scholarly journals Chronic Myeloid Leukemia: Molecular Monitoring of Residual Disease by Genomic DNA Compared to Conventional mRNA Analysis in Follow-Ups up to 8 Years

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1687-1687
Author(s):  
Ilaria Stefania Pagani ◽  
Orietta Spinelli ◽  
Adelaide Bussini ◽  
Tamara Intermesoli ◽  
Francesco Pasquali ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Minimal Residual Disease ◽  
Genomic Dna ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Patient Specific ◽  
Imatinib Treatment ◽  
Molecular Monitoring ◽  
Qrt Pcr ◽  
Minimal Residual

Abstract Abstract 1687 Background: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder resulting from the t(9;22)(q34;q11) balanced reciprocal translocation within a pluripotent stem cell (SC). The resulting Philadelphia (Ph) chromosome produces BCR-ABL1 fusion gene coding for a deregulated Abl tyrosine-kinase with constitutive and tumorigenic activity. The first line therapy of CML is imatinib mesylate, which targets Bcr-Abl protein, inhibiting proliferation pathways. Complete cytogenetic response can be achieved in 95% of patients treated in the early chronic phase (CP)1. Molecular monitoring of minimal residual disease is crucial to detect poor responses to imatinib and optimizing treatment with second generation tyrosine-kinase inhibitors or allogeneic stem cell transplantation. Residual leukemia is assessed by a quantitative manner evaluating levels of BCR-ABL1 transcripts by real-time reverse transcriptase PCR (qRT-PCR). Although qRT-PCR detects mRNA levels in a very sensitive manner, the negative result is difficult to interpret, because undetectable levels of chimeric transcript can reflect either an effective elimination of leukemia cells, or the presence of a quiescent leukemia SC transcriptionally silent. Methods: We developed a novel highly sensitive method to identify quiescent leukemic cells through quantitative real-time PCR (Q-PCR) targeting the genomic breakpoint sequence1. In CML each patient shows a unique genomic fusion sequence1, that need to be characterized in order to design a specific genomic assay. We selected 14 patients with CML diagnosed in the early CP. We identified junctions sequences by long-range PCR2. We carried out Q-PCR assay using common primer forward and probe in BCR, and 2 different primers reverse, in ABL or BCR, used as control1. The percentage of leukemic cells (LCs) was calculated using the derivation of the δCt formula1: LC= [100*(2/2δCt+1)]/n], where δCt is the difference between amplification cycles of BCR-ABL1 and BCR reactions, and n is the number of experimental replicates. We tested the sensitivity and the efficiency of the method on K562 cell line. According to the human C value, K562 were diluted in normal commercial genomic DNA until 10−4 dilutions. Eight CML patients in early CP were the object of this study. A patient specific Q-PCR assay was performed on DNA obtained at diagnosis and subsequently applied to monitor minimal residual disease during Imatinib treatment for up to 8 years, for a total of 61 samples. In parallel the same peripheral blood samples were tested by standard qRT-PCR, and the percentage of residual disease (international scale) measured by mRNA was compared with DNA analysis. Results: Positive levels of mRNA were obtained in 79% of samples analyzed by qRT-PCR,while we detected Ph-positive cells in 92% of samples. In all positive samples for chimeric transcript we measured positive levels of corresponding genomic DNA, confirming the sensitivity of the Q-PCR method. In 13% of samples, with undetectable levels of mRNA, we observed the persistence of quiescent leukemic cells, transcriptionally silent like shown by patient 2 in figure 1. This could probably indicate the presence of pluripotent LSCs or progenitor cells, that does not respond to imatinib treatment. Finally undetectable levels of mRNA were confirmed by a correspondent DNA negativity in 8,2% of the samples. This datum should be investigated further in order to establish if the disease was been eradicated. Patients negative by mRNA detection in several consecutive follow-ups could be candidates to stop imatinib therapy. The development of a DNA base technique could be a powerful tool to evaluate the effective presence/absence of leukemic cells. Patient 8 resulted negative at 70 months monitored by RNA and DNA technique could be a candidate to stop the therapy (figure 2). Conclusion: Although the initial characterization of genomic breakpoint sequence is still time consuming, it may provide a patient-specific DNA biomarker that can be used to detect the presence of quiescent leukemic cells otherwise undetectable using a conventional qRT-PCR. The DNA genomic Q-PCR could be a very sensitive and direct technique to detect minimal residual disease in CML patients treated with tyrosine-kinase inhibitors and allogeneic transplantation. We thank AIL Varese and Bergamo. Disclosures: No relevant conflicts of interest to declare.

Qualitative and Quantitative Molecular Follow up of Minimal Residual Disease after Non Myeloablative Allografting for Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5128-5128 ◽  
Author(s):  
B. Bruno ◽  
M. Ladetto ◽  
M. Astolfi ◽  
L. Veneziano ◽  
L. Cimolin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Minimal Residual Disease ◽  
Nested Pcr ◽  
Genomic Dna ◽  
Residual Disease ◽  
Patient Specific ◽  
Molecular Remission ◽  
Post Transplant ◽  
Minimal Residual

Abstract New allogeneic transplant protocols with non myeloablative conditioning regimens for treatment of multiple myeloma (MM) have been developed in the attempt to reduce the transplant related toxicity associated with myeloablation. Preliminary data have been encouraging with remarkable clinical response rates (Maloney et al, Blood 2003). However, data on the achievement of molecular remission, prerequisite for eventual cure, are still lacking. We implemented a tandem transplant approach consisting of high dose melphalan (200 mg/sqm) with autografting followed by non myeloablative low dose (2.0 Gy) total body irradiation and G-CSF mobilized PBSC infusion from HLA-identical siblings. The curative potential relies exclusively upon a potent graft versus myeloma (GVM) effect through donor T cells. At diagnosis, patient specific clonal markers were generated based upon the rearrangement of the immunoglobulin heavy chain (IgH) genes and used for nested polymerase chain reaction (PCR) detection of minimal residual disease after transplant. Molecular remission was defined as the disappearance of the molecular marker post transplant in both bone marrow and blood. The sensitivity of the nested PCR-based assay was 1 in 100000 cells. A patient specific marker was generated in 11/15 (73%) patients who entered the study. After a median follow up of 16 months (range 5–50), molecular follow up post transplant showed that 3/11 (27%) reached molecular remission at 1, 3 and 7 months post allografting, respectively. Of the remaining 8 patients, 3/8 and 5/8 reached clinical complete remission, defined as the disappearance of the monoclonal paraprotein by immunofixation, and partial remission, respectively. However, minimal residual disease by nested PCR could be detected at all timepoints. The molecular remissions have been durable at 7, 30, and 48 months post transplant, respectively. In 1 case the remission was achieved and sustained in the absence of graft versus host disease (GVHD) which is consistent with the notion that GVHD is not essential for GVM. Furthermore, in 4/11 patients real-time quantification of IgH rearrangements was performed on genomic DNA samples using tumor specific primers and consensus probes. All patients showed a considerable tumor burden reduction post autografting. Samples from two patients became negative by real time PCR at 3 months post allografting, but became PCR-negative by nested PCR at 3 and 7 months, respectively. This discrepancy is explained by the greater sensitivity of nested PCR and the larger amount of IgH copies which are expected in cDNA compared to genomic DNA. The remaining two patients only obtained a clinical partial response throughout the study period. This report indicates that the tandem auto-allo transplant approach can lead to molecular remission in MM. Prospective quantitative monitoring of disease response may be helpful to design individual additional immunotherapeutic manoeuvres, such as donor lymphocyte infusions, to enhance GVM. Longer follow up on a larger series of patients is needed to determine the frequency and durability of molecular remissions.

FLT3-ITD Positive AMLs Harbouring A Second MRD Molecular Marker May Benefit From a Minimal Residual Disease (MRD) Guided Transplant Decision

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2621-2621
Author(s):  
Joanna Schiller ◽  
Michael Hallek ◽  
Karl-Anton Kreuzer
Keyword(s):  
Complete Remission ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Research Funding ◽  
Residual Disease ◽  
Patient Specific ◽  
Consolidation Therapy ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
Minimal Residual

Abstract Introduction The FLT3 internal tandem duplication (FLT3-ITD) occurs in 15%-35% of all AMLs. FLT3-ITD-positive AMLs are associated with high relapse rates after reaching complete remission. Therefore these patients are considered for allogeneic stem cell transplantation (allo-SCT). Data shows that allo-SCT does not influence the overall survival (OS) of these patients. Minimal residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) can predict relapse clearly in advance and therefore allows early therapeutic intervention. Studies on MRD monitoring have shown a positive influence on OS in AML patients. Due to the high sequence variability of individual FLT3-ITD a universal PCR approach has a low sensitivity (approx. 1: 5x102) and therefore cannot be used for MRD monitoring. Methods We developed a novel cDNA-based, highly sensitive, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay for the detection of the FLT3-ITD mutation level. On the basis of individual FLT3-ITD, mutation- specific forward or reverse primers were designed. The expression of FLT3-ITD was determined using complementary DNA samples at different points in time during diagnosis and subsequent treatment. Results Retrospectively we studied 53FLT3-ITD-positive AML patients. 10 patients received palliative treatment, seven died during induction therapy; three patients are still in induction course of treatment. 32 patients achieved complete remission and 12 of them had undergone allo-SCT. For 47 patients we designed patient-specific qRT-PCR with mutation-specific forward and reverse primers. 41 (87%) assays were highly specific (1:104 - 1:106) and yielded similar results when compared to other high sensitive assays for molecular markers like NPM1 or PML-RARA.The median of follow-up time was 754 days (68-2546 days). MRD status was available for 23 patients after consolidation therapy. In 16 (81%) of these patients, FLT3-ITD negativity was demonstrated. MRD negativity predicted lasting remission independent of allo-SCT (N = 4) or non-allo-SCT (N=12). 3 patients relapsed after reaching MRD negativity. Only one patient relapsed without molecular relapse. 7 out of 32 patients stayed MRD positive after consolidation therapy. 5 of them underwent allo-SCT, nevertheless 3 of them stayed MRD positive (molecular non-responders) and finally relapsed. Furthermore we compared paired PB and BM samples at diagnosis and after induction therapy in 5 cases. The differences in FLT3-ITD expression were not statistically significant (p=0.8) which is in line with recent studies. Conclusion We conclude that highly sensitive detection of individual FLT3-ITD possesses equal prognostic power in AML like established molecular MRD markers. Using this approach MRD guided treatment decisions appear to be justified and should be incorporated in future studies. Disclosures: Hallek: Janssen: Research Funding; Gilead: Research Funding; Roche: Research Funding. Kreuzer:Roche: Honoraria; Mundipharma: Honoraria.

Quantitative DNA Real-Time PCR Compared To mRNA and Cytogenetic Assays To Monitor Minimal Residual Disease In Chronic Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1316-1316
Author(s):  
Ilaria Stefania Pagani ◽  
Orietta Spinelli ◽  
Cristina Pirrone ◽  
Diana Pigni ◽  
Sara Lupoli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Cancer Stem Cells ◽  
Minimal Residual Disease ◽  
Cytogenetic Analysis ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Qrt Pcr ◽  
Minimal Residual

Abstract Introduction Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia (CML), effectively prolonging overall survival. Because discontinuation of treatment is associated with molecular relapse, IM is required indefinitely to maintain operational cure. To evaluate the degree of response to therapy and to highlight the persistence of the disease after treatment, patients should be monitored routinely. The gold standard for diagnosing CML is the cytogenetic analysis, a direct not-sensitive method to detect Ph-positive cells. Quantitative real-time RT-PCR (qRT-PCR) provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells and cannot detect transcriptionally silent leukemic stem cells. Methods Here we will propose a new sensitive approach to directly detect the number of leukemic cells using a DNA-based biomarker specific for each patient. We applied targeted next-generation sequencing for the identification of genomic BCR-ABL1 fusion junctions, and we developed a sensitive new approach to detect the number of leukemic cells by a DNA Q-PCR assay based on the genomic break-point, with a formula to calculate the number of Ph+ cells. The percentage of the leukemic cells (LC) was calculated using the following formula: %LC= (2/(2Δct+1))*100, where ΔCt is the difference between the amplification cycles of the BCR-ABL1 and BCR reactions. The number of LC was calculated by multiplying the total number of cells analyzed in each sample by the percentage of LC calculated by the ΔCt formula. We then defined a limit of quantization and a limit of sensitivity in the evaluation of minimal residual disease (MRD), as described by guidelines for the detection of MRD by genomic Q-PCR in acute lymphoblastic leukemia (ALL). We defined a “quantitative range” of detection, the portion of the standard curve in which the MRD levels can be quantified reproducibly and accurately, and we defined the “limit of sensitivity”, the lowest MRD level that still can be detected, although not in all replicates. We thus calculated the exact number of leukemic cells only when the MRD fell within the range of quantization. The detection of MRD at the limit of sensitivity was indicated as positive but not quantified. Results We monitored eight CML patients treated with Imatinib for 8 years. We tested the same samples by patient specific Q-PCR, and in parallel by cytogenetic analysis and by standard qRT-PCR. In all samples positive for chimeric transcripts we measured corresponding chimeric genomic DNA (gDNA) by Q-PCR, confirming the sensitivity of the Q-PCR method. According to conventional criteria, undetectable levels of BCR-ABL1 mRNA assessed by qRT-PCR are indicative of complete molecular response (CMR), but in 33.3% (45/135) samples with undetectable levels of mRNA, we detected the persistence of transcriptionally-silent leukemic cells. However, we never found samples negative by gDNA Q-PCR and positive by RNA-based qRT-PCR (Figure 1). Thirty-six of 135 samples were also analyzed cytogenetically until the achievement of CCyR. As expected, Ph+cells were detected only in 25% (9/36) and 22,2% (8/36) of samples by CBA and I-FISH, respectively, whereas BCR-ABL1 mRNA was detected by qRT-PCR in 83.3% (30/36) of samples and Ph+ cells were detected by genomic Q-PCR in 91.7% (33/36) of samples (Figure 1). Finally, the separation of different cell populations from blood and bone marrow revealed the presence of a population of transcriptionally silent cancer stem cells. The gDNA based Q-PCR analysis performed on highly purified (immunomagnetically selected ) CD34+and CD3+ cells confirmed the presence of a population of transcriptionally silent cancer stem cells. Conclusions The demonstration of positive gDNA Q-PCR in 33.3% of samples negative for the RNA qRT-PCR could partially explain why some patients lose MMR and CMR and others do not, when IM is discontinued for brief periods. The gDNA based Q-PCR could be used to supplement conventional techniques, providing clinicians with additional information about disease status and response in determining whether to stop or alter therapy. Acknowledgments to AIRC and AIL. Disclosures: No relevant conflicts of interest to declare.

Patient-specific microRNA expression profiles as a marker for minimal residual disease in acute myeloid leukemia

Hematology ◽  
2013 ◽  
Vol 19 (1) ◽  
pp. 18-21 ◽  
Author(s):  
Velizar Shivarov ◽  
Angel Stoimenov ◽  
Branimir Spassov ◽  
Svetlana Angelova ◽  
Monika Niagolov ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Expression Profiles ◽  
Microrna Expression ◽  
Patient Specific ◽  
Minimal Residual ◽  
Acute Myeloid

Moving Beyond Autologous Transplantation in Multiple Myeloma: Consolidation, Maintenance, Allogeneic Transplant, and Immune Therapy

2016 ◽  
pp. 210-221 ◽  
Author(s):  
Amrita Krishnan ◽  
Ravi Vij ◽  
Jesse Keller ◽  
Binod Dhakal ◽  
Parameswaran Hari
Keyword(s):  
Multiple Myeloma ◽  
Disease Control ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
The Other ◽  
Novel Agents ◽  
Patient Specific ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Minimal Residual

For multiple myeloma, introduction of novel agents as part of the front-line treatment followed by high-dose chemotherapy and autologous hematopoietic stem cell transplantation (ASCT) induces deep responses in a majority of patients with this disease. However, disease relapse is inevitable, and, with each relapse, the remission duration becomes shorter, ultimately leading to a refractory disease. Consolidation and maintenance strategy after ASCT is one route to provide sustained disease control and prevent repeated relapses. Though the consolidation strategy remains largely confined to clinical trials, significant data support the efficacy of consolidation in improving the depth of response and outcomes. There are also increasing rates of minimal residual disease–negativity with additional consolidation therapy. On the other hand, maintenance with novel agents post-transplant is well established and has been shown to improve both progression-free and overall survival. Evolving paradigms in maintenance include the use of newer proteasome inhibitors, immunotherapy maintenance, and patient-specific maintenance—a concept that utilizes minimal residual disease as the primary driver of decisions regarding starting or continuing maintenance therapy. The other approach to overcome residual disease is immune therapeutic strategies. The demonstration of myeloma-specific alloimmunity from allogeneic transplantation is well established. More sophisticated and promising immune approaches include adoptive cellular therapies, tumor vaccines, and immune checkpoint manipulations. In the future, personalized minimal residual disease–driven treatment strategies following ASCT will help overcome the residual disease, restore multiple myeloma–specific immunity, and achieve sustained disease control while minimizing the risk of overtreatment.

scholarly journals Immunophenotyping Investigation of Minimal Residual Disease Is a Useful Approach for Predicting Relapse in Acute Myeloid Leukemia Patients

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2465-2470 ◽  
Author(s):  
J.F. San Miguel ◽  
A. Martı́nez ◽  
A. Macedo ◽  
M.B. Vidriales ◽  
C. López-Berges ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Multidrug Resistance ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Rhodamine 123 ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 × 10−3 residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 × 10−3 residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 × 10−3 leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine-123 assay. Patients with ≥5 × 10−3 residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% ± 24%) than those with less than 5 × 10−3 residual cells (mean, 32% ± 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

Sign in / Sign up

Export Citation Format

Share Document