cell Transplant Recipients-Correlation with Flow Cytometric MRD Assessment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1717-1717
Author(s):  
Maya Danielle Hughes ◽  
Rong Zeng ◽  
Kristen L. Miller ◽  
Soheil Meshinchi
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Flow Cytometric ◽  
Detection Assay ◽  
Minimal Residual

Abstract Abstract 1717 FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutation that is associated with therapy resistance in acute myeloid leukemia (AML). Early data demonstrated low sensitivity for this assay, thus limiting its utility to the evaluation of diagnostic specimens, and precluding its utility in remission samples. We inquired whether the standard FLT3/ITD assay can be modified to enable its utility to detect presence of residual disease in remission specimens. Enhanced FLT3/ITD assay sensitivity was accomplished by altering annealing temperature, increasing the number of cycles as well as amount and concentration of the product that was subjected to capillary electropheresis. To assess the sensitivity of the enhanced assay, FLT3/ITD positive cells M4V11 were serially diluted in a population of ITD negative cells (HL60). The concentration of M4V11 cells in each sample ranged from 10% to 0.0001%. PCR product was subjected to capillary electropheresis and the appropriate region of the electropherogram was examined for the presence of the appropriate mutant product length. Appropriate FLT3/ITD signal was detected in dilutions down to 0.01%, validating our ability to detect extremely low levels of FLT3/ITD. We subsequently examined the remission marrows from patients with a history of FLT3/ITD who had undergone stem cell transplantation. Available bone marrow specimens (N = 51) from patients who underwent stem cell transplantation for FLT3/ITD-positive AML were analyzed and the result was correlated with the available standard PCR as well as the available MRD assessment by muti-dimensional flow cytometry; samples negative for FLT3/ITD by standard assay (N=11) were then subjected to the enhanced PCR methodology. Available ITD length for each patient was used for examination of the appropriate region of the electropherogram in each case. Of the available 51 bone marrow specimens analyzed, 23 specimens had FLT3/ITD detectable by standard PCR protocol. Using our modified PCR method and capillary electrophoresis, an additional 13 specimens had identifiable FLT3/ITD. In 6/11 patients, where initial FLT3/ITD was negative by standard methodology, enhanced assay identified FLT3/ITD signal. In each case, detection of FLT3/ITD by the enhanced assay was followed by morphologic or immunophenotypic emergence of disease, prompting therapeutic intervention. We further evaluated the ability to detect FLT3/ITD in patients with minimal residual disease by flow cytometry. 33 of the bone marrow specimens analyzed had a less than 5% abnormal blast population as detectable via flow cytometry. Among these samples, 7 had FLT3/ITD detectable using standard detection techniques. An additional 11 samples had detectable FLT3/ITD when our modified protocol was employed. Of the specimens that had less than 1% abnormal blast population as detectable via flow cytometry (N = 27), 4 had FLT3/ITD detectable using the standard detection assay; when our modified protocol was employed, an additional 6 samples had detectable FLT3/ITD. 17 bone marrow specimens had no abnormal blast cells detectable via flow cytometry; of these samples 1 had detectable FLT3/ITD using the standard detection assay, while an additional 3 had detectable FLT3/ITD using our modified assay. In four patients, FLT3/ITD was detected in bone marrow specimens found to have flow cytometric MRD of 0% (N=2), 0.1% (N=1) and 0.4% (N=1). In two patients with no detectable disease by MDF, both had emergence of morphologic (60% blast) or immunophenotypic disease by MDF (1.1%) within 4–6 weeks of detection of FLT3/ITD by enhanced assay. In this study, we demonstrate that simple modifications to the FLT3/ITD genotyping assay significantly increases its sensitivity and provides a highly sensitive and very specific assay for identifying this disease associated mutation in remission specimens. The enhanced assay can be incorporated into the standard evaluation of remission status for patients with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection of Minimal Residual Disease by Flow Cytometry for Patients with Multiple Myeloma Submitted to Autologous Hematopoietic Stem Cell Transplantation

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Suzane Dal Bó ◽  
Annelise Pezzi ◽  
Bruna Amorin ◽  
Vanessa Valim ◽  
Rosane Isabel Bittencourt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Hematopoietic Stem ◽  
Minimal Residual

The treatment strategy in multiple myeloma (MM) is to get complete remission followed by high-dose chemotherapy and autologous Hematopoietic Stem Cell Transplantation (HSCT). Neoplastic Plasma Cells (NPCs) are CD45-/dim, CD38+high, CD138+, CD19−, and  CD56+high in most cases. The description of this immunophenotype is of major importance as it leads to the correct identification of minimal residual disease (MRD). Samples from 44 Patients were analyzed prospectively in this study. We analyzed if the presence of MRD at three months after HSCT was predictive of relapse or death. There were 40 evaluable patients of whom 16/40 patients had MRD at three moths after HSCT and there were none in cytological relapse. The mean overall survival (OS) was 34 months and disease-free survival (RFS) was 28 months after HSCT. There was no significant difference in the log rank analysis comparing OS and the presence of MRD (P=0,611) and RFS (P=0,3106). Here, we demonstrate that three color flow cytometry (FCM) is more sensitive for MDR evaluation than cytological analyzes. However, based in our data we can not affirm that MRD is a good predictor of MM relapse or death. In conclusion, our results could be attributed to a short followup, small sample size, and over most to the inability of a three-color FCM to detect the NPC population.

scholarly journals Multiparameter flow cytometry for assessment of minimal residual disease in patients with myelodysplastic syndromes treated with allogeneic stem cell transplantation

2020 ◽  
Vol 51 (2) ◽  
pp. 88-94
Author(s):  
Katarzyna Wiśniewska-Piąty ◽  
Joanna Dziaczkowska-Suszek ◽  
Agata Wieczorkiewicz-Kabut ◽  
Karolina Chromik ◽  
Anna Koclęga ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Myelodysplastic Syndromes ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Minimal Residual

AbstractBackgroundMyelodysplastic syndromes (MDSs) are a heterogeneous group of clonal myeloid neoplasms. Allogeneic stem cell transplantation (allo-SCT) remains the curative method for MDS treatment. Little is known about the monitoring of minimal residual disease (MRD) in patients with MDS after allo-SCT.AimWe aimed to evaluate the significance of leukemia-associated immunophenotypes (LAIPs) identified in acute myeloid leukemia (AML) for MRD monitoring in patients with MDS after allo-SCT.Material and methodsSeven males and 4 females with a median age of 55 years were included. The diagnosis of MDS was established according to 2016 World Health Organization (WHO) criteria. The significance of eight LAIPs in bone marrow samples using multiparameter flow cytometry (MFC) was evaluated for MRD.ResultsEight patients were positive for several LAIPs before allo-SCT. The identified LAIPs included the presence of aberrant lymphoid antigens on myeloblasts and lack of CD33 expression on myeloblasts. All studied MDS patients were negative for LAIPs at Day +30 after the procedure. This was followed by full-donor chimerism in all cases. The Ogata score after allo-SCT decreased in all patients in whom it was indicative for MDS before allo-SCT.ConclusionsMFC could be useful in monitoring MRD in MDS patients after allo-SCT. Further studies in this field are needed.

Flow Cytometric Detection of Minimal Residual Disease One Year Post Allogeneic Stem Cell Transplantation Predicts Outcome in Patients with B-CLL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 202-202 ◽  
Author(s):  
Uta Oelschlaegel ◽  
Martin Bornhäuser ◽  
Alexander Kiani ◽  
Uwe Platzbecker ◽  
Christoph Röllig ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Residual Disease ◽  
First Year ◽  
Flow Cytometric ◽  
One Year ◽  
Minimal Residual

Abstract Abstract 202 Introduction: In a completed study (NCT00337519), patients with advanced B-CLL received allogeneic stem cell transplantation (SCT) after cytoreductive treatment with alemtuzumab followed by a wash-out period for the antibody and conditioning with fludarabine/busulfan. Aim of the present investigation was to correlate flow cytometric levels of minimal residual disease (MRD) in the peripheral blood at different time points after transplantation with patient outcome. Patients and Methods: In 58 CLL patients 900 flow cytometric MRD investigations (at least 4 measurements/patient: at day 30, between days 31–100, 101–180, and 181–365 after SCT) were performed measuring the following CLL phenotype: CD19posCD5posCD20dimCD79bneg. Therefore, a 4-color-approach (FACSCalibur, until 2006) or an 8-color-approach then in combination with T- and NK cell antigens (FACSCanto) was performed. A patient was defined as MRD negative if less than 0.05% CLL cells were detectable or as relapse if more than 0.05% CLL cells were again redetectable in at least two successive investigations. For assessment of progression-free survival (PFS), clinical progress was defined according to the NCI criteria. Results: The median follow up time after SCT was 536 days (range: 44d –1758d). Considering all 58 transplanted patients the probability of one-year overall survival (OS) including the 95% confidence interval was 83% ± 10% (2-year OS: 76%±12%) and of one-year PFS 74% ± 12% (2-year PFS: 50%±16%). In the majority of cases flow cytometric MRD negativity was achieved within the first year post SCT with a cumulative incidence of 36%±13% at day 100 and of 73%±12% at one year, respectively. Only two additional patients became MRD negative within the second year post SCT. Patients who achieved MRD negativity until day 365 showed a significantly better 2-year OS compared to the MRD positive group (96%±7% vs. 56%±49%; p=0.009). Remarkably, the 2-year PFS of patients achieving flow cytometric MRD negativity until day 365 was also significantly better than in the MRD positive cases (83%±16% vs. 0%, 3-year: 75%±20% vs. 0%; p=0.002). Of note, early flow cytometric MRD negativity until day 100 was not informative concerning OS or PFS at one year (88%±13% vs. 79%±15% and 77%±18% vs. 72%±18%). The flow cytometric MRD status was one trigger to speed the taper of cyclosporine or to give DLI. Interestingly, this kind of immunomodulation resulted in flow cytometric MRD negativity in eight out of nine patients after a median of 130 days. The probability of relapse in the investigated patient cohort was 15%±10% after 1 year and 31%±14% after 2 years. Thus, 8/14 patients showed a clinical relapse in parallel with flow cytometric MRD positivity. Two patients featured an isolated flow relapse with MRD at two successive investigations. Four patients showed a mere nodal relapse, all but one occurring within the first year post SCT. Conclusion: In summary, the present flow cytometric MRD study in B-CLL patients elucidates the dynamics of remission induction and relapse in the first year post SCT. In the majority of patients MRD is eradicated between day +100 and day +365 which is the time interval when chronic GvHD occurs in most cases. Therefore, close monitoring of MRD status in the first year after SCT is necessary. Once patients are flow cytometrically MRD negative at day 365, they seem to have a high probability of long term survival. Disclosures: Schetelig: Schering-Bayer: Research Funding.

scholarly journals Minimal residual disease and stem cell transplantation outcomes

Hematology ◽  
2019 ◽  
Vol 2019 (1) ◽  
pp. 617-625 ◽  
Author(s):  
Jacqueline Cloos ◽  
Gert J. Ossenkoppele ◽  
Richard Dillon
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Clinical Decision Making ◽  
Residual Disease ◽  
Response To Therapy ◽  
Multicolor Flow Cytometry ◽  
Minimal Residual

Abstract Risk classification and tailoring of treatment are essential for improving outcome for patients with acute myeloid leukemia or high-risk myelodysplastic syndrome. Both patient and leukemia-specific characteristics assessed using morphology, cytogenetics, molecular biology, and multicolor flow cytometry are relevant at diagnosis and during induction, consolidation, and maintenance phases of the treatment. In particular, minimal residual disease (MRD) during therapy has potential as a prognostic factor of outcome, determination of response to therapy, and direction of targeted therapy. MRD can be determined by cell surface markers using multicolor flow cytometry, whereas leukemia-specific translocations and mutations are measured using polymerase chain reaction–based techniques and recently using next-generation sequencing. All these methods of MRD detection have their (dis)advantages, and all need to be standardized, prospectively validated, and improved to be used for uniform clinical decision making and a potential surrogate end point for clinical trials testing novel treatment strategies. Important issues to be solved are time point of MRD measurement and threshold for MRD positivity. MRD is used for stem cell transplantation (SCT) selection in the large subgroup of patients with an intermediate risk profile. Patients who are MRD positive will benefit from allo-SCT. However, MRD-negative patients have a better chance of survival after SCT. Therefore, it is debated whether MRD-positive patients should be extensively treated to become MRD negative before SCT. Either way, accurate monitoring of potential residual or upcoming disease is mandatory. Tailoring therapy according to MRD monitoring may be the most successful way to provide appropriate specifically targeted, personalized treatment.

Clinical significance of minimal residual disease, as assessed by different techniques, after stem cell transplantation for chronic lymphocytic leukemia

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4563-4569 ◽  
Author(s):  
Carol Moreno ◽  
Neus Villamor ◽  
Dolors Colomer ◽  
Jordi Esteve ◽  
Eva Giné ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Autologous Transplantation ◽  
Lymphocytic Leukemia ◽  
Minimal Residual

AbstractWe analyzed minimal residual disease (MRD) by consensus polymerase chain reaction (PCR), quantitative PCR (qPCR), and flow cytometry in 40 patients with chronic lymphocytic leukemia (CLL) who underwent stem cell transplantation; 97.4%, 89%, and 100% of the patients could be studied by consensus PCR, qPCR, and flow cytometry, respectively. Overall, 164 of 248 samples were negative for MRD by consensus PCR. Among those, CLL cells were detected by qPCR and by flow cytometry in 77 (47%) and 39 (23%) of the 164 samples, respectively. All 84 samples positive on PCR had detectable CLL cells by qPCR and flow cytometry. A good correlation was seen between MRD levels by flow cytometry and by qPCR (n = 254; r = 0.826; P < .001). Fifteen of 25 patients receiving autografts suffered a relapse, with increasing levels of MRD being observed before relapse in all of them. MRD detection within the first 6 months after autologous transplantation identified patients with a high relapse risk. In contrast, in allografted patients (n = 15) MRD did not correlate with outcome. In conclusion, quantitative methods to assess MRD (flow cytometry and qPCR) are more accurate than consensus PCR to predict clinical evolution. These results might be useful to investigate treatments aimed at preventing relapse in patients with CLL who have received an autograft.

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