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.

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.

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

WT1 Monitoring of Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3695-3695 ◽  
Author(s):  
Michele Malagola ◽  
Crisitina Skert ◽  
Enrico Morello ◽  
Francesca Antoniazzi ◽  
Erika Borlenghi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Newly Diagnosed ◽  
End Of Treatment ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Background: Although a complete remission (CR) can be achieved in 70-80% of newly diagnosed acute myeloid leukemia (AML) patients, relapses occur in up to the 50% of cases. Thus, minimal residual disease (MRD) monitoring is a major issue for early detection of patients at high-risk of treatment failure and relapse. Aim: to dynamically evaluate WT1 pan-leukemic molecular marker of MRD in patients with AML. Matherial and methods: 107 newly diagnosed AML patients consecutively treated between 2010 and 2013 were monitored with quantitative WT-1 from bone marrow (BM) and peripheral blood (PB) at baseline, after induction, after the first consolidation course, before allogeneic stem cell transplantation (allo-SCT), at the 3rd and the 6th month after transplantation Results: At diagnosis, 104/107 (97%) had increased PB and BM WT1 levels assessed according to the ELN assay. Eighty-eight out of 107 patients (82%) achieved a complete remission (CR) after induction, 30/88 (34%) relapsed during follow up and 24/107 (22%) were addressed to allogeneic stem cell transplantation (allo-SCT). By univariate analysis, PB-WT > 50x10^4/ABL and BM-WT1 > 250x10^4/ABL after induction (PB: p=0.02; BM: p=0.04), after consolidation (PB: p=0.003), at the end of treatment (PB and BM: p=0.001), at 3rd month of follow up (PB and BM: p=0.005) and at 6th month of follow up (PB: p=0.005) were associated with a reduced overall survival (OS). By multivariate analysis, a BM-WT1 > 250 x 10^4/ABL at the end of treatment was significantly associated with a reduced OS. In order to adapt the cut-off of WT1 in our series of patients, we considered WT1 levels as continuous variables and categorized them at approximately the 25th, 50th, and 75th percentile. A cut-off of PB-WT1 > 25x10^4/ABL and BM-WT1 > 125x10^4/ABL at the end of the treatment program was identified as correlated with reduced leukemia-free survival (LFS) and OS (p=0.001). Similarly, and restricting the analysis on the 24 patients allo-transplanted in CR, 8/11 (73%) with pre-transplant PB-WT1 ≥ 5 and 4/13 (31%) with PB-WT1 < 5 relapsed, respectively (p=0.04). The incidence of relapse was higher in AML patients with PB-WT1 ≥ 5 measured at 3rd (56% vs 38%; p=0.43) and 6th month (71% vs 20%; p=0.03) after allo-SCT. Interestingly, 5/5 (100%) patients with pre-transplant PB-WT1 ≥ 5 who never reduced this level at 3rd or 6th month after allo-SCT experienced a disease recurrence. Conclusions: our data, although retrospectively collected, show that WT1 monitoring may be useful to predict the relapse in AML patients. Acknowledgments: This work was supported in part by Banca di Credito Cooperativo di Pompiano e Franciacorta and Lions Club Bassa Bresciana Association. Disclosures Russo: Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy.

scholarly journals WT1 Peptide Vaccine Is Able to Induce WT1-Specifc Immune Response with TCR Clonal Enrichment to Control Minimal Residual Disease in Patients with Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3984-3984 ◽  
Author(s):  
Hongtao Liu ◽  
Yuanyuan Zha ◽  
Gregory Malnassy ◽  
Noreen Fulton ◽  
Margaret Green ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Cd8 T Cell ◽  
Advisory Committees ◽  
Cell Responses ◽  
Qrt Pcr ◽  
Vaccine Site ◽  
Minimal Residual

Abstract Background: Immunotherapy has the potential for clinical efficacy in patients with myeloid leukemia, especially in the setting of minimal residual disease. WT-1, aberrantly expressed in both myeloid and lymphoid leukemia, is associated with adverse risk in AML. WT1 is highly antigenic and is an attractive target for immunotherapy. The optimal strategy for vaccination to induce CD8+ T cell responses against WT1 is not known. Methods: We performed a pilot randomized study of HLA-A02+ patients to receive vaccination with WT1 126-134 peptide (RMFPNAPYL) in Montanide or in poly ICLC (Hiltonol from Oncovir, a TLR3 agonist) to explore the novel immune adjuvant in patients with myeloid leukemia (NCT01842139). The vaccine was administered q 2 weeks X 6 during the induction phase followed by monthly booster vaccinations X 6 months. Enrollment: Seven patients (4 males, 3 female ages 39 to 73) were randomized. Four patients received WT1 in Montanide (3 AML, 1 CML myeloid blast phase, 2 s/p allo-SCT), and three with WT1 in poly ICLC (2 AML, one MDS RAEB2 s/p allo-SCT). Five patients were in morphologic remission (3 in CR1) and two had very low burden of residual morphologic disease at study entry. Toxicities: All patients finished the induction phase without any major toxicity except mild transient local injection reaction. One patient post allo-SCT on the Montanide arm developed transverse myelitis with evidence of bacterial meningitis following the first monthly booster vaccination. Another patient on the Montanide arm developed aseptic ulceration at the 12th vaccine site followed by inflammation at the 11th WT1 vaccine site, and persistent erythema at the 1st induction vaccine site about 4 weeks after the completion of all 12 WT1 vaccinations. The aseptic ulcers eventually healed with wound care without antibiotics. Efficacy: Three of 4 patients on the Montanide arm had decease of WT1 qRT-PCR levels after WT1 vaccination, and two of them demonstrated generation of WT1-specific cytotoxic CD8+ T cell responses with biased TCR beta chain enrichment. Three patients from who cells were available for TCR alpha and beta CDR3 sequencing had TCR clonal enrichment after WT1 vaccination. In contrast, no obvious WT1-specific immune responses were detected in 2 patients on the poly ICLC arm, nor was there clonal enrichment by TCR alpha/beta sequencing; however, these patients did have a decrease in WT1 qRT-PCR levels and remained in remission 3 years after the initiation of WT1 vaccination. Thus, WT1 peptide in poly ICLC may induce anti-leukemia immune response not detected by our current assays. The third patient on the poly ICLC arm was later found to have A0202 instead of A0201, and thus could serve as negative control. Not surprisingly, this patient did not have a decrease of WT1 qRT-PCR levels nor TCR clonal evolution during vaccination. The patient tolerated the vaccine well without injection reactions and had stable AML for 12 weeks, but the disease progressed before the first monthly WT1 vaccination. Conclusions: WT1 peptide vaccine with Montanide as an adjuvant induces WT1-specific CD8+ T cell responses with TCR clonal and specific TCR beta CDR3 enrichment, which may be capable of controlling leukemia recurrence in the setting of minimal residual disease. Future investigation to combine checkpoint inhibitors with peptide vaccination might further enhance efficacy in patients with myeloid leukemia. Disclosures Liu: Karyopharm: Research Funding; BMS: Research Funding. Salazar:Oncovir Inc: Employment. Odenike:Suneisis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Algeta: Honoraria, Membership on an entity's Board of Directors or advisory committees; Spectrum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Research Funding; CTI/Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gajewski:Abbvie: Consultancy; Celldex: Consultancy, Research Funding; Jounce: Consultancy; Incyte: Consultancy, Research Funding; Evelo: Patents & Royalties: Patent application; BMS: Research Funding; Merck: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding; Bayer: Consultancy; Aduro: Patents & Royalties: Patent application.

scholarly journals The Presence of Minimal Residual Disease, As Determined By Highly Sensitive Quantitation of NPM1-Mutatation, Provided Powerful Prognostic Information in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5097-5097
Author(s):  
Atsushi Marumo ◽  
Hiroki Yamaguchi ◽  
Yuho Najima ◽  
Kensuke Usuki ◽  
Shinichi Kako ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Normal Karyotype ◽  
Npm1 Mutation ◽  
Minimal Residual ◽  
Hematological Remission ◽  
Acute Myeloid

Background: As recurrence of acute myeloid leukemia (AML) is difficult to predict, it is important to detect it by measuring minimal residual disease (MRD). PML-RARA, RUNX-RUNX1T1, CBFB-MYH11 are regarded as the reliable MRD markers. However, in AML with normal karyotype and many other forms, no MRD markers have been established. NPM1 mutations, occurring in approximately 30% of adult AML cases, and 50-60% of AML cases with normal karyotype, represent one of the most frequent mutations in AML. Recently, NPM1 mutation is reported to be useful in assessing MRD. We undertook a retrospective and prospective investigation of the usefulness of NPM1 mutation as an MRD marker in Japanese patients with AML. Methods: The subjects were 38 NPM1-mutated AML patients with first hematological remission at several hospitals related to our institution between 2001 and 2018. This study was approved by the ethics committee of Nippon Medical School and the informed consents were obtained from all patients, according to the Declaration of Helsinki. We analyzed peripheral blood cells or bone marrow cells at diagnoses, and evaluated only bone marrow cells after diagnoses. Detection of NPM1 mutation was carried out using allele-specific real time PCR following creation of a complementary primer. After dilution of the samples, sensitivity to TCTG, CATG, and CCTG was found to be 0.001%. The NPM1 mutant copies were qualified only at successful amplification of internal control. Results: The median age of the patients was 58 years (18-79 years). There were 32 cases with intermediate cytogenetic prognosis and 6 cases with unclear chromosomal profile. Of the 38 cases, 14 cases (37%) were FLT3-ITD-positive and allogeneic hematopoietic stem cell transplantation was carried out in 14 cases (37%). The base sequence was TCTG in 36 cases and CCTG in 2 cases. Persistence of NPM1-mutatation was present in 25 patients with first hematological remission (66%). Compared with patients with MRD negative, patients with MRD positive were associated with DNMT3A mutation (MRD positive 12/25 vs MRD negative 0/13, p=0.003). The rate of relapse in patients with MRD positive was significantly higher than those of in patients with MRD negative (MRD positive 76% vs MRD negative 23%, p=0.004). The rates of relapse free survival (RFS) and overall survival (OS) in patients with MRD positive were significantly lower than those in patients with MRD negative (RFS at 2 years: MRD positive 14% vs MRD negative 86% p=0.003; Figure 1, OS at 2 years: MRD positive 25% vs MRD negative 93%, p<0.001). In FLT3-ITD negative group, the rates of RFS in patients with MRD positive were significantly lower than those in patients with MRD negative. (RFS at 2 years: MRD positive 21% vs MRD negative 92% p=0.001; Figure 1). Conclusion: The presence of MRD with NPM1 mutation is significantly associated with relapse and it is useful to decide their treatment strategy. Especially, there is the usefulness of NPM1 mutation as an MRD marker in NPM1 positive Flt3-ITD negative AML patients who are generally classified as favorable risk. According to previous reports, it is known that NPM1-mutated AML sometimes relapse with losing NPM1 mutations. However, in this study, all NPM1-mutated AML relapse without losing NPM1 mutations. We need to collect more patients and are going to confirm whether there are patients who relapse with losing NPM1 mutations or not. We plan to analyze the genetic background of MRD positive and negative patients with next-generation sequencing. We are going to announce the genetic characteristics in addition to this result at ASH. Disclosures Usuki: Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Kako:Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

scholarly journals Molecular Minimal Residual Disease Detection in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-30-SCI-30
Author(s):  
Peter Valk
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Patient Specific ◽  
Molecular Abnormalities ◽  
Minimal Residual ◽  
Risk Of Relapse ◽  
Acute Myeloid

Abstract Minimal residual disease (MRD) detection based on the standardized molecular monitoring of the t(9;22)-related BCR-ABL1 fusion transcript is well established for patients with chronic myeloid leukemia (CML). The levels of BCR-ABL1 serve as a guide to tailor treatment of the CML patient. In acute myeloid leukemia (AML) MRD detection based on polymerase chain reaction (PCR) approaches targeted towards the acquired molecular abnormalities is less well established. MRD measurement of the CBFB-MYH11 and RUNX1-RUNX1T1 fusion transcripts after induction therapy has been shown to be of some clinical importance. However, these transcripts can persist during long term complete remission, without having an effect on treatment outcome. In contrast, sequential MRD monitoring of the PML-RARA fusion transcript in acute promyelocytic leukemia (APL) is a strong predictor of relapse. Initial molecular MRD studies were limited to these favorable AML subtypes. Due to the discovery of novel recurrent abnormalities in AML the potential of molecular MRD detection has increased substantially. Although, certain acquired mutations, such as those in NPM1, are known for a number of years, only recently the application of these molecular abnormalities for MRD detection has been investigated in larger clinical trials. By NPM1 mutant MRD detection we can now recognize patients with higher risk of relapse. Highly sensitive targeted detection of the hotspot mutations in AML subsets is feasible by means of real-time PCR, but detection of patient specific mutations with this technology is still challenging. Next generation sequencing (NGS) revealed that AML is an extremely heterogeneous disease, as illustrated by the multitude of acquired mutations, but this technology has also opened possibilities for detection of MRD in virtually every patient. With NGS there is no need for patient specific assays since practically all mutations are detected. These molecular abnormalities, as single marker or in combination, will most certainly improve MRD monitoring of AML. However, it remains yet to be determined how MRD levels are assessed and which combination of markers in a MRD detection result in clinically relevant information, requiring extensive validation in large clinical AML trials. Smaller studies already demonstrated the variable dynamics of MRD during treatment and associations between somatic mutations persistence and risk of relapse. However, clonal hematopoiesis of undetermined potential, i.e., preleukemic mutations that may persist after treatment, provides an extra layer of complexity to the applicability of MRD detection. For example, the clinical applicability of MRD detection in the setting of mutant DNMT3A and IDH mutations is likely less effective due to the persistent DNMT3A and IDH mutant preleukemic cells following treatment. However, should all mutations be cleared after treatment or can preleukemic mutations in otherwise normal hematopoiesis persist without resulting in relapse? Taken together, there is need for molecular approaches to understand the dynamics of residual disease in AML during treatment. Disclosures No relevant conflicts of interest to declare.

Quantitative Molecular Expression of the Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase in Acute Myeloid Leukemia Cells as a Possible Marker for Minimal Residual Disease Detection.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4229-4229
Author(s):  
Michela Aluigi ◽  
Antonio Curti ◽  
Emanuela Ottaviani ◽  
Giovanna Lucchetti ◽  
Valentina Salvestrini ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mrna Expression ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemia Cells ◽  
Qrt Pcr ◽  
Acute Myeloid Leukemia Cells ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract The expression of the catalytic enzyme of tryptophan, indoleamine 2,3 dioxygenase (IDO) has been identified as a T-cell inhibitor effector pathway in different normal and neoplastic cells. We have recently shown that normal bone marrow (BM) cells, including hematopoietic CD34+ cells, express IDO mRNA only upon IFN-γ stimulation, whereas in a subset of human acute myeloid leukemia cells (AML) IDO is constitutively expressed both at molecular and protein level and induces immunological escape by promoting the generation of T-reg cells. To investigate whether the IDO transcript can be used as a marker for minimal residual disease (MRD) detection, we used a sensitive real-time reverse transcription-polymerase reaction assay (qRT-PCR) to quantify IDO mRNA levels in peripheral blood (PB) and BM samples of newly diagnosed AML patients. The level of IDO transcript was evaluated as IDO copy number/104 ABL copies. As control samples, we used normal PB and BM mononuclear cells (MNCs). Our data showed that normal BM and PB cells expressed minimal amount of IDO mRNA (range 100–1000). Among AML samples, we identified three subsets of patients according to IDO mRNA expression: 1) 33/74 (44.6%) IDO negative (i.e. range < 100), 28/74 (37.84%) IDO low (range 100–1000) and 13/74 (17.6%) IDO high (i.e. range > 1000). BM and PB AML blasts gave similar results. Assessment of protein expression and enzymatic activity was in accordance with molecular results. Some patients were evaluated for IDO mRNA expression before and after induction chemotherapy and the IDO levels were found to correlate with the reduction of BM blasts. Taken together, our qRT-PCR data demonstrate that normal PB and BM cells are negative for IDO mRNA expression, which, in turn, is significantly up-regulated in a subset of AML patients (IDO high) and IDO mRNA expression correlates with tumor burden, thus suggesting its possible role in the detection of MRD in IDO high patients.

Monitoring of minimal residual disease in acute myeloid leukemia with frequent and rare patient-specific NPM1 mutations

2010 ◽  
Vol 85 (12) ◽  
pp. 926-929 ◽  
Author(s):  
Dana Dvorakova ◽  
Zdenek Racil ◽  
Ivana Jeziskova ◽  
Ivo Palasek ◽  
Marketa Protivankova ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Patient Specific ◽  
Minimal Residual ◽  
Acute Myeloid

Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B–ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2315-2323 ◽  
Author(s):  
Tomasz Szczepański ◽  
Marja J. Willemse ◽  
Bas Brinkhof ◽  
Elisabeth R. van Wering ◽  
Mirjam van der Burg ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Patient Specific ◽  
Gene Rearrangements ◽  
Secondary Acute Myeloid Leukemia ◽  
Minimal Residual ◽  
Selection Of ◽  
Acute Myeloid

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecular studies in 96 childhood precursor-B–ALL at diagnosis and at relapse (n = 91) or at presumably secondary acute myeloid leukemia (n = 5). Clonal Ig and TCR targets for MRD detection were identified in 94 patients, with 71% of these targets being preserved at relapse. The best stability was found for IGK-Kde rearrangements (90%), followed byTCRG (75%), IGH (64%), and incompleteTCRD rearrangements (63%). Combined Southern blot and PCR data for IGH, IGK-Kde, and TCRDgenes showed significant differences in stability at relapse between monoclonal and oligoclonal rearrangements: 89% versus 40%, respectively. In 38% of patients all MRD-PCR targets were preserved at relapse, and in 40% most of the targets (≥ 50%) were preserved. In 22% of patients most targets (10 cases) or all targets (10 cases) were lost at relapse. The latter 10 cases included 4 patients with secondary acute myeloid leukemia with germline Ig/TCR genes. In 5 other patients additional analyses proved the clonal relationship between both disease stages. Finally, in 1 patient all Ig/TCR gene rearrangements were completely different between diagnosis and relapse, which is suggestive of secondary ALL. Based on the presented data, we propose stepwise strategies for selection of stable PCR targets for MRD monitoring, which should enable successful detection of relapse in most (95%) of childhood precursor-B–ALL.

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