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.

Highly Sensitive Monitoring of Minimal Residual Disease in FLT3-itd-Positive Acute Myeloid Leukemia Patients with a Mutation Specific Quantitative-PCR Method,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3530-3530
Author(s):  
Joanna Schiller ◽  
Inka Praulich ◽  
Michael Hallek ◽  
Karl-Anton Kreuzer
Keyword(s):  
Myeloid Leukemia ◽  
Research Funding ◽  
Tandem Duplication ◽  
Residual Disease ◽  
Patient Specific ◽  
Base Pairs ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Abstract 3530 Minimal residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) can predict relapse clearly in advance and therefore allows early therapeutic intervention. Moreover, recent studies have highlighted the significance of personalized treatment on the basis of MRD status for improving outcome in AML. The FLT3 internal tandem duplication (FLT3 -ITD) is one of the most frequent mutations in AML patients occurring in 15–35% of all cases and approximately in 20–30% of cytogenetic normal (CN) AML. Clinically, FLT3 -ITDs have been strongly associated with high leukocytosis, high blast counts, normal karyotype and, most importantly, poor clinical outcome. However, due to the high sequence variability of individual FLT3 -ITD commonly a universal PCR approach is applied which has a low sensitivity (approx. 1: 5×102). For this reasons FLT3 -ITD is regarded as not suitable for longitudinal MRD measurements. The aim of this study was to develop 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. Furthermore, the prognostic value of FLT3 -ITD-based MRD detection in AML patients was compared with other genetic markers like NPM1 mutations, MLL -partial tandem duplication (MLL -PTD) and the expression of PML /RARα fusion gene or WT1 over-expression. To design patient-specific qRT-PCR, FLT3 -ITDs were amplified with universal primers, purified corresponding bands from agarose gel and directly sequenced. On the basis of the FLT3 -ITD, individual mutation-specific primers were designed. The expression of FLT3 -ITD was determined using complementary DNA samples at different points in time diagnosis and subsequent treatment. For estimation of the sensitivity and specificity of this approach we used a dilution of FLT3 -ITD cDNA in a pool of FLT3 -unmutated cDNA. From a total of 394 newly diagnosed AML patients 55 (14%) were FLT3 /ITD positive. Retrospectively we analyzed ITD mutation of FLT3 in 39 available cases. The length of ITD ranged from 3 to 144 base pairs (median 46). Nine patients had extra insertions of 2–38 base pairs between two repeats. For the FLT3 -ITD quantification we developed patient-specific qRT-PCR for 29 individuals with mutation-specific forward primers and studied 83 peripheral blood and 61 bone marrow samples. Three cases with WT1, fifteen with NPM1, three with MLL -PTD and five with PML /RARα fusion genes expression levels were compared with FLT3 -ITD expression levels. 26 of 29 assays (90%) were highly specific (1:104 − 1:105) and yielded similar results when compared to other high sensitive assays for molecular markers like NPM1 or PML /RARα. In three cases (10%) a co-amplification of the wild-type could not be avoided resulting in lower sensitivity (1:103). We could show that FLT3 -ITD positivity reliably predicted relapse up to 10 months in advance. 92% patients, who achieved FLT3 -ITD negativity with our assay, did not relapse. Furthermore we compared paired PB and BM samples at diagnosis and after induction therapy in 5 cases. The difference in FLT3 -ITD expression were not statistically significant (p=0.8) which is in line with recent studies. We conclude that highly sensitive detection of individual FLT3 -ITD posses 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: Hoffmann-la Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kreuzer:Alexion: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Shire: Honoraria, Research Funding.

CD11b is a therapy resistance– and minimal residual disease–specific marker in precursor B-cell acute lymphoblastic leukemia

Blood ◽  
2010 ◽  
Vol 115 (18) ◽  
pp. 3763-3771 ◽  
Author(s):  
Peter Rhein ◽  
Rita Mitlohner ◽  
Giuseppe Basso ◽  
Giuseppe Gaipa ◽  
Michael N. Dworzak ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Consolidation Therapy ◽  
Cd11b Expression ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

Abstract A consistently increased mRNA expression of the adhesion receptor CD11b is a hallmark of the reported genomewide gene expression changes in precursor B-cell acute lymphoblastic leukemia (PBC-ALL) after 1 week of induction therapy. To investigate its clinical relevance, CD11b protein expression in leukemic blasts has been prospectively measured at diagnosis (159 patients) and during therapy (53 patients). The initially heterogeneous expression of CD11b inversely correlated with cytoreduction rates measured at clinically significant time points of induction therapy in the ALL–Berlin-Frankfurt-Münster 2000 protocol. CD11b positivity conferred a 5-fold increased risk of minimal residual disease (MRD) after induction therapy (day 33) and of high-risk group assignment after consolidation therapy (day 78). In the multivariate analysis CD11b expression was an independent prognostic factor compared with other clinically relevant parameters at diagnosis. During therapy, CD11b expression increased early in most ALL cases and remained consistently increased during induction/consolidation therapy. In more than 30% of MRD-positive cases, the CD11b expression on blast cells exceeded that of mature memory B cells and improved the discrimination of residual leukemic cells from regenerating bone marrow. Taken together, CD11b expression has considerable implications for prognosis, treatment response monitoring, and MRD detection in childhood PBC-ALL.

Assessment of Minimal Residual Disease (MRD) In Relapsed CLL Patients Treated with Fludarabine and Cyclophosphamide with or without Rituximab (REACH)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1390-1390
Author(s):  
Annika Dufour ◽  
S. K Bohlander ◽  
Karsten Spiekermann ◽  
Stephanie Schneider ◽  
Jan Braess ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
Complete Response ◽  
Small Sample ◽  
Patient Specific ◽  
Molecular Response ◽  
Igh Rearrangement ◽  
Minimal Residual

Abstract Abstract 1390 Introduction: Levels of minimal residual disease (MRD) have been shown to correlate with PFS in previously untreated patients with CLL (CLL8, Boettcher et al. Leukemia, 2009). Patients who remain MRD positive after treatment have a higher risk of relapse. Eradication of MRD is therefore a desirable clinical endpoint of treatment. We were interested to assess this correlation in REACH, a randomized international clinical study in previously treated CLL patients, randomized 1:1 for treatment with rituximab, fludarabine and cyclophosphamide© R-FC (276 patients) or FC alone (276 patients); (Robak et al. JCO 2010). Methods: While MRD quantification by flow cytometry requires an identifiable stable phenotype and fresh blood samples, PCR based methods can be performed centrally on frozen samples. We have therefore developed a Realtime Quantitative (RQ) PCR method, using patient-specific IgVH (immunoglobulin variable heavy chain) gene rearrangements as targets. Briefly, genomic DNA was isolated from CD19 sorted B-cells. ASO (allele-specific oligonucleotide) primers were designed matching the hypervariable N-D-N region of the patient-specific leukemic clone and used with reverse consensus primers and hydrolysis probes annealing to the family-specific joining region of the IGH rearrangement (Brüggemann et al., Leukemia, 2004). Maximum sensitivity and quantitative range were defined for every RQ-PCR. Patients were categorized as molecular responders (MRD negative) if there was no detectable clonal IgH rearrangement, using a sensitivity cut-off of 1×10-4. Molecular response was assessed at the time of CR confirmation and 6 months later (if CR was maintained). Results: Among the 103 patients who achieved CR during the study, 86 patients had at least one MRD assessment in peripheral blood, 92 patients in bone marrow. Since many patients had a CR confirmation at different time points during the follow-up period, we initially analyzed the MRD levels only in patients who had achieved confirmed complete response at end of treatment +/−3 month (“EOT - period”). The rate of MRD negativity in blood (22 pts: 5(15) FC, 6(7)R-FC) at EOT was 33% for patients treated with FC, and 86% for patients treated with R-FC (p=0.06); In bone marrow at the EOT (61 patients: 5(27) FC, 20(34) R-FC) the rates were 19% and 59%, respectively (p= 0,02), indicating higher efficacy of the Rituximab containing regimen in eradication of residual disease; This is in line with the previously reported results using FACS analysis of MRD in the CLL8 trial; the differences in the detection rate in blood versus bone-marrow, suggest a higher sensitivity for detection of MRD in bone marrow. We therefore compared the levels of MRD negativity in samples from blood and bone marrow in patients where both samples were taken at the same time point. Results were concordant in 8/9 patients, one patient had a positive result in bone marrow with no detectable signal in blood. This supports the notion that assessment of MRD in bone marrow of CLL patients may be more sensitive than assessment in blood only. However, for a definitive statement larger sample size would be needed. We then correlated MRD status at EOT, regardless of treatment arm, with PFS: In line with previous reports, there was a clear trend to longer PFS in patients who had reached MRD negativity (median PFS not reached), while patients with residual disease had shorter PFS; however, due to small sample numbers, statistical significance could not be reached. We also analyzed the correlation of MRD negativity reached at any time during and after treatment with PFS, bearing in mind that this sample set is inherently biased, since patients with early progression will be lost from the analysis; the results are consistent with the EOT findings. Summary: ASO IgVH RQ-PCR is a powerful method to detect residual levels of disease in CLL patients with clinical complete response and undetectable MRD correlates with longer PFS. Among patients in REACH achieving clinical CR on either study arm, a higher percentage achieved MRD negativity on the R-FC arm, consistent with the increased efficacy shown for the Rituximab treatment arm by the REACH clinical data. Disclosures: Mundt: Roche: Employment. Smith:Roche: Employment. Lin:Genentech: Employment. Barrett:Roche: Employment. Hurst:Genentech: Employment. Geisler:Roche: Research Funding, Speakers Bureau. Hiddemann:Roche: Research Funding.

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.

scholarly journals Interim Results of a First in Man Study with the Fc-Optimized FLT3 Antibody Flysyn for Treatment of Acute Myeloid Leukemia with Minimal Residual Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3928-3928 ◽  
Author(s):  
Sabine Kayser ◽  
Jonas S. Heitmann ◽  
Daniela Dörfel ◽  
Felicitas Thol ◽  
Michael Heuser ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Pharmacokinetic Analysis ◽  
Shareholder Interest ◽  
Minimal Residual ◽  
Acute Myeloid

Background: Substantial surface expression of the FLT3 receptor can be measured on blast cells in 70% to 100% of acute myeloid leukemia (AML) patients, while no or only low levels are expressed on healthy cells like monocytes and progenitor stem cells. Thus, FLT3 is a suitable and highly selective target for therapeutic antibodies. FLYSYN is a chimeric Fc-optimized IgG1 antibody and binds specifically and with high avidity to human FLT3 (CD135). Despite achievement of complete remission, roughly half of AML patients display minimal residual disease (MRD) after end of therapy and relapse. Methods: We perform an open-label, single-arm, first in man multicenter trial to assess safety and tolerability as well as preliminary efficacy of FLYSYN as monotherapy in adult (≥18 years) AML patients in complete remission with MRD (NCT02789254). FLYSYN is administered as IV infusion over a 3 hour period. Recruitment started in March 2017 with an estimated maximum number of 31 patients and estimated recruitment until January 2020. The main inclusion criterion was confirmed stable or increasing MRD positivity in two sequential measurements. MRD was measured by central RT-qPCR and/or next generation sequencing (NGS). Sensitivity with RT-qPCR (for NPM1 only) was 10-6 and 10-4 with NGS. Patients with acute promyelocytic leukemia as well as prior hematopoietic stem cell transplantation were excluded. Using a "3 + 3" dose escalation design, five of the planned six cohorts with escalating doses have currently completed treatment (cohort 1: 0.5 mg/m² BSA; cohort 2: 1.5 mg/m² BSA; cohort 3: 5 mg/m² BSA; cohort 4: 15 mg/m² BSA; cohort 5: 45 mg/m²; cohort 6: in total 45 mg/m²; 15 mg/m² on day 1, 15 and 29). Three patients were treated per cohort, except for cohort 4, which was expanded to nine patients. The interim analysis for preliminary efficacy was performed after 18 patients were treated in cohorts 1-4. Response is defined as 1 log MRD reduction. Results: Median age was 60 years (range, 21-80 years). Sixteen patients were MRD positive for NPM1 and one patient each for RUNX1-RUNX1T1 and IDH2. So far, FLYSYN was well tolerated. Only one temporary grade 3 adverse event (AE) occurred (neutrophil decrease on day 3 only) in a patient of cohort 3, which was suspected to be related to FLYSYN treatment. There were no reported dose-limiting toxicities. The most frequently reported AEs were grade 1 and 2 gastrointestinal toxicities and laboratory abnormalities, which all were manageable with supportive care. After treatment, neither human anti-mouse nor anti-human antibodies were detected in any of the patients. Preliminary pharmacokinetic analysis was performed in the first 12 patients of the study and revealed a half-life of FLYSYN of roughly 6.5 days. Regarding preliminary efficacy, 1 patient of cohort 1 achieved permanent MRD negativity in bone marrow (BM; lasting until day 545) and 1 patient a temporary BM MRD reduction (at day 15). One patient of cohort 2 achieved BM MRD negativity (day 22) with MRD progression on day 365, whereas the other 2 patients were BM MRD progressive. None of the patients of cohort 3 achieved a MRD response in BM, but 2 patients achieved a temporary MRD response in peripheral blood. Four patients of cohort 4 achieved non-permanent BM MRD negativity. Overall, 6 patients achieved BM MRD negativity (33 %, 6/18), enduring more than 1 year in 1 patient. Conclusions: Our data suggest that FLYSYN is safe and very well tolerated as monotherapy in MRD positive AML patients. Preliminary efficacy data are promising, and recruiting is ongoing in cohort 6 in which 15 mg/m² FLYSYN is given for three times. Up-dated results will be presented at the ASH meeting. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Steiner:Synimmune: Employment, Other: shareholder interest. Grosse-Hovest:SYNIMMUNE: Employment, Other: shareholder interest. Jung:Synimmune: Other: shareholder interest. Salih:SYNIMMUNE: Consultancy, Research Funding.

scholarly journals Value of Immunohistochemistry-Based Direct Visualization for Localization, Lineage Determination and Monitoring of IDH1 p.R132H Mutant Clones in AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1726-1726
Author(s):  
Habibe Kurt ◽  
Carlos E. Bueso-Ramos ◽  
Joseph D Khoury ◽  
Mark Routbort ◽  
Rashmi Kanagal-Shamanna ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Complete Remission ◽  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
Advisory Board ◽  
Post Treatment ◽  
Immature Cells ◽  
Minimal Residual

Abstract Background Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are important prognostic biomarkers in acute myeloid leukemia (AML). Although the clinicopathologic correlates of IDH mutations have been extensively studied, the distribution of abnormal myeloid cells carrying these mutations has not been studied. Specific localization of cells carrying IDH mutations will be useful in further understanding the pathophysiology and post-treatment biology of IDH mutant cases of AML. This characterization is becoming particularly relevant for identification of minimal residual disease, especially for patients treated with novel IDHinhibitors. In this study, we characterized IDH1 p.R132H clones in bone marrow specimens involved by AML using a mutation specific antibody. Materials and Methods Bone marrow tissue sections (biopsy or clot specimens) from 32 AML cases with IDH1 p.R132H mutation were stained with IDH1 p.R132H-mutation specific antibody. These cases include 20 de novoAML and 12 cases of AML with myelodysplasia-related changes (AML-MRC). We also included 10 AML cases with wild-type IDH1 as a control. After confirmation of the positive IDH1 immunohistochemical (IHC) signal in the primary specimens, follow up bone marrow specimens (n=67) including (a) persistent disease, (b) minimal residual disease by flow cytometry, (3) complete remission by morphology and flow cytometry, but, positive for mutation by PCR, as well as (4) relapsed cases after complete remission were included in the study (in progress). We also included pre- and post-treatment (unresponsive with increasing blast counts, stable disease, persistent disease with decreasing blast counts, complete remission, and relapse) bone marrow specimens (n=72) from 16 patients treated with IDH inhibitors (in progress). Results All the IDH1 wild type AML cases were negative for IDH1 IHC stain showing 100% specificity. Positive signal was detected in all de novo AML and AML-MRC (allelic frequency ranges from 1.8% to 47% by PCR) except one AML case with 8.9% allele burden which was a limited sample; overall sensitivity was 96%. The IHC signal was detected in the cytoplasm of myelomonocytic cells, their precursors, and megakaryocytes. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative. The signal intensity ranged from weak (n=10) to moderate (n=9), to strong (n=13). The positive cells predominantly showed an interstitial distribution in the bone marrow. In the de novo AML group, only the immature cells were positive in 100% of pre-treatment AML cases. However, both mature and immature cells were positive in 7/13 (54%) post-treatment AML cases (6 cases treated with hypomethylating agents). One case was transformed from MPN which also showed positivity in mature and immature cells. In two cases with complete morphologic remission and one case with minimal residual disease detected by flow cytometry, IHC signal was detected in both mature and immature cells; both patients relapsed in 8 and 11 months. In the AML-MRC group, both immature and mature cells were positive in 11/12 (92%) cases of which 2 were not previously treated indicating the possibility that IDH1 mutation is an early event. Since the remaining 9 patients were treated with hypomethylating agents, the positivity of both mature and immature cells as a result of maturation effect versus an early event cannot be assessed. Additional studies for follow-up AML cases, including cases on an IDH inhibitor clinical trial are in progress. Conclusions Our preliminary data indicate that IDH1 IHC is a highly specific and sensitive tool to detect IDH1 R132H mutated cases and can be used as a primary method to localize the population of mutation-bearing cells in the bone marrow. IHC also allows determination of whether the IDH1 mutation in the post-treatment setting is arising from immature or mature cells. IHC provides an opportunity to understand the difference between these two populations and, based on characterization of cell type and distribution, may be helpful to predict whether the risk of relapse is high. Disclosures DiNardo: Agios: Other: advisory board, Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Celgene: Research Funding; Abbvie: Research Funding.

Monitoring of Minimal Residual Disease in NPM1 Mutated Acute Myeloid Leukemia (AML): Results of the AML Study Group (AMLSG)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 699-699
Author(s):  
Jan Krönke ◽  
Richard Schlenk ◽  
Kai-Ole Jensen ◽  
Karina Eiwen ◽  
Marianne Habdank ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Consolidation Therapy ◽  
Dna And Rna ◽  
Pcr Assays ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Background: Mutations in the nucleophosmin 1 (NPM1) gene represent the most frequent gene mutations in acute myeloid leukemia (AML), with highest frequency (50–60%) in cytogenetic normal (CN)-AML and these mutations can be used as molecular markers for monitoring of minimal residual disease (MRD). Aims: To examine the applicability and sensitivity of DNA- and RNA-based real-time quantitative polymerase chain reaction (RQ-PCR) assays and to evaluate whether MRD levels are of prognostic relevance in younger (16 to 60 years) patients (pts) with AML harboring NPM1 mutations (NPM1mut ) type A, B or D. Methods: All pts were treated within the AMLSG 07-04 treatment trial including double induction therapy with ICE (idarubicin, cytarabine, etoposide) followed by three courses of high-dose cytarabine or allogeneic stem cell transplantation from matched related donors as consolidation therapy. Levels of NPM1mut expression ratios, defined as NPM1mut copies/ 104abl copies, were determined by RQ-PCR using TaqMan technology. DNA- and RNA-based RQ-PCR assays were used in parallel. Results: A total of 537 samples [bone marrow (BM) n=463, peripheral blood (PB) n=74] from 88 pts have been analyzed (mean number of samples per patient, n=6;, range, 1–16) at diagnosis (BM, n=75; PB, n=12), after induction therapy (BM, n=190; PB, n=28), during consolidation (BM, n=115; PB, n=16) and during follow-up (BM, n=83; PB n=18). Sensitivity was 10−6 for the RNA- and 10−4 for the DNA-based assays. Both assays were highly specific as no wildtype (wt) NPM1 could be detected. The lower sensitivity of the DNA-based assay was demonstrated by the finding that the DNA-based RQ-PCR became negative in 102 samples while RNA-based RQ-PCR still showed NPM1mut expression in 68 of these samples (range 2.1–11018.1). In addition, comparison of 63 paired (BM and PB) samples revealed a median 6.8 times higher NPM1mut expression in BM with 13 of 15 samples being negative in PB but positive in BM. Based on these results we subsequently performed RNA-based RQ-PCR analysis in BM samples. NPM1 mut expression ratios at diagnosis varied between 1.1 × 104 and 6.4 × 106 with a median of 5.6 × 105. Pretreatment transcript levels were not associated with clinical characteristics (e.g., age, WBC counts, BM blasts) and did not impact on relapse-free (RFS) and overall survival (OS). The median decrease of the MRD level ratio after first and second induction therapy normalized to pretreatment levels was 2.24 log10 (−8.57–1.88 log10) and 3.9 log10 (−8.58–0.67 log10), respectively. There was no difference in FLT3-ITD positive and negative pts (p=0.72 and p=0.20, respectively). During consolidation therapy NPM1mut expression levels in the FLT3-ITD negative pts further decreased after each cycle and were significantly lower compared to FLT3-ITD positive patients (p=0.00008, p=0.05, p=0.003). Of note, FLT3-ITD positive pts had rebounding MRD levels during consolidation therapy in comparison to levels achieved after induction therapy. Achievement of RQ-PCR-negativity in at least 1 BM sample (n=20) during consolidation therapy was associated with superior RFS (p=0.004) of 76.8% (95% CI 13%–61%) after 2 years compared to 29.0% (95% CI 59%–100%) for pts with persistent RQ-PCR positivity (n=37). For this analysis pts with allogeneic transplantation were excluded. Different courses of MRD were identified in 31 relapsed pts. In 19 patients increasing (range 1.0–322.3) or constantly high MRD levels 28 to 518 days (median 66) before occurrence of clinical relapse were detected. For the remaining pts, molecular relapse was not detectable due to loss of the NPM1mut clone in relapse material (n=3), a decrease in MRD levels 71 to 90 days before relapse (n=3) or the lack of samples within 3 months before relapse (n=6). Conclusions: NPM1 mutations are a sensitive marker for MRD monitoring in AML, in particular when RNA-based RQ-PCR assays on BM samples are used. Achievement of MRD negativity appears to predict favorable clinical outcome.

scholarly journals Minimal Residual Disease Assessment of Remission after Induction Therapy Is Superior to Morphologic Assessment for Risk Stratification in Childhood Acute Lymphoblastic Leukemia: A Report from the Children's Oncology Group (COG)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 758-758
Author(s):  
Sumit Gupta ◽  
Meenakshi Devidas ◽  
Si Chen ◽  
Cindy Wang ◽  
Mignon L. Loh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Risk Stratification ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Laboratory Services ◽  
Minimal Residual

Abstract Background: Minimal residual disease (MRD) assessment after initial therapy is integral to modern risk stratification in both precursor B and T lineage acute lymphoblastic leukemia (B-ALL and T-ALL). While MRD is used to determine depth of remission, remission is still defined, both in clinical practice and clinical trials, according to morphological assessment. We aimed to determine the outcomes of children, adolescents and young adults with discordant assessments of remission by morphology vs. by MRD, and in doing so, the extent to which morphologic assessment of remission contributes to risk assessment in this population. Methods: We identified a cohort of patients age 1-30.99 years enrolled on frontline COG trials for B-ALL [standard risk (SR): AALL0331; high risk (HR) AALL0232] and T-ALL (AALL0434) that underwent bone marrow assessment of remission at the end of induction therapy (Day 29). Morphologic response was assessed by local centers and was categorized according to traditional criteria: M1 (<5% leukemic blasts - remission) vs. M2 (5-25%) vs. M3 (>25%). MRD was measured by flow cytometry at one of two central laboratories. We determined predictors of MRD discordance and compared event free survival (EFS) between those with discordant vs. concordant morphology/MRD remission assessments. Results: Day 29 remission assessments and central MRD data were available on 9,350 patients, 7,857 (84%) with B-ALL (AALL0331: N=5049; AALL0232: N=2808) and 1,493 (16%) with T-ALL. Table 1 shows the distribution of end induction marrow morphology vs. flow cytometry results. Few patients with M2/M3 marrows had discordant low MRD values. For example, of 84 patients with M3 morphology, only 2 (2.4%) had MRD <5%. Of 202 patients with M2/M3 morphology, 23 (11.4%) had MRD<1% and 9 (4.5%) had MRD<0.1%. Subsequent analyses of discordance were thus restricted to patients with M1 morphology but flow cytometry consistent with failure to achieve remission (MRD>=5%). Using this definition, discordance was uncommon among subjects with B-ALL (66/7,748; 0.9%) but significantly more common in T-ALL (97/1,400; 6.9%; p<0.0001). Among subjects with B-ALL and M1 morphology, significant predictors of discordance (MRD>=5%) in multivariable regression included variables traditionally associated with poor response: age >=10 years [odds ratio (OR)=1.7, 95th percentile confidence interval (CI) 1.1-2.8; p=0.03), presenting white blood cell count >=50,000/microliter (OR=2.1, CI 1.3-3.6; p=0.004), and unfavorable compared to favorable cytogenetics (OR=31, CI 8.9-109; p<0.0001). In B-ALL, subjects with end induction M1 morphology but discordant MRD (>=5%) had modestly superior 5-year EFS when compared to those with M2 morphology and MRD >=5% (33.1%±6.2% vs. 22.0%±6.9%; p=0.03), but EFS was significantly inferior to those with M1 morphology and concordant MRD (<5%) (33.1%±6.2% vs. 86.8%±0.4%; p<0.0001) (Figure 1). In T-ALL, the 5-year EFS of subjects with M1 morphology/discordant MRD was not significantly different from those with M2 morphology and MRD >=5% (80.3%±7.3% vs. 62.7%±13.5%; p=0.13); outcomes of both groups were superior to their equivalents with B-ALL, in keeping with known slower disease clearance kinetics in T-ALL. Conclusions: Patients in morphologically defined remission but with MRD >=5% have outcomes similar to those who fail to achieve morphological remission. These results suggest that, in addition to measuring depth of remission, MRD should replace morphology in defining remission in subjects with ALL, with consequent implications for risk stratification, treatment assignment and eligibility for experimental agents. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Borowitz:HTG Molecular: Consultancy; Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding; BD Biosciences: Research Funding. Wood:Juno: Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement.

Minimal Residual Disease Negative Status At the End of Induction Therapy Is a Potent Prognostic Marker in Adult Non-Ph Acute Lymphoblastic Leukemia: Results of the ALL MRD2002 Study

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2581-2581
Author(s):  
Koji Nagafuji ◽  
Toshihiro Miyamoto ◽  
Tetsuya Eto ◽  
Tomohiko Kamimura ◽  
Shuichi Taniguchi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Prognostic Factor ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Clinical Indication ◽  
Significant Prognostic Factor ◽  
Consolidation Therapy ◽  
Minimal Residual

Abstract Abstract 2581 Introduction A clinical indication for allogeneic hematopoietic stem cell transplantation (HSCT) in adult Philadelphia-chromosome negative [Ph (−)] acute lymphoblastic leukemia (ALL) patients in complete remission 1 (CR1) remains to be clarified. An international study showed that matched related donors HSCT for ALL in CR1 provides survival benefits for standard-risk ALL patients compared to chemotherapy (Blood. 2008 111:1827). Minimal residual disease (MRD) status has been reported to be a strong prognostic factor in adult ALL patients (Blood. 2006 107:1116, 2009 113:4153). We prospectively monitored MRD status during induction and consolidation therapy in adult Ph (−) ALL patients to determine a clinical indication for HSCT. Materials & Methods From July 2002 to August 2008, 110 adult ALL patients were enrolled in this study. Eligibility criteria included non-L3 ALL, 16–65 years of age, and adequate organ function. Of these patients, 101 were eligible for this study and 59 were Ph (−). The treatment protocol used in this study was modified CALGB 19802. Treatment consisted of 6 courses given in the order of A-B-C-A-B-C, followed by a maintenance phase. Induction chemotherapy (course A) consisted of cyclophosphamide (1,200 mg/m2), daunorubicin (60 mg/m2 × 3), vincristine (VCR) (1.3 mg/m2 (max 2mg) × 4), L-asparaginase (3,000 U/m2 × 6) and prednisolone. Granulocyte colony-stimulating factor was started on day 4 and continued until neutrophil recovery. Consolidation B consisted of mitoxantrone (10 mg/m2 × 2), cytarabine (2,000 mg/m2/day × 4) and intrathecal (IT) administration of methotrexate (MTX). Consolidation C consisted of VCR (1.3 mg/m2 (max 2mg) × 3) and MTX (1,500 mg/m2 × 3) with leucovorin rescue and IT MTX. Patients received maintenance chemotherapy on a monthly basis: prednisolone 60 mg/m2 on days 1–5, VCR (1.3mg/m2 (max 2mg) × 3) on day 1, oral MTX 20 mg/m2 weekly, and oral 6-mercaptopurine 60 mg/m2 daily. MRD status was evaluated after induction therapy (first course A) and after second consolidation therapy (first course C). When MRD statuses after the consolidation were positive, patients were supposed to proceed to HSCT whenever possible. Results A total of 59 patients were Ph (−). MRD status of 41 of these patients (69.5%) could be monitored by the major rearrangement patterns of TCRƒÁ, TCRƒÁ, and IgƒÈ chain genes, and secondarily for IgH gene rearrangements (Blood. 1991 77:331), and chimeric RNA analysis (TCRƒÁ n=14, TCRƒÁ n=9, IgƒÈ n=6, IgH n=1, other n=1, E2A-PBX1 n=3, MLL-AF4 n=1). There were 21 male and 20 female patients. The median age was 31.0 (range 17–63 years). The median white blood cell count at presentation was 10,900/ml (range 1,000–408,700). A total of 36 patients (85.7%) achieved CR. The probability of 3-year overall survival and progression-free survival (PFS) were 59.3 % and 48.9 %, respectively. In terms of CR1 status, patients who were MRD (−) after induction therapy (first course A)(n = 22) had a significantly better 3-year PFS compared with those who were MRD (+)(n = 13)(72.2 % vs. 33.6 %, p = 0.011). Those MRD (−) patients also had a significantly lower 3-year probability of relapse compared with MRD (+) patients (27.8 % vs. 58.0 %, p = 0.031). Patients who were MRD (+) after consolidation therapy (n=3) had an ominous prognosis without HSCT (3-year PFS 0%), while the MRD (+) patients with HSCT (n=3) had 66.7 % 3-year PFS. On multivariate analysis, age (≥35 vs. <35 years, HR 4.535, p = 0.007) and MRD status after induction therapy (+ vs. -, HR 5.250, p = 0.009) were significant prognostic factors, whereas WBC count (≥30,000 vs. <30,000, HR 1.502, p = 0.498) was not. Discussion HSCT for CR1 ALL has the greatest anti-ALL activity. However, HSCT has higher morbidity and mortality rates than chemotherapy. Thus, HSCT should be avoided in patients who have good prognosis with chemotherapy alone. Our results show that patients with molecular remission after induction therapy have an excellent PFS without HSCT, and patients who are MRD (+) after several consolidation therapies have very poor PFS without HSCT. The present study indicates that MRD status after induction and consolidation therapies is a significant prognostic factor. MRD monitoring is useful to determine a clinical indication of HSCT for patients who achieve CR1. For CR1 patients with MRD (+) status after several consolidation therapies, further intensification of chemotherapy may be possible when HSCT is not a suitable option. Disclosures: No relevant conflicts of interest to declare.

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